Integrating gene expression, ecology and mycotoxin production by Fusarium and Aspergillus species in relation to interacting environmental factors

Environmental factors, such as water availability (water activity, aw), temperature and their interactions, have a significant impact on the life cycle of mycotoxigenic fungi. Growth and mycotoxin production are influenced by these interacting factors resulting in a broader range of aw × temperature conditions for germination, than growth or mycotoxin production. The biosynthetic genes are mostly clustered together and by using microarrays with sub-arrays for specific mycotoxins, such as trichothecenes, fumonisins and aflatoxins it has been possible to examine the relationship between interacting aw × temperature conditions on growth, toxin gene cluster expression and relate these to phenotypic toxin production. The data for groups of biosynthetic genes (Fusarium culmorum/Fusarium graminearum; Fusarium verticillioides; Aspergillus flavus) were integrated with data on growth and mycotoxin production under different aw × temperature conditions using a mixed growth model. This was used to correlate these factors and predict toxin levels which may be produced under different abiotic stress conditions. Indeed, the relative importance of the different genes could be examined using ternary diagrams of the relative expression of 3 genes at a time in relation to aw, temperature and mycotoxin production to identify the most important relationships. The effect of three-way interacting environmental factors representative of climate change (CC) scenarios (water stress × temperature (+2-4 °C) × elevated CO2 (350-400 vs 650 and 1000 ppm) on growth and mycotoxin production by A. flavus and by species of the Aspergillus section Circumdati and section Nigri have been determined. These studies on maize grain and coffee, respectively, suggest that while growth may not be significantly affected, mycotoxin production may be stimulated by CC factors. This approach to integrate such data sets and model the relationships could be a powerful tool for predicting the relative toxin production under extreme stress conditions, including CC scenarios

Impact of interacting climate change factors on growth and ochratoxin A production by Aspergillus section Circumdati and Nigri species on coffee

The objectives of this study were to evaluate the effect of interacting climate change (CC) factors (water stress [water activity, aw; 0.99-0.90]); temperature [30, 35 °C]; and elevated CO2 [400 and 1000 ppm] on (1) lag phases prior to growth, (2) growth and (3) ochratoxin A (OTA) production by species of Aspergillus sections Circumdati and Nigri on coffee-based media and stored coffee beans. The lag phases, prior to growth, of all strains/species were slightly increased as aw, temperature and CO2 were modified. The interacting CC factors showed that most strains/species examined grew well at 30 °C and slightly less so at 35 °C except for Aspergillus niger (A 1911) which could tolerate the higher temperature. In addition, the interaction of elevated CO2 (1000 ppm) + temperature (35 °C) increased OTA production when compared with 30 °C but only for strains of Aspergillus westerdijkiae (B2), Aspergillus ochraceus (ITAL 14) and Aspergillus steynii (CBS 112814). Most of the strains had optimum growth at 0.95 aw at 35 °C, while at 30 °C the optimum was at 0.98 aw. On stored coffee beans there was only a significant stimulation of OTA production by A. westerdijkiae strains in elevated CO2 (1000) at 0.90 aw. These results suggest differential effects of CC factors on OTA production by species in the Sections Circumdati and Nigri in stored coffee and that for most species there is a reduction in toxin production

Environmental stress and elicitors enhance taxol production by endophytic strains of Paraconiothyrium variabile and Epicoccum nigrum

This study examined the effect of different elicitors (seven, different concentrations) and environmental factors (water activity (aw), pH) on taxol production by strains of two endophytic fungi, Paraconiothyrium variabile and Epicoccum nigrum, isolated from temperate yew trees. A defined liquid broth medium was modified with elicitors, solute aw depressors at different pH values. For P. variabile, the best elicitor was salicylic acid at 50 mg/l which gave a taxol yield of 14.7 ± 4.8 μg/l. The study of synergistic effects between elicitor, aw and pH on taxol production showed that the highest yield of taxol (68.9 ± 11.9 μg/l) was produced under modified ionic stress of 0.98 aw (KCl) at pH 5 when supplemented with 20 mg/l of salicylic acid. For E. nigrum, serine was the best elicitor which increased yield significantly (29.6 fold) when KCL was used as the aw depressor (0.98 aw) at pH 5.0 with 30 mg/l of serine. The maximum taxol yield produced by E. nigrum was 57.1 ± 11.8 μg/l. Surface response models were used to build contour maps to determine the conditions for maximum and marginal conditions for taxol yield in relation to the best elicitor and aw, and the best pH for the first time. This will be beneficial for identifying key parameters for improvement of taxol yields by endophytic fungi

Evaluation of the risk of fungal spoilage when substituting sucrose with commercial purified Stevia glycosides in sweetened bakery products

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 aw 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 aw 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

Relationship between environmental conditions, carbon utilisation patterns and Niche Overlap Indices of the mycotoxigenic species Fusarium verticillioides and the biocontrol agent Clonostachys rosea

Recently, it was shown that a strain of the fungal antagonist Clonostachys rosea 016 was able to inhibit fumonisin B1 mycotoxin production by Fusarium verticillioides FV1 when using different ratios of spores of each species in vitro. The objectives of the present work were therefore to: (a) compare the nutritional utilisation patterns and rates of uptake of key C-sources in maize by the antagonist C. rosea 016 and that by the pathogen F. verticillioides FV1; (b) examine their Niche Overlap Indices (NOI) under different interacting environmental conditions; and (c) evaluate whether the rate of utilisation of key maize C-sources influenced the competitiveness of either species using the Bioscreen®. It was found that water potential (Ψ) × temperature interactions had significant impacts on C-source utilisation patterns by C. rosea 016 and the pathogen. The NOIs, based on the utilisation of the C-sources by each strain divided by those utilised in common, showed that the antagonist and the pathogen occupied similar niches at −0.70 MPa Ψ+30 °C and −2.8 MPa Ψ+25 °C. Under the other conditions tested, they appeared to occupy separate niches suggesting niche exclusion. Temporal C-source utilisation patterns were then compared under different Ψ × temperature treatments. This showed that the dominant maize-based C-sources utilised by the pathogen and the antagonist were different. The pathogen F. verticillioides FV1 utilised carbohydrates rapidly followed by amino acids and then one fatty acid, palmitic acid. The antagonist C. rosea 016 utilised both carbohydrates and amino acids at a similar rate but more slowly than the pathogen. There were also differences in the utilisation of some individual amino acids and carbohydrates which might explain the occupation of different niches under some interacting environmental conditions. These findings are discussed in the context of why some competitors are able to inhibit mycotoxin production while others cannot

Comparison of dry matter losses and aflatoxin B1 contamination of paddy and brown rice stored naturally or after inoculation with Aspergillus flavus at different environmental conditions

The objective of this study was to compare the effect of different storage moisture conditions (0.70, 0.85, 0.90 and 0.95 water activity, aw) and temperatures (20, 25, 30 °C) on (a) respiration rates (R) and dry matter loss (DML) of paddy and brown rice and (b) quantify aflatoxin B1 (AFB1) production by isolates of Aspergillus flavus from the rice samples and (c) inoculation of both rice types with A. flavus under these storage conditions on R, DML and AFB1 contamination. There was an increase in temporal CO2 production with wetter and warmer conditions in naturally contaminated rice. Higher R and consequently, % DML, were generally found in the brown rice (21%) while in paddy rice this was only up to 3.5% DML. From both rice types, 15 (83.3%) of 18 A. flavus isolates produced detectable levels of AFB1 in a range 2.5–1979.6 μg/kg. There was an increase in DML in both rice types inoculated with A. flavus as temperature and aw were increased. Interestingly very little AFB1 was detected in paddy rice, but significant contamination occurred in the brown rice. The %DML in the control and A. flavus inoculated rice increased with temperature and aw at both 25 and 30 °C from 1-2% to 15–20% DML at 30 °C and 0.95 aw. All the inoculated rice samples had AFB1 levels above the EU legislative limits for contamination in other temperate cereals and products derived from cereals (=2 μg/kg). Even samples with % DML as low as 0.2% had AFB1 contamination levels twice the limits for other cereals. These results suggest that the mycotoxin contamination risk in staple commodities like rice, is influenced by whether the rice is processed or not, and that measurement of R rates can be used to predict the relative risk of AFB1 contamination in such staple commodities

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

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

Impact of environmental factors on growth and satratoxin G production by strains of Stachybotrys chartarum

The black mould Stachybotrys chartarum and its mycotoxins have been linked to damp building-associated illnesses. The objective of this study was to determine the effects of water availability (water activity, aw) and temperature on growth and production of satratoxin G (SG) by a macrocyclic trichothecene-producing strain (IBT 7711) and non-producing strain (IBT 1495) of S. chartarum. Growth studies were carried out on potato dextrose agar modified with glycerol to 0.995-0.92 aw at 10-37 °C. Growth extension was measured and the cultures were extracted after 10 days and a competitive enzyme-linked immunosorbent assay (ELISA) method used to quantify the SG content. Growth was optimal at 25 to 30 °C at 0.995 aw, but this was modified to 0.98 aw at 30 °C for both strains (1.4- 1.6 mm/day, respectively). The ELISA method revealed that, in contrast to growth, SG production was maximal at 20 °C with highest production at 0.98 aw (approximately 250 μg/g mycelia). When water was freely available (0.995 aw), SG was maximally produced at 15 °C and decreased as temperature was increased. Interestingly, the strain classified as a non-toxigenic produced very low amounts of SG (<1.6 μg/g mycelia) that were maximal at 25 °C and 0.98 aw. Contour maps for growth and SG production were developed from these data sets. These data have shown, for the first time, that growth and SG production profiles are very different in relation to key environmental conditions in the indoor environment. This will be very useful in practically determining the risk from exposure to S. chartarum and its toxins in the built env

Climate change, food security and mycotoxins: do we know enough?

Climate change (CC) scenarios are predicted to have significant effects on the security of staple commodities. A key component of this impact is the infection of such crops by mycotoxigenic moulds and contamination with mycotoxins. The impacts of CC on mycotoxigenic fungi requires examination of the impacts of the three-way interactions between elevated CO2 (350–400 vs 650–1200 ppm), temperature increases (+2–5 °C) and drought stress on growth/mycotoxin production by key spoilage fungi in cereals and nuts. This review examines the available evidence on the impacts of interacting CC factors on growth and mycotoxin production by key mycotoxigenic fungi including Alternaria, Aspergillus, Fusarium and Penicillium species. Aspergillus flavus responsible for producing aflatoxin B1 (AFB1) is a class 1A carcinogen and its growth appears to be unaffected by CC factors. However, there is a significant stimulation of AFB1 production both in vitro and in vivo in maize. In contrast, studies on Aspergillus section Circumdati and Nigri species responsible for ochratoxin A contamination of a range of commodities and F. verticillioides and fumonisins suggest that some species are more resilient than others, especially in terms of mycotoxin production. Acclimatisation of mycotoxigenic fungal pathogens to CC factors may result in increased disease and perhaps mycotoxin contamination of staple cereals. Predictive modelling approaches to help identify regions where maximum impact may occur in terms of infection by mycotoxigenic fungi and toxin contamination of staple crops is hindered by the lack of reliable inputs on effects of the interacting CC factors. The present available knowledge is discussed in the context of the resilience of staple food chains and the impact that interacting CC factors may have on the availability of food in the future

Assessment of intraspecies variability in fungal growth initiation of Aspergillus flavus and aflatoxin B1 production under static and changing temperature levels using different initial conidial inoculum levels

Intraspecies variability in fungal growth and mycotoxin production has important implications for food safety. Using the Bioscreen C we have examined spectrophotometrically intraspecies variability of A. flavus using 10 isolates under different environments, including temperature shifts, in terms of growth and aflatoxin B1 (AFB1) production. Five high and five low AFB1 producers were examined. The study was conducted at 5 isothermal conditions (from 15 to 37 °C) and 4 dynamic scenarios (between 15 and 30 °C). The experiments were carried out in a semisolid YES medium at 0.92 aw and two inoculum levels, 102 and 103 spores/mL. The Time to Detection (TTD) of growth initiation was determined and modelled as a function of temperature through a polynomial equation and the model was used to predict TTD under temperature upshifts conditions using a novel approach. The results obtained in this study have shown that a model can be developed to describe the effect of temperature upshifts on the TTD for all the studied isolates and inoculum levels. Isolate variability increased as the growth conditions became more stressful and with a lower inoculum level. Inoculum level affected the intraspecies variability but not the repeatability of the experiments. In dynamic conditions, isolate responses depended both on the temperature shift and, predominantly, the final temperature level. AFB1 production was highly variable among the isolates and greatly depended on temperature (optimum temperature at 30–35 °C) and inoculum levels, with often higher production with lower inoculum. This suggests that, from an ecological point of view, the potential isolate variability and interaction with dynamic conditions should be taken into account in developing strategies to control growth and predicting mycotoxin risks by mycotoxigenic fungi