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There is interest in understanding the relationship between naturally contaminated commodities and the potential for the production of different useful and toxic secondary metabolites (SMs). This study examined the impact of interacting abiotic stress parameters of water availability and temperature of stored naturally contaminated maize on the SM production profiles. Thus, the effect of steady-state storage water activity (aw; 0.80–0.95) and temperature (20–35 °C) conditions on SM production patterns in naturally contaminated maize was examined. The samples were analysed using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) to evaluate (a) the total number of known SMs, (b) their concentrations, and (c) changes under two-way interacting environmental stress conditions. A total of 151 metabolites were quantified. These included those produced by species of the Aspergillus, Fusarium and Penicillium genera and other unspecified ones by other fungi or bacteria. There were significant differences in the numbers of SMs produced under different sets of interacting environmental conditions. The highest total number of SMs (80+) were present in maize stored at 20–25 °C and 0.95 aw. In addition, there was a gradation of SM production with the least number of SMs (20–30) produced under the driest conditions of 0.80 aw at 20–30 °C. The only exception was at 35 °C, where different production patterns occurred. There were a total of 38 Aspergillus-related SMs, with most detected at >0.85 aw, regardless of the temperature in the 50–500 ng/g range. For Fusarium-related SMs, the pattern was different, with approx. 10–12 SMs detected under all aw × temperature conditions with >50% produced at 500 ng/g. A total of 40–45 Penicillium-related SMs (50–500 ng/g) were detected in the stored maize but predominantly at 20–25 °C and 0.95 aw. Fewer numbers of SMs were found under marginal interacting abiotic stress storage conditions in naturally contaminated maize. There were approx. eight other known fungal SM present, predominantly in low concentrations (<50 ng/g), regardless of interacting abiotic conditions. Other unspecified SMs present consisted of <20 in low concentrations. The effect of interacting abiotic stress factors for the production of different suites of SMs to take account of the different ecological niches of fungal genera may be beneficial for identifying biotechnologically useful SMs. [ABSTRACT FROM AUTHOR]

Zearalenone (ZEN) contamination from Fusarium graminearum colonization is particularly important in food and feed wheat, especially during post-harvest storage with legislative limits for both food and feed grain. Indicators of the relative risk from exceeding these limits would be useful. We examined the effect of different water activities (aw; 0.95-0.90) and temperature (10-25 °C) in naturally contaminated and irradiated wheat grain, both inoculated with F. graminearum and stored for 15 days on (a) respiration rate; (b) dry matter losses (DML); (c) ZEN production and (d) relationship between DML and ZEN contamination relative to the EU legislative limits. Gas Chromatography was used to measure the temporal respiration rates and the total accumulated CO2 production. There was an increase in temporal CO2 production rates in wetter and warmer conditions in all treatments, with the highest respiration in the 25 °C × 0.95 aw treatments + F. graminearum inoculation. This was reflected in the total accumulated CO2 in the treatments. The maximum DMLs were in the 0.95 aw/20-25 °C treatments and at 10 °C/0.95 aw. The DMLs were modelled to produce contour maps of the environmental conditions resulting in maximum/minimum losses. Contamination with ZEN/ZEN-related compounds were quantified. Maximum production was at 25 °C/0.95-0.93 aw and 20 °C/0.95 aw. ZEN contamination levels plotted against DMLs for all the treatments showed that at ca. <1.0% DML, there was a low risk of ZEN contamination exceeding EU legislative limits, while at >1.0% DML, the risk was high. This type of data is important in building a database for the development of a post-harvest decision support system for relative risks of different mycotoxins. [ABSTRACT FROM AUTHOR]

Changes in environmental stress impact on secondary metabolite (SM) production profiles. Few studies have examined targeted SM production patterns in relation to interacting environmental conditions in stored cereals. The objectives were to examine the effect of water activity (aw; 0.95-0.90) x temperature (10-25 °C) on SM production on naturally contaminated stored wheat and that inoculated with Fusarium graminearum. Samples were analysed using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) on (a) total number of known SMs, (b) their concentrations and (c) changes under environmental stress. 24 Fusarium metabolites were quantified. Interestingly, statistical differences (ChisSq., p < 0.001) were observed in the number of SMs produced under different sets of interacting environmental conditions. The dominant metabolites in natural stored grain were deoxynivalenol (DON) and nivalenol (NIV) followed by a range of enniatins (A, A1, B, B1), apicidin and DON-3-glucoside at 10 °C. Increasing temperature promoted the biosynthesis of other SMs such as aurofusarin, moniliformin, zearalenone (ZEN) and their derivatives. Natural wheat + F. graminearum inoculation resulted in a significant increase in the number of metabolites produced (ChisSq., p < 0.001). For ZEN and its derivatives, more was produced under cooler storage conditions. Fusarin C was enhanced in contrast to that for the enniatin group. The relative ratios of certain groups of targeted SM changed with environmental stress. Both temperature and aw affected the amounts of metabolites present, especially of DON and ZEN. This study suggests that the dominant SMs produced in stored temperate cereals are the mycotoxins for which legislation exists. However, there are changes in the ratios of key metabolites which could influence the relative contamination with individual compounds. Thus, in the future, under more extreme environmental stresses, different dominant SMs may be formed which could make present legislation out of step with the future contamination which might occur. [ABSTRACT FROM AUTHOR]

The objectives of this study were to compare the effect of different Stevia-based sugar substitutes (S1–S3), sucrose alone and a mixture of sucrose + S1 on: (a) humectant properties, (b) relative colonisation rates of sponge cake slices at 0.90 a w by strains of Aspergillus flavus , Eurotium amstelodami , Fusarium graminearum and Penicillium verrucosum at 20 and 25 °C and (c) shelf-life periods in days prior to visible growth. Results showed that sucrose, S1 commercial sugar substitute and the mixture of sucrose + S1 in water solutions were able to reach water activity levels similar to those of glycerol and glucose mixtures. The S2 and S3 commercial sugar substitutes were unable to reduce a w levels significantly. At 25 °C, colonisation of sponge cake slices by E. amstelodami , A. flavus and P. verrucosum occurred in all the treatments. Growth of F. graminearum only occurred on sponge cake slices containing S2 and S3 Stevia-based products at both temperatures. The best control of growth (30 days) was achieved in cake slices modified with sucrose or S1 Stevia treatments inoculated with A. flavus and in the sucrose treatment for E. amstelodami at 20 °C. F. graminearum growth was completely inhibited when sucrose alone, S1 or sucrose + S1 treatments were used at both temperatures. This study suggests that, as part of a hurdle technology approach, replacing sucrose with low calorie sugar substitutes based on Stevia glycosides needs to be done with care. This is because different products may have variable humectant properties and bulking agents which may shorten the potential shelf-life of intermediate moisture bakery products. [ABSTRACT FROM AUTHOR]

This investigated the roles of water activity (aw) and fungicides on the competitiveness of two Fusarium verticillioides strains against other spoilage fungi commonly present in maize (F. proliferatum, Aspergillus niger, A. flavus, A. ochraceus and Penicillium verrucosum). Fungal strains were inoculated on artificial media containing maize flour. The effects were determined of three aw levels (0.99, 0.98 and 0.95) and three fungicides (tebuconazole, procloraz and prothioconazole) on fungal interactions, the Index of Dominance (ID) of isolates and fumonisin B1+B2 (FBs) production. The two strains of F. verticillioides showed similar behaviour in conditions where water was freely available (0.99 aw); at 0.98 and 0.95 aw both F. verticilliodes strains had the lowest total ID scores (8-6 and 10-12, respectively). They showed the same ability to compete against other fungi having the highest ID scores against P. verrucosum and A. ochraceus and the lowest against A. niger and A. flavus. The lowest water activity gave (0.95 aw) was the most conducive for fumonisin production with significant differences to 0.98 and 0.99 aw. In a co-inoculation experiment, only FBs production from P. verrucosum was greater in the presence of the F. verticilliodes strains other fungi. The use of fungicides reduced Indices of Dominancy (ID) for both F. verticilliodes strains. A significant reduction in F. verticilloides growth was observed when combining water stress and fungicide treatments. This information provides increased understanding of the colonisation patterns of F. verticillioides in relation to other mycobiota and to both environmental and chemical stresses, and has implications in relation to future climate change scenarios. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of calcium propionate, water activity (aw) and incubation time on the total fungal count and aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) production in the broiler finisher feed. The feed was added with calcium propionate (5 g kg–1), adjusted to 0.85, 0.90 and 0.95 aw and stored for 28 days at 25°C, analysing for mould growth and aflatoxins production every 7 days. Analysis of variance indicated that all the factors (preservative, aw and storage time) alone and in combination significantly (p < 0.001) affected the total fungal count and aflatoxins production in the feed. Minimum total fungal counts (1.99 × 102 CFU g–1) were observed in calcium propionate feed at 0.85 aw on day 1 and the highest (4.36 × 109 CFUs g–1) in control sample at 0.95 aw on day 28 of storage. During the storage period, AFB1 content in control samples increased from 11.35 to 73.44, from 11.58 to 81.81 and from 11.54 to 102.68 ng g–1, whereas in preserved feed the content of B1 increased from 11.47 to 37.83, from 11.54 to 49.07 and from 11.20 to 53.14 ng g–1 at 0.85, 0.90 and 0.95 aw, respectively. Similar patterns were noted for AFB2, AFG1 and AFG2 contents. All the aflatoxins readily increased over storage time; however, the increase was much slower in preserved feed that contained a lower amount of available water. This study reveals that calcium propionate addition to poultry litter along with water activity amelioration is an effective tool for controlling mould incidence and aflatoxin production in poultry feed. [ABSTRACT FROM AUTHOR]

Two new species of Aspergillus (A. aculeatinus, A. sclerotiicarbonarius) were previously isolated from coffee in Thailand. The objective of this study was to examine the effect of interacting environmental factors of water availability (water activity, aw) and temperature on lag phases prior to growth, growth and potential for ochratoxin A (OTA) production by three strains of each species on a green coffee-based medium for the first time. This showed that overall the growth of the three strains of each species was similar over the 20-37 °C and 0.85-0.99 aw ranges. The lag phase prior to growth was < 1 day at 0.95-0.98 aw and 25-37 °C and increased to 2-3 days at marginal temperatures and aw levels. The growth of strains of the uniseriate species A. aculeatinus was optimum at 0.98 aw and 30-35 °C. For the biseriate A. sclerotiicarbonarius strains this was 0.99 aw and 30 °C. This species was not able to grow at 37 °C. None of the strains of the two species grew at 0.85 aw, regardless of temperature. Integrated profiles based on the data from three strains of each species have been developed to show the optimum, maximum and marginal conditions of interacting aw and temperature conditions for growth. None of the strains produced OTA on a green coffee-based medium. This information is important as these species are part of the mycobiota of coffee and may influence OTA contamination by other ochratoxigenic species during coffee processing. [ABSTRACT FROM AUTHOR]

This review considers the available information on the potential impact of key environmental factors and their interactions on the molecular ecology, growth and aflatoxin production by Aspergillus flavus in vitro and in maize grain. The recent studies which have been carried out to examine the impact of water activity × temperature on aflatoxin biosynthesis and phenotypic aflatoxin production are examined. These have shown that there is a direct relationship between the relative expression of key regulatory and structural genes under different environmental conditions which correlate directly with aflatoxin B1 production. A model has been developed to integrate the relative expression of 10 biosynthetic genes in the pathway, growth and aflatoxin B1 (AFB1) production which was validated under elevated temperature and water stress conditions. The effect of interacting conditions of aw × temperature × elevated CO2 (2 × and 3 × existing levels) are detailed for the first time. This suggests that while such interacting environmental conditions have little effect on growth they do have a significant impact on aflatoxin biosynthetic gene expression (structural aflD and regulatory aflR genes) and can significantly stimulate the production of AFB1. While the individual factors alone have an impact, it is the combined effect of these three abiotic factors which have an impact on mycotoxin production. This approach provides data which is necessary to help predict the real impacts of climate change on mycotoxigenic fungi. [ABSTRACT FROM AUTHOR]

Abstract: The effects of temperatures (20–30 °C) and water activity (0.90–0.99 a w) on the lag phase duration, mycelial growth, and nutritional utilisation patterns of two toxigenic (AFL1+ & AFL2+) and three atoxigenic (AFL1−, AFL2−, & AFL3−) Aspergillus flavus strains were evaluated in vitro. Both temperature and a w and their interactions had a significant influence on the growth and nutritional utilisation patterns (p < 0.05). There were no significant differences between toxigenic and atoxigenic strains in terms of lag phase prior to growth and mycelial growth rates. Based on carbon source (CS) utilisation patterns, toxigenic and atoxigenic strains' niche size was greater at higher temperatures and in wetter conditions. Additionally, based on niche overlap indices (NOIs), regardless of temperature, when water was freely available, atoxigenic and toxigenic strains co-existed. However, under moisture stress, the nutritional competitiveness was variable. Temporal carbon utilisation sequences (TCUS) of toxigenic and atoxigenic strains were compared. At 0.99 a w most CS sources were utilised by the strains and the time to detection (TTD) of each strain was shortest on monosaccharides at the same level of a w. Conversely, under moisture stress the least number of CS was utilised. The current study has demonstrated that carbon utilisation patterns are equally important as are other determinants of competitiveness and that growth rate alone is not a key attribute which determines competitiveness. [Copyright &y& Elsevier]

This study examined the relationship between storage environmental factors (water activity (aw) (0.89–0.97) and temperature (15°C–30°C)), colonisation of wheat and maize by Fusarium graminearum and F. verticillioides respectively and the dry matter losses (DMLs) caused and quantified by contamination with deoxynivalenol (DON), zearalenone (ZEA) and fumonisins (FUMs) during storage. Fungal growth was assessed by the amount of CO2 produced under different interacting conditions of aw and temperature. DMLs were quantified using the cumulative CO2 data, and these were shown to increase as temperature and aw increased. The amount of DON, ZEA (wheat for human consumption) and FUMs (feed maize) produced was significantly affected by the storage conditions. The three toxins however showed different patterns of production. Optimum for DON was at the wettest conditions (0.97aw) and the highest temperature assessed (30°C), whereas for ZEA this shifted to 25°C. FUMs were produced in higher amounts in maize at 30°C and 0.97aw; however, at intermediate aw levels (0.955aw), the highest production occurred at 25°C followed by 20°C. Polynomial models were developed for the effect of the storage factors on DMLs and toxin production. DMLs under different environmental conditions were significantly correlated with DON and FUMs. DON contamination was above the EU limits in at least 80% of the wheat samples with DMLs >1%, whereas at least 70% of the same samples contained ZEA above the respective EU legislative limits. Similarly, at least 75% of the maize samples with DMLs ≥ 0.9% exceeded the EU limits for the sum of FUMs in feed. These results show that it may be possible to use temporal CO2 production during storage of grains as an indicator of the level of contamination of the grain with mycotoxins. [ABSTRACT FROM AUTHOR]

The efficacy of calcium propionate at 2 different doses (0.5% and 1%) against growth and aflatoxins production by Aspergillus flavus (A-2092) was investigated in vitro on Czapek yeast extract agar at different levels of water activity (a w) in the range of 0.94 to 0.996a w. A. flavus spores germinated on all calcium propionate and a w treatments; however, 1% calcium propionate at 0.94 a w delayed the germination process for up to 10 d. The growing rate of mycelia was slower (0.28 mm/d) at 1% calcium propionate and 0.94 a w. Aflatoxins (B1, B2, G1, and G2) were also produced minimally (36.1, 1, 1.86, and 1.01 ng/g of media, respectively) at the aforementioned dose rate of calcium propionate and water activity. It was concluded that addition of calcium propionate and a w amelioration can prove effective tools for suppressing the germination, growth rate, and aflatoxins production by A. flavus in substrate. [ABSTRACT FROM AUTHOR]

Abstract: This study examined the utilization patterns of key carbon sources (CS, 24: including key sugars, amino acids and fatty acids) in maize by strains of Aspergillus flavus and Fusarium verticillioides under different water activity (a w, 0.87–0.98 a w) and temperature (20–35 °C) values and compared the niche overlap indices (NOI) that estimate the in vitro CS utilization profiles [Wilson, M., Lindow, S.E., 1994. Coexistence among epiphytic bacterial populations mediated through nutritional resource partitioning. Applied and Environmental Microbiology 60, 4468–4477.]. The ability to grow in these key CS in minimal media was studied for 120 h in 12 h steps. The NOI was calculated for inter-species (F. verticillioides–A. flavus) and for intra-species (A. flavus–A. flavus) using CS utilization patterns over the range of interacting environmental conditions. 30 °C, over the whole a w range examined, was found to be optimal for utilization of the maximum number of CS by A. flavus. In contrast, for F. verticillioides this was more so at 20 °C; 25 °C allowed a suboptimal usage of CS for both species. NOIs confirmed the nutritional dominance of A. flavus at 30 °C, especially at lower a w levels and that of F. verticillioides at 20 °C, mainly at 0.95 a w. In other conditions of a w, based on CS utilization patterns, the data indicated that A. flavus and F. verticillioides occupied different ecological niches. The variability in nutritional sources utilization between A. flavus strains was not related to their ability to produce aflatoxins (AFs). This type of data helps to explain the nutritional dominance of fungal species and strains under different environmental conditions. This could be useful in trying to find appropriate natural biocontrol microorganisms to compete with these mycotoxigenic species. [Copyright &y& Elsevier]

The aim of this study was the production of high numbers of M. anisopliae blastospores with enhanced germination efficiency under conditions of water-stress (ecological fitness). Different nitrogen sources and concentrations were screened for their ability to induce blastospore production while keeping pH and water activity ( a w ) at fixed levels (6.8, and 0.98 a w , respectively). After optimum nitrogen status was determined (cornsteep solid (CS) + yeast extract (YE); or cottonseed flour (CF) + YE), the effect of interaction of nitrogen source, pH (3.5, 5, 6.8, 8, 9 and 10) and solutes for a w adjustment (KCl, NaCl, PEG 200) on blastospore production, endogenous polyol content (glycerol, erythritol, arabitol and mannitol) and total protein, were determined. For both ionic (NaCl, KCl) and non-ionic solutes (PEG 200), optimum blastospore production (between 4×10 7 and 2×10 8 blastospores ml -1 ) and growth occurred in the pH range 6.8–8, with the CF + YE nitrogen profile giving higher yields than CS + YE. Optimum conditions for high erythritol and total protein endogenous concentrations (40.37–73.44 and 14.33–18.90 mg g -1 fresh weight, respectively) occurred between pH 6.8 and 8 by ionic a w modification (KCl, NaCl) and with the CF + YE nitrogen profile. Germination of blastospores produced under these cultural conditions was between 62 and 89% under conditions of water-stress (0.96 a w ). On the other hand, blastospores with lower amounts of erythritol and total protein content had decreased germination (8–67%). These results could have significant implications for developing a liquid fermentation medium for the production of high numbers of fungal propagules with enhanced efficacy under non-optimum environmental conditions. [ABSTRACT FROM AUTHOR]

The major species of fungi responsible for ochratoxin production (OTA) in a range of commodities are Penicillium verrucosum , Aspergillus ochraceus and Aspergillus section Nigri , especially A. carbonarius. P. verrucosum is particularly important in northern Europe where damp cooler conditions occur and where drying regimes need to be efficient and effective for preventing post-harvest contamination with OTA. A. ochraceus can infect cereals including barley, maize, coffee, cocoa and edible nuts. A. carbonarius has been identified as the key species responsible for OTA contamination of grapes, wine and vine fruits. Recent studies have identified the environmental regimes, especially of temperature and humidity, which are conducive to growth and OTA production by these species pre- and post-harvest and during transport. The optimum and marginal interacting conditions for growth and OTA contamination often vary considerably. This has to be borne in mind when effective preventative management strategies are being implemented. Recent studies with cereals have suggested that isolation frequency can be related to OTA contamination of cereals. A logistical model has been developed and identified that 1000&ThickSpace;CFUs&ThickSpace;g -1 grain of P. verrucosum (CFUs) is the threshold limit at which the probability of exceeding the EU legislative limit of 5&ThickSpace;µg&ThickSpace;kg -1 in cereal grain can be predicted under different storage regimes. Controlled atmospheres&ThickSpace;>50% C0 2 is required to effectively prevent OTA accumulation in damp cereals. With regard to grapes, preharvest contamination with A. carbonarius has been shown to be a good indicator for regional risk in southern Europe from OTA contamination. The ecological conditions for optimum growth and OTA production have been shown to differ with the optima being 30–35°C and 15–25°C and 0.98–0.99 and 0.93–0.95 water activity, respectively. Studies on vine fruits (drying currants) suggest that OTA contamination and increase contamination levels occur during this drying process of 7–14 days. This will be influenced by prevailing weather conditions and drying rates. Minimizing OTA contamination in these and other commodities including coffee and cocoa require clear guidelines on safe moisture and temperature regimes pre- and post-harvest for the development of effective management strategies based on ecological criteria. [ABSTRACT FROM AUTHOR]

The impact of five fungicides (prochloraz, propioconazole, epoxiconazole, tebuconazole and azoxystrobin, 0.5–50 μg g−1) on growth of two Fusarium graminearum was evaluated in relation to water activity (aW, 0.99, 0.97, 0.95) and temperature (15°C and 25°C) on wheat-based media (in vitro). All fungicides reduce growth rates when compared to the control, and this reduction increased as the fungicide concentration increased. In general, none of the isolates was able to grow in the presence of any fungicide treatments at concentrations >15 μg ml−1, regardless of the aW/temperature regime. The same fungicides were used in the second study on wheat grain (in situ), in order to evaluate the effect of two concentration (0.5, 5 μg ml−1), three aW levels (0.995, 0.99 and 0.97) and two temperatures (15°C and 25°C) and their interaction on growth rate and deoxynivalenol (DON) production by F. graminearum. All fungicides showed inhibition of growth at both concentrations in most conditions. The fungicides tested were less effective on grain in controlling growth than in in vitro studies. It was noticeable that at 15°C and 0.99 and 0.97 aW, two of the fungicides (tebuconazole and epoxiconazole) showed growth stimulation. All fungicides showed DON stimulation or reduction in at least one of the conditions assayed. Our results show that stimulation or reduction in DON production in the presence of fungicides is influenced by complex interactions between aW, temperature, fungicide concentration and time of incubation in both strains of F. graminearum studied. Such information is critical for effective fungicide control of Fusarium head blight of wheat. [Copyright &y& Elsevier]

Maize grown in both North and South America are now predominantly genetically modified (GM) cultivars with some resistance to herbicide, pesticide, or both. There is little information on the relative colonisation and aflatoxin B 1 (AFB 1) production with maize meal-based nutritional matrices based on kernels of non-GM maize and isogenic GM-ones by strains of Aspergillus flavus. The objectives were to examine the effect of interacting conditions of temperature (25–35 °C) and water availability (0.99–0.90 water activity, a w) on (a) mycelial growth, (b) AFB 1 production and (c) develop contour maps of optimum and marginal conditions of these parameters for four strains of A. flavus on three different non-GM and isogenic GM-maize based nutritional media. The growth of the four strains of A. flavus (three aflatoxigenic; one non-aflatoxigenic) was relatively similar in relation to the temperature × a w conditions examined on both non-GM and GM-based matrices. Optimum growth overall was at 30–35 °C and 0.99 a w for all four strains. Under water stress (0.90 a w) growth was optimum at 35 °C. Statistically: non-GM, GM cultivars, temperature and a w all significantly affected growth rates. For AFB 1 production, all single and interacting factors were statistically significant except for non-GM × GM cultivar. In conclusion, colonisation of GM- and non-GM nutritional sources was similar for the different A. flavus strains examined. The contour maps will be very useful for understanding the ecological niches for both toxigenic and non-toxigenic strains in the context of the competitive exclusion of those producing aflatoxins. • Aspergillus flavus strains grew similarly on GM/non-GM nutritional media under interacting abiotic factors • Contour maps of optimum and marginal conditions for growth were developed for 4 strains of A. flavus • Aflatoxin B 1 production was optimal at 0.99 water activity and 30 °C, regardless of strain. • Optimum and marginal toxin production contour maps were determined for toxigenic strains [ABSTRACT FROM AUTHOR]

Abstract: A rapid method for screening antifungal compounds and performing ecophysiological studies with filamentous fungi has been developed by the use of specific semi-solid media and spectrophotometric/turbidimetric measurements using the Bioscreen C with 2×100 microtitre well plates. The medium composition and preparation, inoculum size, medium volumes, and incubation parameters for measuring initial germination and growth dynamics have been optimised. These have been applied to assess the effectiveness of 18 concentrations of propyl propane thiosulfinate (PTS) against Aspergillus flavus in YES medium under different environmental regimes. Minimum inhibitory concentrations (MIC), and non-inhibitory concentration (NIC) values, and a newly developed MIC50, have been calculated for the efficacy of these concentrations of PTS under four different environmental conditions (0.995 and 0.95 water activity (a w); 20 and 25°C) over 7d periods with automated measurements every 20min. Data were modelled using the Lambert–Pearson Model, a mathematical modelling approach previously used for bacterial inhibition. Results have been compared with traditional growth rate data and Lethal Dose50 (LD50) values. This approach could have major implications for rapid screening assays for growth and secondary metabolite production by filamentous organisms and has major advantages in terms of time reduction, culture medium volumes required, measurement and the ability to integrate and model key parameters for comparing efficacy. [Copyright &y& Elsevier]

Abstract: A microarray analysis was performed to study the effect of varying combinations of water activity and temperature on the activation of aflatoxin biosynthesis genes in A spergillus flavus grown on YES medium. Generally A. flavus showed expression of the aflatoxin biosynthetic genes at all parameter combinations tested. Certain combinations of a w and temperature, especially combinations which imposed stress on the fungus resulted in a significant reduction of the growth rate. At these conditions induction of the whole aflatoxin biosynthesis gene cluster occurred, however the produced aflatoxin B1 was low. At all other combinations (25°C/0.95 and 0.99; 30°C/0.95 and 0.99; 35°C/0.95 and 0.99) a reduced basal level of cluster gene expression occurred. At these combinations a high growth rate was obtained as well as high aflatoxin production. When single genes were compared, two groups with different expression profiles in relation to water activity/temperature combinations occurred. These two groups were co-ordinately localized within the aflatoxin gene cluster. The ratio of aflR/aflJ expression was correlated with increased aflatoxin biosynthesis. [Copyright &y& Elsevier]

Abstract: The objective of this study was to determine the effects of water activity (a W; 0.900–0.995), temperature (5, 15, 25 and 30 °C), time of incubation (7–49 days) and their interactions on mycelial growth and deoxynivalenol (DON) production on irradiated wheat grain by two strains of Fusarium graminearum isolated from wheat ears in Argentina. Optimal a W levels for growth were in the range 0.950–0.995 with a temperature optima of 25 °C. Maximum growth rates were obtained at the highest a W (0.995) and 25 °C for both strains. No growth was observed at 5 °C regardless of the a W levels assayed. Both strains were able to growth at the lowest a W assayed (0.900), although the temperature ranges allowing growth at this minimal a W was 15–25 °C. DON was produced the most rapidly (7 days) when incubated at 25 °C and 0.995 a W. All other conditions required 7–14 days before DON was produced on grain. Maximum amounts of DON for both strains were produced at the highest a W treatment (0.995) after 6 weeks at 30 °C. The range of DON concentrations varied considerably (5 to 140,000 ng g−1) depending on a W and temperature interaction treatments. Production of DON occurred over a narrower range of a W (0.995–0.95) than that for growth (0.995–0.90). DON was more rapidly produced at 25 °C but the maximum amount produced was at 30 °C. Two-dimensional profiles of a W ×temperature were developed from these data to identify areas where conditions indicate a significant risk from DON accumulation. [Copyright &y& Elsevier]

Abstract: Studies were conducted to examine the effect of water activity (aw, 0.97, 0.95, 0.93), pH (4.5, 6.0) and preservative type and concentration (potassium sorbate (KS), calcium propionate (CaP), 300 and 3000 ppm) on colonisation and ochratoxin A (OTA) production by three strains of Penicillium verrucosum on bread analogues. At 25 °C, colony extension from a point source was similar for all three strains at the different environmental treatments examined, with optimum growth at 0.97 and 0.96 aw and pH 6. Temporal OTA production over 36 days varied significantly with strain, pH and aw level. Maximum OTA was produced at approximately 28–36 days for two of the strains with an optimum at 0.93 aw at pH 6.0. One strain produced 75–80 μg g−1 analogue under these conditions. However, at pH 4.5, optimum OTA was produced at 0.95 aw after a similar time period on bread analogues, with maximum amounts of about 25–35 μg g−1 bread. KS and CaP incorporation inhibited growth completely at pH 4.5 and 3000 ppm at all three aw levels. However, at suboptimal concentration, little growth inhibition occurred. At pH 6, even with 3000 ppm, growth of all three strains occurred. Where effective inhibition of growth was achieved (3000 ppm/pH 4.5, all aw levels), no OTA was detected. However, when 300 ppm was used, up to 7 μg g−1 was detected in bread analogues at pH 4.5. At pH 6, OTA was produced at all treatment conditions by all three strains, although preservative-treated bread had less present. Statistical analysis showed that many of the one-, two- and three-way interactions had a significant effect on growth and OTA production. These results suggest that there is a significant risk from OTA contamination if lower concentrations of existing preservatives are used in wheat-based bakery products contaminated by spoilage mycotoxigenic moulds such as P. verrucosum. [Copyright &y& Elsevier]

The aim was to decipher the temporal impact of key interacting climate change (CC) abiotic factors of temperature (30 vs 37 °C), water activity (a w ; 0.985 vs 0.930) and CO 2 exposure (400 vs 1000 ppm) on (a) growth of Aspergillus flavus and effects on (b) gene expression of a structural (aflD) and key regulatory gene (aflR) involved in aflatoxin B 1 (AFB 1) biosynthesis and (c) AFB 1 production on a yeast extract sucrose medium over a period of 10 days. A. flavus grew and produced AFB 1 very early with toxin detected after only 48 h. Both growth and toxin production were significantly impacted by the interacting abiotic factors. The relative expression of the aflD gene was significantly influenced by temperature; aflR gene expression was mainly modulated by time. However, no clear relationship was observed for both genes with AFB 1 production over the experimental time frame. The optimum temperature for AFB 1 production was 30 °C. Maximum AFB 1 production occurred between days 4–8. Exposure to higher CO 2 conditions simulating forecasted CC conditions resulted in the amount of AFB 1 produced in elevated temperature (37 °C) being higher than with the optimum temperature (30 °C) showing a potential for increased risk for human/animal health due to higher accumulation of this toxin. [ABSTRACT FROM AUTHOR]

Effects of Aspergillus flavus colonization of maize kernels under different water activities (a w ; 0.99 and 0.91) and temperatures (30, 37 °C) on (a) aflatoxin B 1 (AFB 1 ) production and (b) the transcriptome using RNAseq were examined. There was no significant difference ( p = 0.05) in AFB 1 production at 30 and 37 °C and 0.99 a w . However, there was a significant ( p = 0.05) increase in AFB 1 at 0.91 a w at 37 °C when compared with 30 °C/0.99 a w . Environmental stress effects using gene ontology enrichment analysis of the RNA-seq results for increasing temperature at 0.99 and 0.91 a w showed differential expression of 2224 and 481 genes, respectively. With decreasing water availability, 4307 were affected at 30 °C and 702 genes at 37 °C. Increasing temperature from 30 to 37 °C at both a w levels resulted in 12 biological processes being upregulated and 9 significantly downregulated. Decreasing a w at both temperatures resulted in 22 biological processes significantly upregulated and 25 downregulated. The interacting environmental factors influenced functioning of the secondary metabolite gene clusters for aflatoxins and cyclopiazonic acid (CPA). An elevated number of genes were co-regulated by both a w and temperature. An interaction effect for 4 of the 25 AFB 1 genes, including regulatory and transcription activators occurred. For CPA, all 5 biosynthetic genes were affected by a w stress, regardless of temperature. The molecular regulation of A. flavus in maize is discussed. [ABSTRACT FROM AUTHOR]

The objectives of this study were to examine the efficacy of four bacterial antagonists against Aspergillus flavus using 50:50 ratio of bacterial cells/conidia for the control of aflatoxin B 1 (AFB 1 ) production on two different nutritional matrices, nutrient and maize-based media at different water availabilities (0.98, 0.94 water activity (a w ) on nutrient medium; 0.995, 0.98 a w on maize meal agar medium) at 35 °C. The indicators of efficacy used were the relative expression of one structural and regulatory gene in the biosynthetic pathway ( aflD and aflR respectively) and the production of AFB 1 . These studies showed that some of the bacterial species could significantly inhibit the relative expression of the aflD and aflR genes at both 0.98 and 0.94 a w on nutrient agar. On maize-based media some of the bacterial antagonists reduced the activity of both genes at 0.94 a w and some at 0.995 a w . However, the results for AFB 1 production were not consistent with the effects on gene expression. Some bacterial species stimulated AFB 1 production on both nutrient and maize-based media regardless of a w . However, some bacterial treatments did inhibit AFB 1 production significantly when compared to the control. Overall, this study suggests that temporal studies are required on the biosynthetic genes under different environmental and nutritional conditions to evaluate the potential of antagonists to control AFB 1 . [ABSTRACT FROM AUTHOR]

Abstract: The potential for using modified atmospheres of 25–75% CO2 (balanced with N2) and water activity (a w, 0.95, 0.92) to control Aspergillus flavus development and aflatoxin B1 production has been evaluated (a) on synthetic medium and (b) on maize grain during storage for up to 21 days at 25 °C. On agar medium up to 75% CO2 at both 0.95 and 0.92 a w significant inhibition of growth was obtained (P <0.05). In stored grain inoculated with spores of A. flavus there was significantly higher populations of the species at 0.95 a w than 0.92 a w. Up to 75% CO2 resulted in an inhibition of the populations of A. flavus isolated from the grain. Contrasting aflatoxin B1 production was obtained on agar and in stored maize grain. On agar, greatest amounts were produced at 0.92 a w, while more was produced at 0.95 a w on maize grain. Overall, the efficacy of controlled atmospheres× a w showed that treatment with 25% CO2 could be sufficient to efficiently reduce A. flavus development but at least 50% CO2 was required to obtain a significant reduction of aflatoxin synthesis. [Copyright &y& Elsevier]

Abstract: There is significant interest in trying to understand the relationship between environmental factors, preservative concentration and expression of genes involved in mycotoxin production. However, little information is available on the link between physiological stress factors and expression of genes responsible for mycotoxin production. This study has examined the effect of interactions between ionic and non-ionic water availability modified with glycerol or NaCl (a w, 0.98, 0.95 and 0.93) and sub-optimal concentrations of calcium propionate and potassium sorbate (150, 300 ppm) at 25 °C on growth, ochratoxin A (OTA) and otapksPV gene expression by the mycotoxigenic species Penicillium verrucosum. Growth was inhibited between 25–35% by the preservatives at each a w level. However, OTA production was stimulated by 150 and 300 ppm of both preservatives, especially at 0.95 and 0.93 a w. If water activity as a single stress factor was changed, a typical OTA production and otapksPV expression profile occurred, indicating that OTA biosynthesis is activated under two conditions, i.e., under optimal growth conditions and under weak stress conditions. Temporal analysis of otapksPV expression showed that there was an optimum after 8–9 days incubation. Statistical analyses indicated good correlation between sub-optimal concentrations of preservatives, intermediate a w levels and genotypic and phenotypic gene and toxin production. This is the first time that genotypic information has been linked to phenotypic mycotoxin production in relation to such interacting stress factors. [Copyright &y& Elsevier]

The effect of interactions of water activity (aw) (0.99–0.90), temperature (20, 30 and 35 °C) and modifying aw solute (glycerol, NaCl) on growth and sporulation of a wild-type strain of Aspergillus niger (W) and two genetically engineered lysozyme-producing strains (L11, B1) was examined for the first time. Maximum growth rates were achieved for both strains (L11 and B1) under moderate aW levels. L11 showed a higher growth rate than B1. Fastest growth was achieved at 30 °C, using glycerol as solute. Optimum conditions for growth of strain L11 were estimated by means of contour plot surfaces and found to be 0.965 aw with glycerol as solute at 35 °C. The predicted value of the optimum growth rate was 10.5 mm/day. A value of 10.85 mm/day was obtained experimentally giving a good correlation between the estimated and the measured results. Sporulation was optimum for the W strain at 0.99–0.95 by B1 at 35 °C. Significant higher production of conidia by L11 at 0.97–0.93 aw and at 0.97 aw and 35 °C for B1 strain was observed. Optimum conditions for spore production were different from those for growth. Under similar ecological conditions, the W and both the genetically engineered strains had a different growth and sporulation pattern. [Copyright &y& Elsevier]

In the UK and Northern Europe, ripening oats can become contaminated with T-2 and HT-2 mycotoxins, produced mainly by Fusarium langsethiae. There are indicative levels related to the maximum limits for oat grain for these toxins. The objectives of this study were to examine the effect of interacting conditions of temperature (10–30 °C) and water activity (a w , 0.995–0.90) on (a) lag times prior to growth, (b) growth and (c) T-2 and HT-2 toxins by two strains of F. langsethiae isolated from oats in the UK and compare this with the type strain (Fl201059) which has been genomically sequenced, and (d) develop (and validated with published data) a probabilistic models for impacts of temperature × a w on growth and toxin production. All three strains had an optimum a w range and temperature of 0.995–0.98 and 25 °C for growth. For T-2 + HT-2 production these were 0.995 a w and 20 °C. Overall, the type strain produced higher amounts of T-2 + HT-2 with a HT-2/T-2 ratio of up to 76. Using this study data sets and those from the literature, probabilistic models were developed and validated for growth and T-2 + HT-2 toxin production in relation to temperature × a w conditions. These models, when applied in stored oats, will be beneficial in determining the conditions on the relative level of risk of contamination with these two toxins in the context of the EU indicative maximum levels. • F. langsethiae strains grew optimally at 0.995–0.98 a w and 20/25 °C. • Marginal conditions for growth and T-2/HT-2 toxin production was 0.93 a w /15–25 °C. • Probabilistic model for fungal growth for F. langsethiae developed for the first time • Model for marginal and maximum T-2/HT-2 toxin production by this species developed • Potential for use of these models for evaluating relative risk of toxin contamination in oats [ABSTRACT FROM AUTHOR]