Browsing by Author "Medina-Vayá, Ángel"
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Item Open Access 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(Elsevier, 2018-02-16) Aldars-García, Laila; Marín, Sonia; Sanchis, Vicente; Magan, Naresh; Medina-Vayá, ÁngelIntraspecies 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.Item Open Access Biocontrol of mycotoxins: dynamics and mechanisms of action(Elsevier, 2017-09-18) Medina-Vayá, Ángel; Mohale, Sejakhosi; Samsudin, Nik Iskandar Putra; Rodriguez-Sixtos, Alicia; Rodriguez, Alicia; Magan, NareshThis paper discusses the relationship between biocontrol agents (BCAs) and mycotoxigenic fungi and mycotoxin control. In most cases BCAs are examined for control of growth of fungal pathogens and disease symptoms. However, for mycotoxin control the approach and focus needs to be different. The mechanism of action and the inoculum dose necessary for control of toxin production by Aspegillus, Penicillium and Fusarium species may be different from that for traditional fungal plant pathogens. The mechanisms of action, the relative inoculum potential and the impact that interacting environmental conditions have on control of key components of the life cycle of mycotoxigenic fungi are considered. The practical aspects of production and formulation hurdles are discussed and potential future approaches and strategies which may need to be considered for more effective biocontrol of mycotoxigenic fungi and mycotoxins are presented.Item Open Access Bioproduction of xylitol by oleaginous yeast yarrowia lipolytica.(Cranfield University, 2020-01) Thomas, Dominic James; Medina-Vayá, Ángel; Veerheecke-Vaessen, CarolXylitol is a commercially important chemical with multiple applications in the food and pharmaceutical industries. According to the US Department of Energy, xylitol is one of the top twelve platform chemicals that can be produced from biomass. The chemical method for xylitol synthesis is, however, expensive and energy- intensive. In contrast, the biological route using microbial cell factories offers a potentially cost-effective alternative process. The bioprocess occurs under ambient conditions and makes use of biocatalysts and biomass which can be sourced from renewable carbon originating from a variety of cheap waste feedstocks. In this study, the biotransformation of xylose to xylitol was investigated using Yarrowia lipolytica, an oleaginous yeast which, in this study was firstly grown on a glycerol/glucose medium for the screening of a co- substrate, followed by a media optimisation in shake flasks, scale-up studies in a bioreactor and then downstream studies where done on the processing of xylitol. A two-step medium optimization was employed using a central composite design and an artificial neural network coupled with a genetic algorithm. The yeast amassed a concentration of 53 g/L of xylitol whilst using pure glycerol (PG) and xylose media, with a bioconversion yield of 0.97 g/g. Similar results were obtained when PG was substituted with crude glycerol (CG) from the biodiesel industry (titre: 51 g/L; yield: 0.92 g/g). Even when xylose from sugarcane bagasse hydrolysate was used as opposed to pure xylose, a xylitol yield of 0.54 g/g was achieved. The xylitol was successfully crystallized from the PG/xylose and CG/xylose fermentation broths with a recovery yield of 40 and 35 %, respectively. To the best of the author’s knowledge, this study demonstrates for the first time, the potential of using Y. lipolytica as a microbial cell factory for xylitol synthesis from inexpensive feedstocks. The results obtained are competitive with other xylitol producing organisms.Item Open Access Carbon dioxide mediates the response to temperature and water activity levels in Aspergillus flavus during infection of maize kernels(MDPI, 2017-12-22) Gilbert, Matthew K.; Medina-Vayá, Ángel; Mack, Brian M.; Lebar, Matthew D.; Rodriguez, Alicia; Bhatnagar, Deepak; Magan, Naresh; Obrian, Gregory; Payne, GaryAspergillus flavus is a saprophytic fungus that may colonize several important crops, including cotton, maize, peanuts and tree nuts. Concomitant with A. flavus colonization is its potential to secrete mycotoxins, of which the most prominent is aflatoxin. Temperature, water activity (aw) and carbon dioxide (CO2) are three environmental factors shown to influence the fungus-plant interaction, which are predicted to undergo significant changes in the next century. In this study, we used RNA sequencing to better understand the transcriptomic response of the fungus to aw, temperature, and elevated CO2 levels. We demonstrate that aflatoxin (AFB1) production on maize grain was altered by water availability, temperature and CO2. RNA-Sequencing data indicated that several genes, and in particular those involved in the biosynthesis of secondary metabolites, exhibit different responses to water availability or temperature stress depending on the atmospheric CO2 content. Other gene categories affected by CO2 levels alone (350 ppm vs. 1000 ppm at 30 °C/0.99 aw), included amino acid metabolism and folate biosynthesis. Finally, we identified two gene networks significantly influenced by changes in CO2 levels that contain several genes related to cellular replication and transcription. These results demonstrate that changes in atmospheric CO2 under climate change scenarios greatly influences the response of A. flavus to water and temperature when colonizing maize grain.Item Open Access Climate change, food security and mycotoxins: do we know enough?(Elsevier, 2017-05-18) Medina-Vayá, Ángel; Akbar, Asya; Baazeem, Alaa; Rodriguez, Alicia; Magan, NareshClimate 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.Item Open Access Comparison of different bead-beating RNA extraction strategies: An optimized method for filamentous fungi(Elsevier Science B.V., Amsterdam., 2012-03-01T00:00:00Z) Leite, Goncalo M.; Magan, Naresh; Medina-Vayá, ÁngelMolecular studies, especially in relation to the activity of secondary metabolite gene clusters, require the ability to extract good quality RNA from fungal biomass. This is often hindered by the cell wall structure and endogenous RNase activity in filamentous fungi. There is thus a need for rapid methods for the extraction of good quality RNA for use in microarrays and for quantitative PCR assays. The objective of this study was to examine the use of different systems for the high throughput method to extract intact RNA from filamentous fungi. Two bead beating systems with different motion patterns and speed capacities were tested in the development of the extraction protocol. They were evaluated based on the total RNA yield and overall RNA quality. The high speed bead beating with glass beads associated with an automated purification method gave more than three times higher total RNA yields with less than a quarter of the amount of mycelium required. Furthermore the integrity and overall quality was conserved, with RNA Quality Indicator (RQI) numbers consistently >7.5. This method also reduced cross contamination risks and kept RNA handling to a minimum while still being capable of multiple sample processing, reducing the time required to obtain RNA from filamentous fungi.Item Open Access 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(Elsevier, 2017-07-07) Martin, Sara; Medina-Vayá, Ángel; Magan, NareshThe 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.Item Open Access Comparison of water activity and temperature impacts on growth of Fusarium langsethiae strains from northern Europe on oat-based media(Elsevier Science B.V., Amsterdam., 2010-09-01T00:00:00Z) Medina-Vayá, Ángel; Magan, NareshThis study has examined the effect of water activity (aw, 0.995-0.90) and temperature (10-37°C) on the lag phases prior to growth, growth rates and used models to develop two dimensional profiles for optimum and marginal conditions for two strains of Fusarium langsethiae from four northern European countries (UK, Norway, Sweden, and Finland) on an oat-based medium. Results showed that the optimum aw for growth was at 0.98-0.995 and 25°C. The limit for growth of the strains was at 0.92-0.93 aw with minima of 10°C. No growth occurred at 37°C. The lag phases prior to growth were lowest under optimum conditions and extended to >10days at marginal conditions. Statistical analyses of intra and inter- strain differences in terms of both lag phases prior to growth and growth rates were not statistically significant. However, aw and temperature were statistically significant factors. Two dimensional profiles for strains from each country of origin were built to identify optimum and marginal conditions for F. langsethiae for the first time. These environmental profiles will be beneficial for improving the ecological knowledge of this species which is able to produce trichothecene mycotoxins in a range of temperate cereals.Item Open Access Deciphering the Anti-Aflatoxinogenic Properties of Eugenol Using a Large-Scale q-PCR Approach(MDIP, 2016-04-18) Caceres, Isaura; El Khoury, Rhoda; Medina-Vayá, Ángel; Lippi, Yannick; Naylies, Claire; Atoui, Ali; El Khoury, André; Oswald, Isabelle P.; Bailly, Jean-Denis; Olivier PuelProduced by several species of Aspergillus, Aflatoxin B1 (AFB1) is a carcinogenic mycotoxin contaminating many crops worldwide. The utilization of fungicides is currently one of the most common methods; nevertheless, their use is not environmentally or economically sound. Thus, the use of natural compounds able to block aflatoxinogenesis could represent an alternative strategy to limit food and feed contamination. For instance, eugenol, a 4-allyl-2-methoxyphenol present in many essential oils, has been identified as an anti-aflatoxin molecule. However, its precise mechanism of action has yet to be clarified. The production of AFB1 is associated with the expression of a 70 kB cluster, and not less than 21 enzymatic reactions are necessary for its production. Based on former empirical data, a molecular tool composed of 60 genes targeting 27 genes of aflatoxin B1 cluster and 33 genes encoding the main regulatory factors potentially involved in its production, was developed. We showed that AFB1 inhibition in Aspergillus flavus following eugenol addition at 0.5 mM in a Malt Extract Agar (MEA) medium resulted in a complete inhibition of the expression of all but one gene of the AFB1 biosynthesis cluster. This transcriptomic effect followed a down-regulation of the complex composed by the two internal regulatory factors, AflR and AflS. This phenomenon was also influenced by an over-expression of veA and mtfA, two genes that are directly linked to AFB1 cluster regulation.Item Open Access Different sample treatment approaches for the analysis of T-2 and HT-2 toxins from oats-based media(Elsevier Science B.V., Amsterdam., 2010-08-01T00:00:00Z) Medina-Vayá, Ángel; Valle-Algarra, Francisco M.; Jimenez, Misericordia; Magan, NareshA LC-DAD method is proposed for the determination of the T-2 and HT-2 toxins in cultures of Fusarium langsethiae in oat-based and other in vitro media. Test media consisted of freshly prepared milled oats to which T-2 and HT-2 toxin stock solutions were added. Different mixtures of extraction solvent (acetonitrile:water and methanol water), extraction times (30', 60' or 90') and drying methods were investigated. Results showed that extraction with methanol: water (80:20, v/v) for 90 min, drying with N-2 and subsequent analysis by LC-DAD was the fastest and most user friendly method for detecting HT-2 and T-2 toxins production by F. langsethiae strains grown on oat-based media at levels of 0.459 and 0.508 mg of toxin/kg of agar, respectively. The proposed method was used to investigate toxin production of 6 F. langsethiae strains from northern Europe and provided clear chromatograms with no interfering peaks in media with and without glycerol as water activity modifier. (C) 2010 Elsevier B.V. All rights reserved.Item Open Access Ecophysiological approaches to enhance production of the anti-cancer drug taxol by paraconiothyrium variabile and epiccocum nigrum and lysozyme by pichia pastoris(Cranfield University, 2013-07) Somjaipeng, Supunnika; Magan, Naresh; Medina-Vayá, ÁngelThis study has investigated the interaction of ecophysiological factors such as water activity (aw), temperature, pH and solute types on (a) the production of the anti-cancer drug taxol by the endophytic fungi Parachoniothyrium variabile and Epcoccum nigrum, and (b) the production of human lysozyme by a recombinant strain of the methanogenic yeast Pichia pastoris. Of more than 200 isolates from fresh health twigs of Taxus baccato trees on the Cranfield University Campus, only two strains of endophytic fungal species, P. variabile and E. nigrum, were able to produce taxol. P. variabile could produce 0.53 to 1.75 g/l taxol and E. nigrum could produce up to 1.32 µg/l in a defined M1D liquid medium. Ecological studies showed the profiles for growth and taxol production. The growth rate of P. variabile were generally higher at 0.99 aw and 25 C, but optimal at 0.99-0.98 aw. The average growth rate was faster when sorbitol was used as the aw depressor when compared with glycerol, glucose and salt-amended media. Statistical analysis indicated that all the studied stress factors significantly affected radial growth rate of P. variabile on a taxol conducive M1D agar (P<0.05). The optimum pH was 5.0 regardless of the aw x temperature conditions (P<0.05). The combined effects of aw, solute types and temperature on taxol production by P. variabile was determined using a 5x5x3 factorial design. The maximum amount of taxol was 7.11 g/l when cultured with M1D using KCl to modified media to 0.98 aw and 25 C. In contrast, on un- amended M1D medium at 25C yielded about 1.75 g/l of taxol. For E. nigrum, optimal conditions for growth were observed at 0.99-0.98 aw and 20-25 C. Growth on medium imposed with sorbitol was significantly faster (P<0.05). All three stress factors significantly affected radial growth rate of this strain (P<0.05). This strain grew faster at pH 5.0. The attenuation of taxol for this strain was observed during examination of the combined effects on taxol yield. Because of the attenuation and unstable production of taxol by the strain of E. nigrum, it was decided to try and use different elicitors to enhance production of taxol. Cont/d.Item Open Access Ecophysiology and water relations of growth and ochratoxin A production by Penicillium verrucosum adn Aspergillus westerdijkiae, impacts of climate change and control using preservatives.(2019) Abdel Mohsen, Shaimaa Ibrahim; Magan, Naresh; Medina-Vayá, ÁngelPenicillium verrucosum and Aspergillus westerdijkiae contaminate cereal grains and coffee beans with ochratoxin A (OTA) for which legislative limits exist. These fungi reside in soil and contaminate grain during harvesting and post-harvest storage, and post-fermentation during drying of coffee beans. There is a lack of information on the impacts of environmental conditions which influence the inoculum potential in soil and on cereal grain and coffee beans and potential control of OTA during post-harvest storage phases. The objectives of this project were to: (a) examine the water and temperature relations of strains of P. verrucosum (two strains) and A. westerdijkiae (three strains) in relation to growth and OTA production, (b) compare the sensitivity and tolerance of one strain of each species to solute (ionic and non-ionic) and matric stress on growth kinetics, expression of key genes in the OTA cluster (otapks, otanrps) and phenotypic OTA production for the first time, (c) examine the impact of three way interacting climate change related abiotic environmental factors (water activity, temperature, and CO₂ levels) on growth, gene expression and OTA production by strains of the two species, and (d) screen a range of potential preservatives in vitro control of growth and OTA production for in situ control of contamination of grain and coffee under different aw and temperature conditions. The ecological studies revealed that both strains of P. verrucosum (OTA11; straw21) could grow over a wide range of aw levels and temperatures. However, under drier conditions (0.90 aw) growth was much slower. For OTA production, optimum production was at 25°C and 0.98 aw for strain OTA11 when compared to the other strain on a conducive YES medium. On wheat-based media, both strains were able to grow efficiently over a range of aw levels (0.98, 0.95 and 0.90 aw) at 15, 20 and 25°C. On wheat-based matrices, the OTA production was much lower than on the conducive YES medium, regardless of aw and temperature for both strains after 10 days incubation. However, after 20 days, the strain Straw21, produced higher amounts of OTA under water stress condition (0.90 aw) at 25°C. For A. westerdijkiae strains growth occurred over a wide range of aw levels and different temperatures on the conducive YES media. However, the strain A. westerdijkiae CECT produced the highest amount of OTA at 0.98 aw and 25-30°C. Comparison of growth under solute and matric stress showed that the growth of P. verrucosum strain OTA11 is more tolerant of matric potential stress than Straw21 over a wide range of water potentials at 25°C. The strain OTA11 grew well under all non-ionic solute potential stress and under matric stress. However, under ionic solute stress no growth occurred under extreme water stress of -19.6 MPa (=0.86 aw). Upon examination of the expression pattern of the otapks gene under such stress conditions, there was an increase in the gene expression in the non-ionic (glycerol) solute stress-imposed treatments when compared with ionic solute (NaCl) and matric stress treatments. This suggests that this is a key gene involved in OTA biosynthesis. There were some parallels with the phenotypic OTA production. For A. westerdijkiae species, both tested strains (CECT and CCT) grew well under matric stress at all stress levels imposed (-1.4—19.6 MPa = 0.99-0.86 aw). As for P. verrcuosum, this strain was more sensitive to ionic solute stress imposed with NaCl with growth inhibited at -19.6 MPa (=0.86 aw). For this species, the gene expression of otapks was significantly increased under moderate stress conditions -9.8 MPa (=0.95 aw) modified with the non-ionic solute (glycerol). However, under matric stress this expression was significantly reduced when compared to solute stress. For OTA production, this was increased at -9.8 MPa (=0.95 aw) under non-ionic solute stress when compared to the other treatments. Overall, the impact of climate change related abiotic factors, especially elevated CO₂ levels (400 vs 1000 ppm CO₂) had no significant effect on the growth of P. verrucosum when compared with existing conditions under matric stress. However, under non-ionic stress (glycerol imposed), no growth was reported at -2.8 MPa (=0.95 aw) at 1000 ppm and 30°C. Overall, the growth pattern in non-ionic solute stress was lower under elevated levels of CO₂ than in matric stress conditions when compared with existing conditions. For the otapks gene, expression was increased under elevated CO₂ levels in matric stress treatments when compared to existing conditions. This pattern was paralleled with production of OTA under these conditions. With regard to A. westerdijkiae, surprisingly no growth occurred at 37°C in all the conditions tested. However, at 30°C, the elevated levels of CO₂ had no significant impact on growth under matric and non-ionic stress when compared with existing CO₂levels. For the otapks gene expression, this increased in matric imposed water stress in all conditions examined. However, there was no gene expression in non-ionic stress conditions, and this paralleled the OTA production pattern. Initial screening of six potential preservatives showed that for both growth and OTA control by one strain of each species SM, TCA and PP were the most effective compounds. They inhibited growth of P. verrucosum at 250 ppm on wheat-based matrices. While, for FE, it was the least effective treatment as ED50 and ED90 values show that 1000 mg/l and 2700 mg/l are required for controlling the growth rate at both water stress levels (0.95 and 0.95 aw) respectively and for the MIC for toxin production. However, on stored wheat grains, some growth and OTA was produced in the SM and TCA treatments at 250 ppm treatment. For A. westerdijkiae, the most effective compounds inhibiting growth were at 500 and 1000 ppm of SM, TCA and PP at 0.95 and 0.98 aw on coffee-based media. With PP, no toxin was produced at 100 ppm at both water stress levels although some growth occurred. Also, FE was the least effective treatment with ED50 and ED90 values of 1000 and 1530 mg/l respectively at both water stress levels. In stored coffee beans, the results were different with some growth found at 1000 ppm in treatments of TCA and SM at both aw levels. In addition, high amounts of OTA were produced in the 1000 ppm treated and stored coffee beans at both aw levels. Overall, in vitro efficacy was not an accurate guide to in situ efficacy, especially in relation to toxin control in both stored wheat and coffee beans under different aw levels.Item Open Access Effect of climate change on Aspergillus flavus and aflatoxin B-1 production(Frontiers Media, 2014-07-22) Medina-Vayá, Ángel; Rodriguez, Alicia; Magan, NareshThis 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.Item Open Access Efficacy of different caffeine concentrations on growth and ochratoxin A production by Aspergillus species(Wiley, 2016-06-03) Akbar, Asya Hussain; Medina-Vayá, Ángel; Magan, NareshThe 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.Item Open Access Efficacy of fungal and bacterial antagonists for controlling growth, FUM1 gene expression and fumonisin B 1 production by Fusarium verticillioides on maize cobs of different ripening stages(Elsevier, 2017-02-09) Samsudin, Nik Iskandar Putra; Rodriguez Sixtos Higuera, Alicia; Medina-Vayá, Ángel; Magan, NareshThis study was carried out to examine the efficacy of two biocontrol agents (Clonostachys rosea 016, BCA1; Gram-negative bacterium, BCA5) for control of FUM1 gene expression and fumonisin B1 (FB1) production by F. verticillioides FV1 on maize cobs of different ripening stages: R3, Milk (0.985 aw); R4, Dough (0.976 aw); R5, Dent (0.958 aw). Initially, temporal studies on FUM1 gene expression and FB1 production were performed on maize kernels for up to 14 days. This revealed that day 10 was optimum for both parameters, and was used in the biocontrol studies. Maize cobs were inoculated with 50:50 mixtures of the pathogen:antagonist inoculum and incubated in environmental chambers to maintain the natural aw conditions for ten days at 25 and 30 °C. The growth rates of F. verticillioides FV1, the relative expression of the FUM1 gene and FB1 production were quantified. It was found that, aw × temp had significant impacts on growth, FUM1 gene expression and FB1 production by F. verticillioides FV1 on maize cobs of different maturities. The fungal antagonist (BCA1) significantly reduced FB1 contamination on maize cobs by > 70% at 25 °C, and almost 60% at 30 °C regardless of maize ripening stage. For the bacterial antagonist (BCA5) however, FB1 levels on maize cobs were significantly decreased only in some treatments. These results suggest that efficacy of antagonists to control mycotoxin production in ripening maize cobs needs to take account of the ecophysiology of the pathogen and the antagonists, as well as the physiological status of the maize during silking to ensure effective control.Item Open Access Environmental stress and elicitors enhance taxol production by endophytic strains of Paraconiothyrium variabile and Epicoccum nigrum(Elsevier, 2016-05-04) Somjaipeng, Supunnika; Medina-Vayá, Ángel; Magan, NareshThis 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.Item Open Access Evaluation of the risk of fungal spoilage when substituting sucrose with commercial purified Stevia glycosides in sweetened bakery products(Elsevier, 2016-04-27) Rodriguez, Alicia; Magan, Naresh; Medina-Vayá, ÁngelThe 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.Item Open Access Fungal diversity, pest damage and biocontrol of aflatoxins in GM and conventional Brazilian maize cultivars under existing and future climate change scenarios.(Cranfield University, 2018-11) Marcon Gasperini, Alessandra; Magan, Naresh; Medina-Vayá, ÁngelThis study (a) evaluated the fungal biodiversity, toxigenic mycobiota and mycotoxin profiles associated with conventional (non-GM) and genetically modified (GM) isogenic maize cultivars (cvs) from Brazil, (b) studied the ecology of the isolated strains of toxigenic Aspergillus flavus using non-GM and the isogenic GM cv as substrates under different water activity(aw) and temperature interactions in vitro, (c) screened mycobiota for potential biocontrol agents (BCAs) and compared the interactions between atoxigenic (AFL⁻) and toxigenic (AFL⁺ ) A. flavus strains and other antagonistic species for in vitro control of aflatoxins (AFs) using different spore inoculum ratios, (d) examined the best potential BCAs to apply in situ in stored GM and non-GM isogenic maize cvs on AFs production and related expression of structural (aflD) and regulatory (aflR) toxin biosynthetic genes, and (e) examined the resilience of the biocontrol efficacy under simulated pest damage and climate change (CC) scenarios. The majority of the GM and isogenic non-GM cvs analysed (20 samples; 10 each type) had moisture content (%MC) and aw levels within the safety range for safe storage (<0.70 aw). Fusarium and Penicillium spp were the predominant genera identified with a low percentage of isolation of A. flavus strains in the maize cultivars examined. There was no significant difference (p<0.05) in the frequency of isolation between non-GM and GM cvs. A total of 22 A. flavus strains were isolated, of which 15 were non-aflatoxin producers, and 7 were aflatoxin B₁ (AFB₁) producers. Six of these strains were from non-GM maize cvs. Six pairs of isogenic GM- and non-GM cvs (n=12) out of the 20 used in this work were selected and analysed in more detail using LC-MS/MS. The mycotoxin profiles showed 29 compounds present, with higher amounts of Fusarium toxins than any other, which paralleled the high isolation frequency of Fusarium spp. AFs were not detected, while Fumonisins (B₁ or B₂) were present in 10 out of the 12 cvs, with only 2 non-GM cultivars having contamination levels above the EU legislative limits (4000 µg kg⁻1). The distribution of the mycotoxins indicated differences between the non-GM and GM cvs (p<0.05) with the latter having lower overall concentrations of mycotoxins. Subsequently, from the 22 isolated strains of A. flavus 4 were selected (3 toxigenic and 1 atoxigenic) for ecological studies using 3 pairs of GM- and non- GM maize cvs as substrate. The strains were able to colonize and grow on maize- based nutritional matrices from both GM (two pesticide and one herbicide + pesticide resistant) and non-GM cvs. The type of cvs did not have a significant effect on the growth of A. flavus, however temperature and aw had a significant effect (p<0.05) on the fungal development. The optimal conditions for growth were slightly different from those for AFB₁ production. Optimal growth occurred at 30-35ᴼ C and 0.99 aw, whereas AFB₁ production was optimal at 25-35ᴼ C and 0.99 aw. Each strain showed a different pattern of AFB₁ production and there was a shift in the optimal conditions depending on the combination of aw × To C × maize cv. In vitro a total of 8 atoxigenic (AFL⁻) and 8 other strains from different genera were tested as BCAs against 5 toxigenic strains (AFL⁺ ). This showed that A. flavus was highly dominant in vitro. One yeast strain (Y6) was able to compete against A. flavus on malt extract agar (MEA) at 0.98 aw but when it was co- cultivated in milled-maize agar (MMA) against the toxigenic AFL⁺ strain resulted in an increase in AFB₁ when compared to the control. The interaction of the toxigenic AFL⁺ × atoxigenic AFL⁻ strains were mutual intermingling on both MEA and MMA. On MMA for co-cultivation of different inoculum ratios the screening was only done against 3 AFL⁺ toxigenic strains to examine effects on AFB₁ control. The overall control of AFB₁ ranged from 29 to 100%. The most effective ratio of spores of the atoxigenic vs toxigenic strains was found to be a mixture of 50:50 mixed conidial inoculum of each strain. Based on the in vitro screening for potential BCAs, the atoxigenic A. flavus strains were examined to determine whether they had a deletion in biosynthetic genes involved in AFs and cyclopiazonic acid (CPA) production using multiplex PCR. Five atoxigenic strains (AFL⁻) were found to have large deletions of genes in the AFs cluster. While 3 atoxigenic strains amplified most of the markers in the AF cluster, however they were still unable to produce AFs. The strain selected for in situ biocontrol studies (Af53H – AFL4⁻ ) had a large deletion of AF markers but had all the CPA markers. The AFL4⁻ was able to significantly reduce AFB₁ when paired with toxigenic strains in a 50:50 spore ratio in stored GM and non-GM maize cvs. The relative gene expression of aflD and aflR in one of the toxigenic strains (AFLb⁺ ) used as pathogen was significantly inhibited by the chosen BCA. The correlation of gene expression ´ AFB₁ was positive indicating that suppression in the gene expression pathway contributed to the lower toxin levels. The overall biocontrol action seems to have been most effective when used in stored GM maize cultivars. Different levels of simulated pest damage (0, 5 and 15%) showed that AFB₁ production did not increase with a higher level of damage regardless of whether pesticide resistance or herbicide + pesticide resistance cvs were compared with non-GM isogenic ones. The toxin production in 15% damaged maize grain was lower or equal to that with no or 5% damage. The gene expression of aflR and aflD involved in AFs biosynthesis showed differences between the maize cvs. However, the correlation of gene expression × AFB₁ was not significantly positive. The BCA showed resilience under TᴼC × CO₂ × aw × simulated pest damage conditions with similar control levels of AFB₁ which was achieved under existing environmental conditions. The use of a GM cvs showed better results for biocontrol under water stress (0.95 aw) and elevated CO₂ at 35ᴼ C when the kernels were undamaged. However, biocontrol in conventional maize was better when there were damaged kernels at 0.95 aw × 35ᴼ C ×1000 ppm CO₂.Item Open Access Fungal interactions and control of aflatoxins in maize, pre-and post-harvest under different climate change scenarios.(2017-03) Rodriguez Sixtos Higuera, Alicia; Magan, Naresh; Medina-Vayá, ÁngelAspergillus flavus is a ubiquitous fungus that contaminates maize, the main risk from infection is the production of the carcinogenic mycotoxin aflatoxin B₁ (AFB₁). One strategy to control A. flavus contamination is the use of biocontrol agents (BCAs). The aim of this project was to examine the fungal diversity of Mexican maize cultivars and isolate potential BCAs which could control AFB₁ contamination of maize under existing and future climate change scenarios. The four Mexican maize cultivars had low moisture content, below that which would cause any mould spoilage. A. flavus and other associated mycobiota were enumerated, isolated and identified. Eight candidate BCAs were screened for potential antagonism and dominance of toxigenic strains of A. flavus including a type strain. This showed that the Index of Dominance of the BCAs vs A. flavus was influenced by strain and water activity (aw). On maize-based media, at 50:50 inoculum ratios four potential BCAs, an atoxigenic Afl- MEX02, T. atroviride MEX03, T. funiculosus MEX05 and C. rosea 016 were effective in reducing AFB₁ production. The atoxigenic Afl- MEX02 A. flavus strain decreased AFB₁ production by >95% by the toxigenic strain. These BCAs were then tested in more detail with different inoculum ratios including the atoxigenic A. flavus strain. The 50:50 ratios were used to analyse the expression of two key genes of the aflatoxin biosynthetic pathway, aflR (regulatory) and aflD (structural). For type strain of A. flavus (NRRL 3357) aflD relative gene expression was stimulated by the BCAs at 0.98 and 0.93 aw. The toxigenic MEX01 strain had aflD expression down-regulated at 0.98 aw in the presence of all the BCAs. The atoxigenic strain isolated from Mexican maize was the most effective at inhibiting AFB₁production under all aw x temperature conditions examined on maize-based media giving >90% control. Additionally, the potential reduction of inoculum potential of A. flavus by the best four BCA candidates on senescent maize leaves was examined under different aw levels. None of the four BCAs were able to reduce the conidial production by the toxigenic strain of A. flavus. The best candidate BCAs, atoxigenic Afl- MEX02 and C. rosea 016 were examined for efficacy in stored maize grain under different antagonist: pathogen ratios of 25:75; 50:50 and 75:25 initial inoculum. The relative gene expression of the treatment 50:50 ratio was analysed. The expression of both genes was down-regulated in the presence of the BCAs. Also the atoxigenic A. flavus trains had a lower expression compared to the control. This resulted in >60% control of AFB1 production by the atoxigenic strain under the aw x temperatures tested. For the C. rosea 016 strain this was only affected with relatively freely available water. The two best BCA candidates were examined for efficacy and control of toxigenic A. flavus strain growth and AFB₁ production on maize cobs of different ripening ages which also represented different aw and nutritional levels. Using 50:50 antagonist:pathogen ratios of inoculum this showed that the BCAs down-regulated the expression of the aflD and aflR genes in the aflatoxin biosynthetic pathway. However, there was no effect on growth or AFB₁ production. The resilience of the candidate BCAs was tested under climate change scenarios (aw x temperature x CO₂). This showed that at the different ripening stages A. flavus was able to grow at similar rates to the control and that AFB₁ production was unaffected by the conditions and indeed by the presence of the BCAs examined. The results obtained are discussed in the context of the different minimisation strategies which can be employed to try and reduce exposure of consumers to this carcinogenic mycotoxin.Item Open Access Fusarium graminearum in stored wheat: use of CO2 production to quantify dry matter losses and relate this to relative risks of Zearalenone contamination under interacting environmental conditions(MDPI, 2018-02-17) Garcia-Cela, Esther; Kiaitsi, Elisavet; Sulyok, Michael; Medina-Vayá, Ángel; Magan, NareshZearalenone (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.
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