Fungal interactions and control of aflatoxins in maize, pre-and post-harvest under different climate change scenarios.

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.PhD in Environment and Agrifoo

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

This 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