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

Environmental factors modify carbon nutritional patterns and niche overlap between Aspergillus flavus and Fusarium verticillioides strains from maize.

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 (aw, 0.87–0.98 aw) 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 aw 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 aw levels and that of F. verticillioides at 20 °C, mainly at 0.95 aw. In other conditions of aw, 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

Foreword: mycotoxins in a changing world

This special issue arose because of the changes in the global landscape in relation to the impact and implications of our changing climate on food security and quality, consumer habits, trade and economics, regulations and scientific thinking. The EU green paper (EC, 2007) on climate change (CC) has suggested significant hot spots in different regions where food production will be considerably affected both in quality and quantity. Indeed, a recent UNEP report on ‘Emerging Issues of Environmental Concern’ (UNEP, 2016) has included a section entitled ‘Poisoned chalice: Toxin accumulation in crops in an era of climate change’ which refers to the impact that aflatoxin contamination is having in low and middle income countries (LMICs)

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

Alternaria in food: Ecophysiology, mycotoxin production and toxicology

Alternaria species are common saprophytes or pathogens of a wide range of plants pre- and post-harvest. This review considers the relative importance of Alternaria species, their ecology, competitiveness, production of mycotoxins and the prevalence of the predominant mycotoxins in different food products. The available toxicity data on these toxins and the potential future impacts of Alternaria species and their toxicity in food products pre- and post-harvest are discussed. The growth of Alternaria species is influenced by interacting abiotic factors, especially water activity (a w ), temperature and pH. The boundary conditions which allow growth and toxin production have been identified in relation to different matrices including cereal grain, sorghum, cottonseed, tomato, and soya beans. The competitiveness of Alternaria species is related to their water stress tolerance, hydrolytic enzyme production and ability to produce mycotoxins. The relationship between A. tenuissima and other phyllosphere fungi has been examined and the relative competitiveness determined using both an Index of Dominance (I D ) and the Niche Overlap Index (NOI) based on carbon-utilisation patterns. The toxicology of some of the Alternaria mycotoxins have been studied; however, some data are still lacking. The isolation of Alternaria toxins in different food products including processed products is reviewed. The future implications of Alternaria colonization/infection and the role of their mycotoxins in food production chains pre- and post-harvest are discussed

Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature

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 Aspergillus flavus grown on YES medium. Generally A. flavus showed expression of the aflatoxin biosynthetic genes at all parameter combinations tested. Certain combinations of aw 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

Detection and discrimination between ochratoxin producer and non-producer strains of Penicillium nordicum on a ham-based medium using an electronic nose

The aim of this work was to evaluate the potential use of qualitative volatile patterns produced by Penicillium nordicum to discriminate between ochratoxin A (OTA) producers and non-producer strains on a ham-based medium. Experiments were carried out on a 3% ham medium at two water activities (aw ; 0.995, 0.95) inoculated with P. nordicum spores and incubated at 25°C for up to 14days. Growing colonies were sampled after 1, 2, 3, 7 and 14days, placed in 30-ml vials, sealed and the head space analysed using a hybrid sensor electronic nose device. The effect of environmental conditions on growth and OTA production was evaluated based on the qualitative response. However, after 7days, it was possible to discriminate between strains grown at 0.995 aw, and after 14days, the OTA producer and non-producer strain and the controls could be discriminated at both aw levels. This study suggests that volatile patterns produced by P. nordicum strains may differ and be used to predict the presence of toxigenic contaminants in ham. This approach could be utilised in ham production as part of a quality assurance system for preventing OTA contaminatio

Efficacy of potential biocontrol agents for control of Fusarium verticillioides and fumonisin B1 under different environmental conditions

A mycotoxigenic strain of Fusarium verticillioides previously isolated from Malaysian maize kernels and identified morphologically and molecularly was used in the present work. The objectives were (1) to screen the competitiveness of three potential antagonists isolated from Malaysian maize and three other known candidates for control of growth of F. verticillioides in vitro based on interaction scores, growth rates and hyphal area of F. verticillioides, and (2) to examine the best candidates using different spore/cell ratios on milled maize agar at different water activity conditions on relative control of fumonisin B1 (FB1). Three fungi and three bacteria (BCAs 1-6) were examined for antagonistic effects against F. verticillioides in dual-culture assays. These showed that all fungal candidates intermingled with F. verticillioides while all bacterial candidates inhibited F. verticillioides on contact or at a distance, which in turn decreased the growth rates and hyphal area of F. verticillioides significantly. Although BCA1 (Clonostachys rosea 016) did not inhibit growth or hyphal area of F. verticillioides, it was included in FB1 inhibition studies with other bacterial candidates (BCA4, Streptomyces sp. AS1; BCA5, Gram-negative rod; BCA6, Enterobacter hormaechei) because of its established mycoparasitism. The FB1 inhibition studies were conducted on milled maize agar with different spore/cell ratios of pathogen:antagonist mixtures at 0.95/0.98 water activity (aw) and 25 °C for 14 days. FB1 biosynthesis for all treatments was significantly higher at 0.95 than 0.98 aw. Of the four antagonists tested, the best was BCA1 which inhibited FB1 biosynthesis by 73 and 100% at 0.95 and 0.98 aw, respectively. BCA5 was the next best, resulting in 38 and 78% FB1 inhibition at 0.95 and 0.98 aw, respectively. The pathogen:antagonist ratios for BCA1 and BCA5 showed best results at 50:50 and 25:75. These results were discussed in context of using biocontrol agents to minimise fumonisins in maize

Effects of osmotic and matric potential on radial growth and accumulation of endogenous reserves in three isolates of Pochonia chlamydosporia

For the first time, the effects of varying osmotic and matric potential on fungal radial growth and accumulation of polyols were studied in three isolates of Pochonia chlamydosporia. Fungal radial growth was measured on potato dextrose agar modified osmotically using potassium chloride or glycerol. PEG 8000 was used to modify matric potential. When plotted, the radii of the colonies were found to grow linearly with time, and regression was applied to estimate the radial growth rate (mm day-1). Samples of fresh mycelia from 25-day-old cultures were collected and the quantity (mg g-1 fresh biomass) of four polyols (glycerol, erythritol, arabitol and mannitol) and one sugar (glucose) was determined using HPLC. Results revealed that fungal radial growth rates decreased with increased osmotic or matric stress. Statistically significant differences in radial growth were found between isolates in response to matric stress (P<0.006) but not in response to osmotic stress (P=0.759). Similarly, differences in the total amounts of polyols accumulated by the fungus were found between isolates in response to matric stress (P<0.001), but not in response to osmotic stress (P=0.952). Under water stress, the fungus accumulated a combination of different polyols important in osmoregulation, which depended on the solute used to generate the stress. Arabitol and glycerol were the main polyols accumulated in osmotically modified media, whereas erythritol was the main polyol that was accumulated in media amended with PEG. The results found that Pochonia chlamydosporia may use different osmoregulation mechanisms to overcome osmotic and matric stresses

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

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