PhD, EngD and MSc by research theses (SWEE)

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Browsing PhD, EngD and MSc by research theses (SWEE) by Subject "A. flavus"

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    Ecology, climate change and control strategies for aspergillus flavus colonisation and aflatoxin contamination of pistachio nuts.
    (2017-12) Baazeem, Alaa Abdulaziz; Magan, Naresh
    Pistachio nuts (Pistacia vera L.) have become one of the most important products in the economy of many countries including the USA, Iran, Syria, Greece, Turkey, China, EU and the Middle East. Pistachio nuts are very commonly colonized by spoilage mycobiota especially aflatoxigenic species because they are very hygroscopic and can adsorb water. Aspergillus flavus can contaminate pistachio nuts with aflatoxins (AFs), especially aflatoxin B1 (AFB1) classified as a class 1a carcinogen. The objectives of this project were (a) to examined the mycobiota and the aflatoxin producing strains of Aspergillus section Flavi species in pistachio nuts originating from different countries and sourced in the Kingdom of Saudi Arabia (KSA), (b) investigated the effect of the interactions between temperature and water activity (aw) on the ecology and molecular ecology growth and AFB₁ production by Aspergillus flavus strains in vitro on pistachio nut-based media and in stored raw pistachio nuts (c) evaluate the effect of Climate Change (CC) interacting factors on growth and AFB1 production by strains of A.flavusand on relative genes expression of the aflD and aflR genes involved in the biosynthetic pathway for AFB₁ production, (d) examine whether acclimatisation to 1000 ppm CO₂ of A.flavus strains AB3 and AB10 for 5 generations affected growth and AFB₁ production; and (e) to examine the use of gaseous O₃ for the control of germination, A.flavus populations and AFB₁ contamination of stored pistachio nuts for up to 4 weeks. Pistachio samples were colonized by a range of Aspergillus and Penicillium species. In some samples, typically phyllosphere fungi such as yeasts, Mucor, Rhizopus, Alternaria, Epicoccum and Phoma species were isolated. 10 different species of A.flavus were isolated and molecularly identified. The relative toxigenic nature of strains was evaluated using selective media and HPLC and confirmed using molecular tools. Four strains were used in ecological studies and two (AB3, AB10) in other studies. The ecological studies showed that optimum growth of AB3 and AB10 strainswas at 0.98 or 0.95 aw and 30-35°C. The effect of the same factors on aflR gene expression of the two strains showed optimum condition at 30-35°C and 0.98 aw; with optimum conditions for AFB₁ production at 35°C and 0.98 aw for strain AB3. There was little difference between the effect of using a non-ionic (glycerol) or ionic (NaCl) to modify water stress in in vitro studies. The effect of interacting CC factors on growth of A.flavus colonisation was not significant. However, AFB₁ production was stimulated. With regards to aflD gene expression, at 35°C, the relative expression was higher in current CO₂ conditions (400 ppm) for both strains except that for strain AB3 the gene expression was higher at 1000 ppm CO2 at 0.95 aw. However, at 37°C, the expression was generally higher in the 1000 ppm CO₂ than with existing atmospheric CO₂ levels. The aflR gene expression was higher at 1000 ppm CO₂ at 37°C for both strains. AFB₁ production was higher at 35°C at the two CO₂ levels for both strains. At the same temperature, AFB1 production was significantly increased at 1000 ppm CO₂ and 0.98 aw. At 37°C, AFB₁ production was either decreased in strain AB3 or 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 (35 or 37°C) as in some cases for AFB₁. Acclimatisation influenced growth of one strain while there was no significant effect on another strain when colonising pistachio nuts. For AFB₁, the production was significantly stimulated after ten days colonisation after acclimatisation for one strain, while there was no significant increase for the other strain. This suggests that there may be intra-strain differences in effects of acclimatisation and this could influence mycotoxin contamination of such commodities as mixed population of contaminant fungi often occurs. Exposure of conidia to gaseous O₃ initially had lower germination percentages when compared to the controls at different aw levels. Complete inhibition of germinations was observed after 12 h treatment of 200 ppm O₃ at 0.98 aw. However, spore viability appeared to recover and the germination was increased after 24 h and reached 100% after 48 h. Growth rates of mycelial colonies were decreased with increasing of O₃ dose and colony extension was significantly inhibited by O₃ treatment at 0.98 aw. Variable effects on AFB1 production during exposure to O₃ treatment after in vitro exposure of colonies of A.flavus incubated for ten days at 30°C. The populations of A.flavus were significantly decreased by O₃ exposure; however, there was little difference between 50-200 ppm treatment levels. A reduction in AFB₁ was only observed in the 50 ppm O₃ × 0.98 aw treatment in stored pistachio nuts. The relationship between exposure concentration × time of exposure and prevailing aw level to determine the efficacy in terms of toxin control needs to be better understand.
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    ItemOpen 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, Ángel
    Aspergillus 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.