Staff publications - Cranfield Health

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Open Access
Complex regulation of the aflatoxin biosynthesis gene cluster of Aspergillus flavus in relation to various combinations of water activity and temperature
(Elsevier, 2009-11-15) Schmidt-Heydt, Markus; Abdel-Hadi, Ahmed; Magan, Naresh; Geisen, Rolf
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.
Open Access
Control of Aflatoxin Production of Aspergillus flavus and Aspergillus parasiticus Using RNA Silencing Technology by Targeting aflD (nor-1) Gene.
(MDPI AG, 2011-06-30T00:00:00Z) Abdel-Hadi, Ahmed; Caley, D. P.; Carter, D. R.; Magan, Naresh
Aspergillus ﬂavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B(1 )(AFB(1)), and aflatoxin G(1) (AFG(1)) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB(1) production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB(1 )production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB(1) production by A. flavus EGP9 and AFG(1 )production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG(1) production by A. parasiticus SSWT 2999. Changes in AFB(1) production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technolog
Open Access
Discrimination between aflatoxigenic and non-aflatoxigenic Aspergillus section Flavi strains from Egyptian peanuts using molecular and analytical techniques
(Wageningen Academic Publishers, 2011-02-01T00:00:00Z) Abdel-Hadi, Ahmed; Carter, David; Magan, Naresh
A wide range of Aspergillus section Flavi strains were isolated from Egyptian peanut samples. Eighteen of these strains were compared with two type strains (Aspergillus flavus SRRC G1907 and Aspergillus parasiticus 2747) for aflatoxin production based on (a) qualitative fluorescence using a coconut cream agar medium (CAM), and (b) aflatoxin production on a conducive Yeast Extract-Sucrose (YES) medium using HPLC. These results were validated by using molecular approaches (the structural genes, aflD (nor-1), aflM (ver-1) and aflP (omt A) and the regulatory gene aflR) to discriminate between aflatoxigenic and non- aflatoxigenic strains of the Aspergillus section Flavi group in vitro and on peanut seeds. Overall, 13/18 strains producing aflatoxins B1 and B2 in the range 1.27-213.35 µg/g medium were identified. In addition, 5 non-aflatoxin producing strains were found. The expression of these four genes was assessed using PCR and RT-PCR. PCR showed that all strains contained the four aflatoxin genes examined, regardless of expression profiles. Our results also showed that aflD expression is a reliable marker to discriminate between aflatoxin and non- aflatoxin producers. Interestingly, when an aflatoxin producing strain and three non-aflatoxigenic strains were subsequently grown on peanuts at 0.95 water activity, two of the non-producers were able to initiate aflatoxin biosynthesis. This suggests that growth of strains on the natural food matrix is important for confirming aflatoxigenic production potential
Open Access
Influence of physiological factors on growth, sporulation and ochratoxin A/B production of the new Aspergillus ochraceus grouping
(Wageningen Academic Publishers, 2009-11) Abdel-Hadi, Ahmed; Magan, Naresh
Recently, new species within the Aspergillus section Circumdati group responsible for ochratoxin production were reported. This study has examined the impact of interactions between water activity (aw, 0.99-0.90), temperature (20-35 °C) on growth, asexual spore production and ochratoxin A and B (OTA and OTB) on strains of each of the three species from this new grouping (A. ochraceus, A. steynii, and A. westerdijkiae) for the first time. The maximum growth occurred at 0.95 aw and 30 °C for both A. ochraceus and A. westerdijkiae, while it was at 0.99 aw and 30 °C for A. steynii. No conidial spore production occurred at 0.99 aw in cultures of A. ochraceus and A. steynii but large numbers of spores (2.3×107/cm2) were produced by A. westerdijkiae. Optimum temperature for spore production was 0.95 aw and 30 °C for A. westerdijkiae and A. ochraceus, and 0.95 aw and 35 °C for A. steynii. Quantification of OTA showed that optimum was produced at 0.99 aw, by A. steynii at 30 °C, for A. westerdijkiae at 25 °C and for A. ochraceus at 20 °C. As water stress was imposed (0.95 aw), the temperature for maximum OTA production changed. For example, for A. steynii and A. westerdijkiae this was at 35 °C, for A. ochraceus, 25 °C. Much less OTB was produced relative to OTA, but the production followed the same pattern at all aw levels and temperatures. This is the first detailed study to examine the similarities and differences in ecology of these related species in this important mycotoxigenic group.
Open Access
The production of aflatoxin B1 or G1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression
(Mycotoxin Research, 2010-11-01T00:00:00Z) Schmidt-Heydt, Markus; Rüfer, C. E.; Abdel-Hadi, Ahmed; Magan, Naresh; Geisen, Rolf
The influence of varying combinations of wateractivity (aw) and temperature on growth, aflatoxin biosynthesisand aflR/aflS expression of Aspergillus parasiticuswas analysed in the ranges 17-42°C and 0.90-0.99 aw.Optimum growth was at 35°C. At each temperature studied,growth increased from 0.90 to 0.99 aw. Temperatures of 17and 42°C only supported marginal growth. The externalconditions had a differential effect on aflatoxin B1 or G1biosynthesis. The temperature optima of aflatoxin B1 andG1 were not at the temperature which supported optimalgrowth (35°C) but either below (aflatoxin G1, 20-30°C) orabove (aflatoxin B1, 37°C). Interestingly, the expression ofthe two regulatory genes aflR and aflS showed anexpression profile which corresponded to the biosynthesisprofile of either B1 (aflR) or G1 (aflS). The ratios of theexpression data between aflS:aflR were calculated. Highratios at a range between 17 and 30°C corresponded withthe production profile of aflatoxin G1 biosynthesis. A lowratio was observed at >30°C, which was related to aflatoxinB1 biosynthesis. The results revealed that the temperaturewas the key parameter for aflatoxin B1, whereas it waswater activity for G1 biosynthesis. These differences inregulation may be attributed to variable conditions of theecological niche in which these specie
Open Access
A systems approach to model the relationship between aflatoxin gene cluster expression, environmental factors, growth and toxin production by Aspergillus flavus.
(2012-04-07T00:00:00Z) Abdel-Hadi, Ahmed; Schmidt-Heydt, Markus; Parra, Roberto; Geisen, Rolf; Magan, Naresh
A microarray analysis was used to examine the effect of combinations of water activity (a(w), 0.995-0.90) and temperature (20-42°C) on the activation of aflatoxin biosynthetic genes (30 genes) in Aspergillus flavus grown on a conducive YES (20 g yeast extract, 150 g sucrose, 1 g MgSO(4)·7H(2)O) medium. The relative expression of 10 key genes (aflF, aflD, aflE, aflM, aflO, aflP, aflQ, aflX, aflR and aflS) in the biosynthetic pathway was examined in relation to different environmental factors and phenotypic aflatoxin B(1) (AFB(1)) production. These data, plus data on relative growth rates and AFB(1) production under different a(w) × temperature conditions were used to develop a mixed-growth-associated product formation model. The gene expression data were normalized and then used as a linear combination of the data for all 10 genes and combined with the physical model. This was used to relate gene expression to a(w) and temperature conditions to predict AFB(1) production. The relationship between the observed AFB(1) production provided a good linear regression fit to the predicted production based in the model. The model was then validated by examining datasets outside the model fitting conditions used (37°C, 40°C and different a(w) levels). The relationship between structural genes (aflD, aflM) in the biosynthetic pathway and the regulatory genes (aflS, aflJ) was examined in relation to a(w) and temperature by developing ternary diagrams of relative expression. These findings are important in developing a more integrated systems approach by combining gene expression, ecophysiological influences and growth data to predict mycotoxin production. This could help in developing a more targeted approach to develop prevention strategies to control such carcinogenic natural metabolites that are prevalent in many staple food products. The model could also be used to predict the impact of climate change on toxin production.
Open Access
Temporal monitoring of the nor-1 (aflD) gene of Aspergillus flavus in relation to aflatoxin B-1 production during storage of peanuts under different water activity levels
(Blackwell Publishing Ltd, 2010-12-01T00:00:00Z) Abdel-Hadi, Ahmed; Carter, D; Magan, Naresh
Aims: A relative quantification system (RQ-PCR) was used to monitor the correlations between the activity of the nor-1 (=aflD) gene of Aspergillus flavus using real-time PCR in relation to phenotypic aflatoxin B-1 (AFB(1)) production and populations of A. flavus in stored peanuts at three water activity levels (a(w), 0.95, 0.90 and 0.85) for 6 weeks. Methods and Results: Real-time PCR was used to amplify the nor-1 gene (target gene), and benA56 (beta-tubulin gene) used as a control gene. Expression of three structural genes, nor-1 (=aflD), ver-1 (=aflM), and omtA (=aflP), and the regulatory gene aflR of the aflatoxin biosynthetic pathway were also assayed. There were significant differences between nor-1 gene expression at the three a(w) levels; higher expression at 0.90 a(w) in weeks 1-3, when compared to 0.95. In contrast, in the driest treatment (0.85 a(w)) none or very low nor-1 expression occurred. The populations of A. flavus colony-forming units (CFUs g(-1)) increased over time with the highest at 0.95 a(w). Highest AFB(1) production was at 0.90 and 0.95 a(w) from weeks 3-6. A(w) had a significant effect on aflR transcription at 0.95 a(w) over the 6-week period, while at 0.90 a(w), only in the last 2 weeks. Conclusions: Correlations between different factors showed that log AFB(1) x log CFUs, log AFB(1) x a(w), and log CFUs x a(w) were statistically significant, while log CFUs x RQ-PCR and RQ-PCR x a(w) were not. The AflR gene may not have an important role in the regulation of nor-1 expression in food matrices (e. g. peanuts). Significance and Impact of the study: Determination of correlations between nor-1 expression and aflatoxin production by A. flavus in raw peanuts under different a(w) levels could be helpful to predict potential risk of aflatoxin production during storage of this hygroscopic food product and minimize contamination with the AFB(1).

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