Fungal diversity and metabolomic profiles in GM and isogenic non-GM maize cultivars from Brazil.

There is little knowledge of the microbial diversity, mycotoxins and associated secondary metabolites in GM maize and isogenic non-GM cultivars (cvs). This study has quantified the microbial populations and dominant fungal genera in 6 cvs of each type representative of herbicide, pesticide or stacked resistance to both. The predominant mycotoxins and targeted metabolomics profiles were also compared between the two sets of cvs. This showed that the overall fungal populations were 8.8 CFUs g-1 maize. The dominant genera, isolated from maize samples, whether surface-sterilised or not, in all maize cvs were Fusarium, followed by Penicillium, Aspergillus and occasionally Cladosporium and Alternaria. The analysis of the targeted metabolomics showed that approx. 29 different metabolites were detected. These were dominated by fumonisins and minor Penicillium spp. metabolites (questiomycin A and rugulovasine A). Interestingly, the range and number of mycotoxins present in the GM cvs were significantly lower than in the non-GM maize samples. This suggests that while the fungal diversity of the two types of maize appeared to be very similar, the major contaminant mycotoxins and range of toxic secondary metabolites were much lower in the GM cvs.

Proof of concept: could snake venoms be a potential source of bioactive compounds for control of mould growth and mycotoxin production.

The objective was to screen 10 snake venoms for their efficacy to control growth and mycotoxin production by important mycotoxigenic fungi including Aspergillus flavus, Aspergillus westerdijkiae, Penicillium verrucosum, Fusarium graminearum and F. langsethiae. The Bioscreen C rapid assay system was used. The venoms from the Viperidae snake family delayed growth of some of the test fungi, especially F. graminearum and F. langsethiae and sometimes A. flavus. Some were also able to reduce mycotoxin production. The two most potent crude snake venoms (Naja nigricollis and N. siamensis; 41 and 43 fractions, respectively) were further fractionated and 83/84 of these fractions were able to reduce mycotoxin production by >90% in two of the mycotoxigenic fungi examined. This study suggests that there may be significant potential for the identification of novel fungistatic/fungicidal bioactive compounds as preservatives of raw and processed food commodities post-harvest from such snake venoms.

Efficacy of metabolites of a Streptomyces strain (AS1) to control growth and mycotoxin production by Penicillium verrucosum, Fusarium verticillioides and Aspergillus fumigatus in culture.

The objectives of this study were to determine the efficacy of metabolites of a Streptomyces strain AS1 on (a) spore germination, (b) mycelial growth, (c) control of mycotoxins produced by Penicillium verrucosum (ochratoxin A, OTA), Fusarium verticillioides (fumonisins, FUMs) and Aspergillus fumigatus (gliotoxin) and (d) identify the predominant metabolites involved in control. Initial screening showed that the Streptomyces AS1 strain was able to inhibit the mycelial growth of the three species at a distance, due to the release of secondary metabolites. A macroscopic screening system showed that the overall Index of Dominance against all three toxigenic fungi was inhibition at a distance. Subsequent studies showed that the metabolite mixture from the Streptomyces AS1 strain was very effective at inhibiting conidial germination of P. verrucosum, but less so against conidia of A. fumigatus and F. verticillioides. The efficacy was confirmed in studies on a conducive semi-solid YES medium in BioScreen C assays. Using the BioScreen C and the criteria of Time to Detection (TTD) at an OD = 0.1 showed good efficacy against P. verrucosum when treated with the Streptomyces AS1 extract at 0.95 and 0.99 water activity (aw) when compared to the other two species tested, indicating good efficacy. The effective dose for 50% control of growth (ED50) at 0.95 and 0.99 aw were approx. 0.005 ng/ml and 0.15 µg/ml, respectively, with the minimum inhibitory concentration (MIC) at both aw levels requiring > 40 µg/ml. In addition, OTA production was completely inhibited by 2.5 µg/ml AS1 extract at both aw levels in the in vitro assays. Ten metabolites were identified with four of these being predominant in concentrations > 2 µg/g dry weight biomass. These were identified as valinomycin, cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val) and brevianamide F.

Carbon dioxide production as an indicator of Aspergillus flavus colonisation and aflatoxins/cyclopiazonic acid contamination in shelled peanuts stored under different interacting abiotic factors.

Aspergillus flavus is the main xerophylic species colonising stored peanuts resulting in contamination with aflatoxins (AFs) and cyclopiazonic acid (CPA). This study evaluated the relationship between storage of shelled peanuts under interacting abiotic conditions on (a) temporal respiration (R) and cumulative CO2 production, (b) dry matter losses (DMLs) and (c) aflatoxin B1 (AFB1) and CPA accumulation. Both naturally contaminated peanuts and those inoculated with A. flavus were stored for 7-days under different water activities (aw; 0.77-0.95) and temperatures (20-35°C). There was an increase in the temporal CO2 production rates in wetter and warmer conditions, with the highest respiration at 0.95 aw + A. flavus inoculum at 30°C (2474 mg CO2kg-1h-1). The DMLs were modelled to produce contour maps of the environmental conditions resulting in maximum/minimum losses. Maximum mycotoxin contamination was always at 0.95 aw although optimal temperatures were 25-30°C for AFs and 30-35°C for CPA. These results showed a correlation between CO2 production and mycotoxin accumulation. They also provide valuable information for the creation of a database focused on the development of a post-harvest decision support system to determine the relative risks of contamination with these mycotoxins in stored shelled peanuts.

Influence of storage environment on maize grain: CO2 production, dry matter losses and aflatoxins contamination.

Poor storage of cereals, such as maize can lead to both nutritional losses and mycotoxin contamination. The aim of this study was to examine the respiration of maize either naturally contaminated or inoculated with Aspergillus flavus to examine whether this might be an early and sensitive indicator of aflatoxin (AF) contamination and relative storability risk. We thus examined the relationship between different interacting storage environmental conditions (0.80-0.99 water activity (aw) and 15-35°C) in naturally contaminated and irradiated maize grain + A. flavus on relative respiration rates (R), dry matter losses (DMLs) and aflatoxin B1 and B2 (AFB1-B2) contamination. Temporal respiration and total CO2 production were analysed by GC-TCD, and results used to calculate the DMLs due to colonisation. AFs contamination was quantified at the end of the storage period by HPLC MS/MS. The highest respiration rates occurred at 0.95 aw and 30-35°C representing between 0.5% and 18% DMLs. Optimum AFs contamination was at the same aw at 30°C. Highest AFs contamination occurred in maize colonised only by A. flavus. A significant positive correlation between % DMLs and AFB1 contamination was obtained (r = 0.866, p < 0.001) in the irradiated maize treatments inoculated with A. flavus. In naturally contaminated maize + A. flavus inoculum loss of only 0.56% DML resulted in AFB1 contamination levels exceeding the EU legislative limits for food. This suggests that there is a very low threshold tolerance during storage of maize to minimise AFB1 contamination. This data can be used to develop models that can be effectively used in enhancing management for storage of maize to minimise risks of mycotoxin contamination.

Impact of climate change environmental conditions on the resilience of different formulations of the biocontrol agent Candida sake CPA-1 on grapes.

Biocontrol agents have become components of integrated crop protection systems for controlling economically important fungal pathogens. Candida sake CPA-1 is a biocontrol agent of fungal pathogens of fruits, both pre- and post-harvest. While the efficacy of different formulations have been examined previously, few studies have considered the resilience of different formulations under changing climatic conditions of elevated temperature, drought stress and increased atmospheric CO2 . This study examined the effect of (a) temperature × RH × elevated CO2 (400 vs 1000 ppm) on the temporal establishment and viability of two dry and one liquid C. sake CPA-1 formulations on grape berry surfaces; (b) temperature stress (25 vs 35°C); and (c) elevated CO2 levels. Results indicated that temperature, RH and CO2 concentration influenced the establishment and viability of the formulations but there was no significant difference between formulations. For the combined three-component factors, increased temperature (35°C) and lower RH (40%) reduced the viable populations on grapes. The interaction with elevated CO2 improved the establishment of viable populations of the formulations tested. Viable populations greater than Log 4 CFUs per g were recovered from the grape surfaces suggesting that these had conserved resilience for control of Botrytis rot in grapes. SIGNIFICANCE AND IMPACT OF THE STUDY: The interaction between environmental factors that are expected to occur in response to climate change (CC) will have a significant impact on food security and availability. Little information exists on how elevated temperature, drought stress and increased CO2 will have on the efficacy of biocontrol agents. The impact of these factors on the viability of different formulations of the biocontrol yeast Candida sake on the surface of grapes berries was evaluated for the first time. Such knowledge is critical for projecting the efficacy of biocontrol under climate change conditions and to identify formulations that have the necessary resilience to perform under CC conditions.

Efficacy of different caffeine concentrations on growth and ochratoxin A production by Aspergillus species.

UNLABELLED: The objective of this study was to evaluate the effect of different caffeine concentrations (0-4%) on (i) lag phase prior to growth, (ii) growth rates and (iii) ochratoxin A (OTA) production by strains from the Aspergillus section Circumdati and Aspergillus section Nigri groups, isolated from coffee, when grown on a conducive medium at 0·98 water activity and 30°C. The lag phases prior to growth increased with caffeine concentration. A strain of Aspergillus niger and Aspergillus carbonarius were the most sensitive to caffeine with growth being inhibited by <1% caffeine. For strains of Aspergillus westerdijkiae, Aspergillus ochraceus and Aspergillus steynii, although growth was inhibited significantly, some growth (10-15% of controls) occurred in 4% caffeine. OTA production was significantly inhibited by only 0·5% caffeine for strains of A. westerdijkiae, A. niger and A. carbonarius. For A. steynii at least 1·5% caffeine was required to inhibit OTA production. In contrast, for the strain of A. ochraceus there was a stimulation of OTA at 3% with a reduction at 4% caffeine. These results are discussed in the context of the different concentrations of caffeine found in Arabica and Robusta coffee and the development of minimization strategies. SIGNIFICANCE AND IMPACT OF THE STUDY: Arabic (0·6%) and Robusta coffee (4%) have significantly different amounts of endogenous caffeine. The growth of six ochratoxigenic fungi which contaminate coffee with ochratoxin A (OTA) had differential tolerance/sensitivity to concentrations of caffeine in vitro in this range. However, low concentrations of caffeine (<0·5%) was inhibitory to OTA production. These results are discussed in the context of the potential for using such information for the design of minimization strategies to control mycotoxin production in such products.

Comparison of five different C18 HPLC analytical columns for the analysis of ochratoxin A in different matrices.

The performance of five different C18 chromatography analytical columns with different lengths, particle sizes and porosities were compared for analysis of ochratoxin A (OTA) in fungal cultures and raisin samples. Chromatographic parameters including retention time, limit of detection, limit of quantification, number of theoretical plates and reduced plate height were obtained and compared. This showed that, compared with traditional columns, shorter ones (100 and 75mm×4.6mm) with 2.7µm solid core particles are suitable for analysis of OTA in different matrices and allows a reduction of the total analysis time by approximately 50% without any detrimental effect on performance. This leads to significant reduction in analysis costs by savings in use of organic solvents and increasing the total number of analyses per day. The capability of these columns for analyzing samples, from different matrices, was assessed by analyzing OTA-contaminated samples from cultures of Aspergillus westerdijkiae and Aspergillus niger grown on a defined nutritional media (yeast extract sucrose agar) and from natural and OTA spiked raisins.

Combined effects of benomyl and environmental factors on growth and expression of the fumonisin biosynthetic genes FUM1 and FUM19 by Fusarium verticillioides.

Fusarium verticillioides is predominantly responsible of fumonisin contamination of maize and other cereals in Mediterranean climatic regions. This study examined the interaction of the fungicide benomyl, at ED50 and ED90 concentrations (effective doses of benomyl to reduce growth by 50% and 90%, respectively), with a range of temperatures (20-35 °C) and water potentials (-0.7, -2.8 and -7.0 MPa) compatible with current and foreseen climate change scenarios for these regions on growth and fumonisin biosynthesis in in vitro assays. The expression of fumonisin biosynthetic genes (FUM1 and FUM19) was quantified by real time RT-PCR. FUM1 encodes a polyketide synthase and FUM19 an ABC-type transporter, located both in the fumonisin biosynthetic cluster. The ED50 and ED90 concentrations obtained at 25 °C were 0.93 mg/L and 3.30 mg/L, respectively. Benomyl affected growth and fumonisin gene expression differently but it generally reduced fungal growth and fumonisin biosynthesis and both were significantly affected by temperature and water potential. This indicated that efficacy of benomyl might be compromised at certain conditions, although at similar or lower levels than other fungicides tested. Both fumonisin biosynthetic genes had similar expression patterns in all treatments and their correlation was positive and significant. The results suggested that Mediterranean climatic scenarios might suffer an additional negative impact of climate change by reducing the efficacy of antifungals used to control pathogens and toxigenic fungi.

Comparison of three different C18 HPLC columns with different particle sizes for the optimization of aflatoxins analysis.

In this work we compared the performance of chromatography columns with particles of 5 and 3 µm with the new 2.7 µm solid core particles for the analysis of aflatoxins B1, G1, B2, and G2 using trifluoroacetic acid pre-column derivatization. Three different columns have been used and chromatographic parameters as retention time, resolution, limit of detection (LOD), limit of quantification (LOQ) were obtained from all of them and compared. The results show that comparing with the traditional columns, shorter columns (100 mm × 4.6 mm) with the new solid core particles are suitable for the analysis of these mycotoxins and allowed the reduction of the analysis time by 45.5% and 33.3% with respect to columns with particle size 5 µm (150 mm × 4.6 mm) and 3 µm (150 mm × 4.6 mm) respectively, without any detrimental effect on performance. This leads to the reduction of the analysis costs by saving on organic solvents and increasing the total number of analyses per day. The capability of these columns for analyzing samples, in different culture media, was assessed by analyzing different samples from: yeasts extract sucrose medium, corn meal agar medium and fresh hazelnut media.

Effect of fenpropimorph, prochloraz and tebuconazole on growth and production of T-2 and HT-2 toxins by Fusarium langsethiae in oat-based medium.

Fusarium langsethiae has been isolated from infected cereals in central and northern Europe where it has been identified in the last decade as the main species involved in the occurrence of high levels of T-2 and HT-2 toxins, mainly in oats. The efficacy of three fungicides (prochloraz, tebuconazole, fenpropimorph) for controlling growth of two strains of F. langsethiae isolated from oats was examined at 0.96 and 0.98 a(w) at 15, 20 and 25 °C on oat-based media. The concentrations necessary for 50 and 90% growth inhibition (ED50 and ED90 values) were determined. The effect on the trichothecene type A mycotoxins T-2 and HT-2 was also determined. Without fungicides both strains grew faster at 0.98 than at 0.96 a(w) and the influence of temperature on growth rates was 25>20>15 °C. Prochloraz and tebuconazole were more effective than fenpropimorph against F. langsethiae. Strain, temperature and type of fungicide significantly influenced the ED50 and ED90 values for growth. The concentration ranges under different environmental conditions were: prochloraz (0.03-0.1 and 0.3-1.5), tebuconazole (0.06-0.9 and 1.3-8.2), and fenpropimorph (22-59 and 125-215 mg l⁻¹). Production of T-2 and HT-2 toxins was influenced by temperature, a(w), type of fungicide and dose. Levels of T-2 were usually higher than those of HT-2 under the same conditions. The biosynthesis of T-2 toxin increased after 10 day incubation, but was reduced with decreasing temperature and increasing fungicide dose. At 0.98 a(w) T-2 levels increased in cultures containing fenpropimorph while at 0.96 a(w) the toxin concentrations increased in response to the other two fungicides. Low doses of prochloraz or tebuconazole enhanced toxin production when compared with untreated cultures for strain 2004-59 at 0.96 a(w) and 20-25 °C. HT-2 was hardly detectable in the treatments with prochloraz or tebuconazole at 0.98 a(w). This is the first study on the effect of these anti-fungal compounds on control of growth of F. langsethiae and on production of T-2 and HT-2 toxins in oat-based media.

Temperature and water activity effects on production of T-2 and HT-2 by Fusarium langsethiae strains from north European countries.

This study has examined the effect of ecophysiological factors, water activity (a(w), 0.995-0.90) and temperature (10-37 °C), on the T-2 and HT-2 toxins production by Fusarium langsethiae. Two dimensional profiles for optimum and marginal conditions have been built for two strains from each of four northern European countries (UK, Norway, Sweden, Finland) on an oat-based medium. This showed that the optimum a(w) and temperature conditions for T-2 + HT-2 production was between 0.98-0.995, and 20-30 °C respectively. Kruskal-Wallis analysis of ranks showed a statistically significant differences between the different a(w) levels examined (P < 0.001) but no significant effect of the temperatures examined. The ratio of HT-2/T-2 was investigated and non-uniform distribution of HT-2 toxin was found under different ecological conditions. No statistically significant differences were found for the mean toxin production between strains from the different countries. Intra-strain differences in toxin production was only found for those from Finland (P-value = 0.0247). The growth/no growth and toxin/no toxin conditions in relation to a(w) x temperature have been constructed for the first time. This knowledge will be useful in developing prevention strategies to minimise T-2 and HT-2 toxin contamination by strains of F. langsethiae on important small grain cereals.

Modelling the relationship between environmental factors, transcriptional genes and deoxynivalenol mycotoxin production by strains of two Fusarium species.

The effect of changes in temperature/water activity (a(w)) on growth, deoxynivalenol (DON) production and trichothecene gene cluster expression (18 genes) for strains of Fusarium culmorum and Fusarium graminearum was studied. The expression data for six key transcription genes (TRI4, TRI5, TRI6, TRI10, TRI12 and TRI13) were analysed using multiple regression analyses to model the relationship between these various factors for the first time. Changes in a(w) and temperature significantly (p = 0.05) affected growth and DON. Microarray data on expression of these genes were significantly related to DON production for both strains. Multi-regression analysis was done and polynomial models found to best fit the relationship between actual/predicted DON production relative to the expression of these TRI genes and environmental factors. This allowed prediction of the amounts of DON produced in two-dimensional contour maps to relate expression of these genes to either a(w) or temperature. These results suggest complex interactions between gene expression (TRI genes), environmental factors and mycotoxin production. This is a powerful tool for understanding the role of these genes in relation to environmental factors and enables more effective targeted control strategies to be developed.

Comparisons of water activity and temperature impacts on growth of Fusarium langsethiae strains from northern Europe on oat-based media.

This study has examined the effect of water activity (a(w), 0.995-0.90) and temperature (10-37 degrees C) on the lag phases prior to growth, growth rates and used models to develop two dimensional profiles for optimum and marginal conditions for two strains of Fusarium langsethiae from four northern European countries (UK, Norway, Sweden, and Finland) on an oat-based medium. Results showed that the optimum a(w) for growth was at 0.98-0.995 and 25 degrees C. The limit for growth of the strains was at 0.92-0.93 a(w) with minima of 10 degrees C. No growth occurred at 37 degrees C. The lag phases prior to growth were lowest under optimum conditions and extended to >10days at marginal conditions. Statistical analyses of intra and inter-strain differences in terms of both lag phases prior to growth and growth rates were not statistically significant. However, a(w) and temperature were statistically significant factors. Two dimensional profiles for strains from each country of origin were built to identify optimum and marginal conditions for F. langsethiae for the first time. These environmental profiles will be beneficial for improving the ecological knowledge of this species which is able to produce trichothecene mycotoxins in a range of temperate cereals.

Temporal monitoring of the nor-1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B1 production during storage of peanuts under different water activity levels.

AIMS: A relative quantification system (RQ-PCR) was used to monitor the correlations between the activity of the nor-1 (=aflD) gene of Aspergillus flavus using real-time PCR in relation to phenotypic aflatoxin B(1) (AFB(1) ) production and populations of A. flavus in stored peanuts at three water activity levels (a(w) , 0·95, 0·90 and 0·85) for 6 weeks. METHODS AND RESULTS: Real-time PCR was used to amplify the nor-1 gene (target gene), and benA56 (ß-tubulin gene) used as a control gene. Expression of three structural genes, nor-1 (=aflD), ver-1 (=aflM), and omtA (=aflP), and the regulatory gene aflR of the aflatoxin biosynthetic pathway were also assayed. There were significant differences between nor-1 gene expression at the three a(w) levels; higher expression at 0·90 a(w) in weeks 1-3, when compared to 0·95. In contrast, in the driest treatment (0·85 a(w) ) none or very low nor-1 expression occurred. The populations of A. flavus colony-forming units (CFUs g(-1) ) increased over time with the highest at 0·95 a(w) . Highest AFB(1) production was at 0·90 and 0·95 a(w) from weeks 3-6. A(w) had a significant effect on aflR transcription at 0·95 a(w) over the 6-week period, while at 0·90 a(w) , only in the last 2 weeks. CONCLUSIONS: Correlations between different factors showed that log AFB(1) × log CFUs, log AFB(1) × a(w) , and log CFUs × a(w) were statistically significant, while log CFUs × RQ-PCR and RQ-PCR × a(w) were not. The AflR gene may not have an important role in the regulation of nor-1 expression in food matrices (e.g. peanuts). SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of correlations between nor-1 expression and aflatoxin production by A. flavus in raw peanuts under different a(w) levels could be helpful to predict potential risk of aflatoxin production during storage of this hygroscopic food product and minimize contamination with the AFB(1) .

The future of early disease detection? Applications of electronic nose technology in otolaryngology.

INTRODUCTION: Recent advances in electronic nose technology, and successful clinical applications, are facilitating the development of new methods for rapid, bedside diagnosis of disease. There is a real clinical need for such new diagnostic tools in otolaryngology. MATERIALS AND METHODS: We present a critical review of recent advances in electronic nose technology and current applications in otolaryngology. RESULTS: The literature reports evidence of accurate diagnosis of common otolaryngological conditions such as sinusitis (acute and chronic), chronic suppurative otitis media, otitis externa and nasal vestibulitis. A significant recent development is the successful identification of biofilm-producing versus non-biofilm-producing pseudomonas and staphylococcus species. CONCLUSION: Electronic nose technology holds significant potential for enabling rapid, non-invasive, bedside diagnosis of otolaryngological disease.

Geostatistical analysis of the spatial distribution of mycotoxin concentration in bulk cereals.

Deoxynivalenol (DON) and ochratoxin A (OTA) in agricultural commodities present hazards to human and animal health. Bulk lots are routinely sampled for their presence, but it is widely acknowledged that designing sampling plans is particularly problematical because of the heterogeneous distribution of the mycotoxins. Previous studies have not explicitly looked at the interactions between the spatial distribution of the mycotoxin and the strategy used to take samples from bulk. Sampling plans are therefore designed on the assumption of random distributions. The objective of this study was to analyse the spatial distribution of DON and OTA in bulk commodities with geostatistics. This study was the first application of geostatistical analysis to data on mycotoxins contamination of bulk commodities. Data sets for DON and OTA in bulk storage were collected from the literature and personal communications, of which only one contained data suitable for geostatistical analysis. This data set represented a 26-tonne truck of wheat with a total of 100 sampled points. The mean concentrations of DON and OTA were 1342 and 0.59 microg kg(-1), respectively. The results showed that DON presented spatial structure, whilst OTA was randomly distributed in space. This difference between DON and OTA probably reflected the fact that DON is produced in the field, whereas OTA is produced in storage. The presence of spatial structure for DON implies that sampling plans need to consider the location of sample points in addition to the number of points sampled in order to obtain reliable estimates of quantities such as the mean contamination.

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 (a(w)). METHODS AND RESULTS: A full factorial design was followed between the factors considered. The a(w) levels assayed were 0.850, 0.880, 0.900, 0.920, 0.940, 0.960, 0.980 and the incubation temperatures were 10, 15, 20, 25, 30, 35 and 40 degrees C. Growth and OTA production responses were evaluated for a period of 25 days. Regarding growth boundaries, the degree of agreement between predictions and observations was >99% concordant for both isolates. The erroneously predicted growth cases were 3.4-4.1% false-positives and 0.7-1.4% false-negatives. No growth was observed at 10 degrees C and 40 degrees C for all a(w) levels assayed, with the exception of 0.980 a(w)/40 degrees C, where weak growth was observed. Similarly, OTA production was correctly predicted with a concordance rate >98% for the two isolates with 0.7-1.4% accounting for false-positives and 2.0-2.7% false-negatives. No OTA production was detected at 10 degrees C or 40 degrees C regardless of a(w), and at 0.850 a(w) at all incubation temperatures. With respect to time, the OTA production boundary shifted to lower temperatures (15-20 degrees C) as opposed to the growth boundary that shifted to higher temperature levels (25-30 degrees C). Using two literature datasets for growth and OTA production of A. carbonarius on the same growth medium, the logistic model gave one false-positive and three false-negative predictions out of 68 growth cases and 13 false-positive predictions out of 45 OTA production cases. CONCLUSIONS: The results of this study suggest that the logistic regression model can be successfully used to predict growth and OTA production interfaces for A. carbonarius in relation to temperature and a(w). SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed modelling approach helps the understanding of fungal-food ecosystem relations and it could be employed in risk analysis implementation plans to predict the risk of contamination of grapes and grape products by A. carbonarius.

Impact of mild heat treatments on induction of thermotolerance in the biocontrol yeast Candida sake CPA-1 and viability after spray-drying.

AIMS: The objective of this study was to examine the induction of thermotolerance in the biocontrol agent Candida sake CPA-1 cells by mild heat treatments to enhanced survival of formulations using spray-drying. The possible role of heat-shock proteins (HSPs) biosynthesis in induced thermotolerance and the role of sugars and sugar alcohols were also determined. METHODS AND RESULTS: Studies were conducted on C. sake cells grown in molasses medium and exposed to mild temperatures of 30 and 33 degrees C during mid- (16 h), late-exponential (24 h), early- (30 h) and mid-stationary (36 h) growth phases. The effect on viability was determined both before and after spray-drying. Cycloheximide and chloramphenicol were used to examine the role of HSPs and HPLC was used to analyse the accumulation of sugar and sugar alcohols. The results indicate that both temperatures induced thermotolerance in cells of C. sake. Mild heat-adapted cells at 33 degrees C in the early- or mid-stationary phases had survival values after spray-drying significantly higher (P