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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]

Aflatoxin B 1 (AFB 1) is one of the most hazardous mycotoxins for humans and livestock that mainly produced by members of the genus Aspergillus in a variety of food commodities. In this study, the effect of S. rosmarinus , T. fruticulosum , and T. caucasicum essential oils (EOs) was studied on fungal growth, AFB 1 production and aflR gene expression in toxigenic A. flavus IPI 247. The AFB 1 producer A. flavus strain was cultured in YES medium in presence of various two-fold concentrations of the plant EOs (62.5–500 μg/mL) for 4 days at 28 °C. EO composition of plants was analyzed by Gas Chromatography/Mass Spectrometry (GC/MS). The amount of fungal growth, ergosterol content of fungal mycelia and AFB 1 content of EO-treated and non-treated controls were measured. The expression of aflR gene was evaluated using Real-time PCR in the fungus exposed to minimum inhibitory concentration (MIC 50) of EOs. The main constituents of the oils analyzed by GC/MS analysis were elemicin (33.80 %) and 2,3-dihydro farnesol (33.19 %) in T. caucasicum , 1,8-cineole (17.87 %), trans-caryophyllene (11.14 %), α and ẞ-pinene (10.92 and 8.83 %) in S. rosmarinus , and camphor (17.65 %), bornyl acetate (15.08 %), borneol (12.48 %) and camphene (11.72 %) in T. fruticulosum. The results showed that plant EOs at the concentration of 500 μg/mL suppressed significantly the fungal growth by 35.24–71.70 %, while mycelial ergosterol content and AFB 1 production were inhibited meaningfully by 36.20–65.51 % and 20.61–89.16 %. T. caucasicum was the most effective plant, while T. fruticulosum showed the lowest effectiveness on fungal growth and AFB 1 production. The expression of aflR in T. caucasicum and S. rosmarinus -treated fungus was significantly down-regulated by 2.85 and 2.12 folds, respectively, while it did not change in T. fruticulosum -treated A. flavus compared to non-treated controls. Our findings on the inhibitory activity of T. caucasicum and S. rosmarinus EOs toward A. flavus growth and AFB 1 production could promise these plants as good candidates to control fungal contamination of agricultural crops and food commodities and subsequent contamination by AFB 1. Down-regulation of aflR as the key regulatory gene in AF biosynthesis pathway warrants the use of these plants in AF control programs. Further studies to evaluate the inhibitory activity of studied plants EOs in food model systems are recommended. [Display omitted] • EOs of T. caucasicum , S. rosmarinus and T. fruticulosum analysed by GC/MS inhibited A. flavus growth and AFB 1 production. • Ergosterol content of fungal mycelia was diminished significantly by the plants EOs. • Fungal growth, AFB 1 synthesis and ergosterol production were inhibited by plant Eos time-dependently. • aflR gene expression was strongly suppressed by T. caucasicum and S. rosmarinus. • These bioactive plants would be promising to control fungal growth and subsequent AFB 1 contamination of food and feed. [ABSTRACT FROM AUTHOR]

The objective of this study was to carry out a transcription analysis of eight genes belonging to the aflatoxin (AF) and cyclopiazonic acid (CPA) biosynthesis pathway, and to detect aflatoxin B 1 (AFB 1 ) and CPA production in Aspergillus flavus strains isolated from Brazil nuts. Additionally, these genes were correlated with the different mycotoxigenic profiles of the same strains. Four previously identified A. flavus strains (ICB-01, ICB-151, ICB-161, and ICB-165) were grown on Brazil nut agar at 25 °C for 10 days. Mycotoxins were separated by high-performance liquid chromatography. Transcriptional analysis was performed by real-time RT-PCR using specific primers designed based on the conserved regions of two regulatory genes ( aflR and aflS ), three structural genes of the AFB 1 biosynthesis pathway ( aflH , aflJ and aflP ), and three structural genes of the CPA biosynthesis pathway ( maoA , dmaT and pks-nrps ). The expression of most genes in the A. flavus isolates varied according to the mycotoxin profile of each strain. The most expressed genes in the aflatoxigenic strain ICB-151 were aflJ (77.11%) and aflH (32.75%), while the CPA-producing strain ICB-161 mainly expressed dmaT (100%), maoA (63.72%), aflS (43.52%), and aflR (42.63%). The ICB-01 isolate was a producer of AFB 1 and CPA and the most expressed genes were aflS ( 47.79%), dmaT (42.77%), aflP (39.5%), and aflR (38.02%). ICB-198 did not produce any mycotoxin and exhibited lower expression of almost all genes analyzed. Furthermore, the ratio of aflS / aflR expression was correlated with the biosynthesis of AF and CPA in A. flavus strains producing exclusively AF or CPA or producing both AF and CPA. The ratio of aflS / aflR expression therefore seems to be related to the production of mycotoxins in Brazil nuts. Our results provide important data for the development of innovative and more cost-effective strategies to reduce and prevent AFB and CPA contamination in Brazil nuts. [ABSTRACT FROM AUTHOR]

Variation in grazing intensity determines the way in which mycelial growth and physiology is influenced by invertebrate fungivores. Recently, low-intensity grazing by facultative mesofaunal fungivores, small dipteran larvae and collembola, was found to enhance the activity of fungal defensive secondary metabolism. Here, we ask whether higher intensity grazing by macrofaunal fungivores induces a different fungal response. To test this, we quantified changes in the expression of two anti-fungivore chemical defence marker genes, laeA and aflR , in the ascomycete Aspergillus nidulans exposed to grazing pressure exerted by individuals of the isopod Oniscus asellus . Compared to no and low-intensity grazing pressure, higher intensity grazing significantly reduced the transcript levels of these genes. This finding suggests a negative relationship between grazing pressure intensity and the capacity of a saprotrophic fungus to resist fungivory. [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: Aspergillus oryzae, although closely related to Aspergillus flavus, does not produce aflatoxin (AF). A. oryzae RIB strains can be classified into three groups (group 1–3) based on the structure of the AF biosynthesis gene homolog cluster (AFHC). In group 1 strains, where AFHC is present, the expression level of the aflR gene is extremely low and there is no expression of the other four AF homologue genes (avnA, verB, omtA and vbs). We conducted a detailed structural comparison of AFLR ORF and AFLJ ORF from A. oryzae and A. flavus and identified several amino-acid substitutions. If these substitutions induce inactivation of AFLR and AFLJ, AF biosynthesis of A. oryzae will be doubly inhibited at the transcriptional and translational level. In this study, we transferred aflR and aflJ to A. oryzae RIB67, a group 2 strain where more than half of AFHC is missing. Under control of the pgkA promoter, aflR and aflJ was expressed and avnA, verB, omtA and vbs gene expression were monitored by RT-PCR. We prepared six types of forced-expression vectors, including aflR (from A. oryzae RIB40 or its three mutants) or aflJ (from A. oryzae RIB40 or A. flavus RIB4011). RT-PCR analysis showed that transformants containing aflJ from A. oryzae displayed no expression of AF biosynthetic homologue genes, whereas aflR substitutions had no such effect. These results strongly suggest that the amino-acid substitutions in AFLJ of A. oryzae induce inactivation at the protein level. [Copyright &y& Elsevier]

The molecular regulation of aflatoxin biosynthesis is complex and influenced by several environmental conditions; one of these is temperature. Aflatoxins are produced optimally at 28-30 C, and production decreases as temperatures approach 37 C, the optimum temperature for fungal growth. To better characterize the influence of temperature on aflatoxin biosynthesis, we monitored the accumulation of aflatoxin and the expression of more than 5000 genes in Aspergillus flavus at 28 C and 37 C. A total of 144 genes were expressed differentially (P < 0.001) between the two temperatures. Among the 103 genes more highly expressed at 28 C, approximately 25% were involved in secondary metabolism and about 30% were classified as hypothetical. Genes encoding a catalase and superoxide dismutase were among those more highly expressed at 37 C. As anticipated we also found that all the aflatoxin biosynthetic genes were much more highly expressed at 28 C relative to 37 C. To our surprise expression of the pathway regulatory genes aflR and aflS, as well as aflR antisense, did not differ between the two temperatures. These data indicate that the failure of A. flavus to produce aflatoxin at 37 C is not due to lack of transcription of aflR or aflS. One explanation is that AFLR is nonfunctional at high temperatures. Regardless, the factor(s) sensing the elevated temperatures must be acute. When aflatoxin-producing cultures are transferred to 37 C they immediately stop producing aflatoxin. [ABSTRACT FROM AUTHOR]

Senna (Cassia angustifolia Vahl.) is a medicinal crop with laxative properties, and it has significant demand in the global pharmaceutical market. Senna pods are highly susceptible to aflatoxin contamination, and the successful export of pods is hindered due to the regulatory limits of importing countries. The senna pod water activity (a w) from harvest to storage is the key factor determining AFB 1 accumulation. The temperature conditions from field to warehouse also interact with pod a w , which influences fungal growth and AFB 1 production. The determination of an ideal combination of a w and temperature led to the assessment of the critical control point for AFB 1 synthesis in senna. Hence, this study aimed to evaluate the influence of a w (0.99, 0.96, 0.93, 0.90, and 0.87 a w) and temperature (20, 28, and 37 °C) on fungal growth, gene expression (aflR and aflS), and AFB 1 production by A. flavus in senna agar medium. The fungus showed the longest lag time (7.7 days) at 20 °C with 0.87 a w. We observed that 0.96 a w (P < 0.01) was optimum for the diametric growth rate at 28 and 37 °C. However, the peak expression of regulatory genes (aflR and aflS) and the maximum AFB 1 production were observed only at 28 °C (0.96 a w). The highest growth rate occurred at 37 °C, which did not favor the expression of genes and AFB 1 production. However, at 28 °C, it positively correlated with gene expression and AFB 1 production. The suppressed expression of regulatory genes and a trace amount of aflatoxin B 1 were found at 20 °C with all the tested a w. In our experiments, the low a w (0.87 and 0.90 a w) suppressed the fungal growth, gene expression, and AFB 1 production of A. flavus at all of the tested temperatures (20, 28, and 37 °C). The rapid drying of senna pods with a low water activity (≤0.87 a w) and storage at low temperature (20 °C) are ideal conditions to avoid AFB 1 and ensure the quality of produce for export. • Temperature and a w are the key factors for aflatoxin contamination in senna pods. • flavus had the longest lag phase and slowest growth rate at 20 °C with 0.87 a w. • The optimum condition for AFB 1 production was found at 28 °C with 0.96 a w. • The fastest fungal growth observed at 37 °C was not correlated with higher AFB 1. • Storing pods at 20 °C with ≤0.87 a w is ideal to avoid aflatoxin contamination. [ABSTRACT FROM AUTHOR]

Pistachio nuts are an important economic tree nut crop which is used directly or processed for many food-related activities. They can become colonized by mycotoxigenic spoilage fungi, especially Aspergillus flavus, mainly resulting in contamination with aflatoxins (AFs), especially aflatoxin B1 (AFB1). The prevailing climate in which these crops are grown changes as temperature and atmospheric CO2 levels increase, and episodes of extreme wet/dry cycles occur due to human industrial activity. The objectives of this study were to evaluate the effect of interacting Climate Change (CC)-related abiotic factors of temperature (35 vs. 37 °C), CO2 (400 vs. 1000 ppm), and water stress (0.98–0.93 water activity, aw) on (a) growth (b) aflD and aflR biosynthetic gene expression and (c) AFB1 production by two strains A. flavus (AB3, AB10) in vitro on milled pistachio-based media and when colonizing layers of shelled raw pistachio nuts. The A. flavus strains were resilient in terms of growth on pistachio-based media and the colonisation of pistachio nuts with no significant difference when exposed to the interacting three-way climate-related abiotic factors. However, in vitro studies showed that AFB1 production was significantly stimulated (p < 0.05), especially when exposed to 1000 ppm CO2 at 0.98–0.95 aw and 35 °C, and sometimes in the 37 °C treatment group at 0.98 aw. The relative expression of the structural aflD gene involved in AFB1 biosynthesis was decreased or only slightly increased, relative to the control conditions at elevated CO, regardless of the aw level examined. For the regulatory aflR gene expression, there was a significant (p < 0.05) increase in 1000 ppm CO2 and 37 °C for both strains, especially at 0.95 aw. The in situ colonization of pistachio nuts resulted in a significant (p < 0.05) stimulation of AFB1 production at 35 °C and 1000 ppm CO2 for both strains, especially at 0.98 aw. At 37 °C, AFB1 production was either decreased, in strain AB3, or remained similar, as in strain AB10, when exposed to 1000 ppm CO2. This suggests that CC factors may have a differential effect, depending on the interacting conditions of temperature, exposure to CO2 and the level of water stress on AFB1 production. [ABSTRACT FROM AUTHOR]