Complete genome of the toxic mold Aspergillus pseudotamarii isolate NRRL 25517 reveals genomic instability of the aflatoxin biosynthesis cluster.

Fungi can synthesize a broad array of secondary metabolite chemicals. The genes underpinning their biosynthesis are typically arranged in tightly linked clusters in the genome. For example, ∼25 genes responsible for the biosynthesis of carcinogenic aflatoxins by Aspergillus section Flavi species are grouped in a ∼70 Kb cluster. Assembly fragmentation prevents assessment of the role of structural genomic variation in secondary metabolite evolution in this clade. More comprehensive analyses of secondary metabolite evolution will be possible by working with more complete and accurate genomes of taxonomically diverse Aspergillus species. Here, we combined short- and long-read DNA sequencing to generate a highly contiguous genome of the aflatoxigenic fungus, Aspergillus pseudotamarii (isolate NRRL 25517 = CBS 766.97; scaffold N50 = 5.5 Mb). The nuclear genome is 39.4 Mb, encompassing 12,639 putative protein-encoding genes and 74-97 candidate secondary metabolite biosynthesis gene clusters. The circular mitogenome is 29.7 Kb and contains 14 protein-encoding genes that are highly conserved across the genus. This highly contiguous A. pseudotamarii genome assembly enables comparisons of genomic rearrangements between Aspergillus section Flavi series Kitamyces and series Flavi. Although the aflatoxin biosynthesis gene cluster of A. pseudotamarii is conserved with Aspergillus flavus, the cluster has an inverted orientation relative to the telomere and occurs on a different chromosome.

Micro-climatic variations across Malawi have a greater influence on contamination of maize with aflatoxins than with fumonisins.

This study reports levels of aflatoxin and fumonisin in maize samples (n = 1294) from all agroecological zones (AEZs) in Malawi. Most maize samples (> 75%) were contaminated with aflatoxins and 45% with fumonisins, which co-occurred in 38% of the samples. Total aflatoxins varied across the AEZs, according to mean annual temperature (P < 0.05) of the AEZs. Samples from the lower Shire AEZ (median = 20.8 µg/kg) had higher levels of aflatoxins (P < 0.05) than those from the other AEZs (median = 3.0 µg/kg). Additionally, the majority (75%) of the positive samples from the lower Shire AEZ had aflatoxin levels exceeding the EU regulatory limit (4 µg/kg), whereas 25%, 37%, and 39% of positive samples exceeded the threshold in the mid-elevation, Lake Shore and upper and middle Shire, and highlands AEZs, respectively. The lower Shire AEZ is characterised by higher mean temperatures throughout the year and low erratic rainfall. However, total fumonisins did not show significant variation across AEZs, but all positive samples exceeded 150 µg/kg, required for tolerable daily intake of 1.0 µg/kg body weight per day, established by the European Food Safety Authority Panel on Contaminants in the Food Chain. Therefore, results of this study suggest that contamination of maize with aflatoxin responds to micro-climate more than with fumonisins. In addition, the data will be useful to public health policy-makers and stakeholders to articulate and implement monitoring and mitigation programs.

Temperature Influences on Interactions Among Aflatoxigenic Species of Aspergillus Section Flavi During Maize Colonization.

Fungal species within Aspergillus section Flavi contaminate food and feed with aflatoxins. These toxic fungal metabolites compromise human and animal health and disrupt trade. Genotypically and phenotypically diverse species co-infect crops, but temporal and spatial variation in frequencies of different lineages suggests that environmental factors such as temperature may influence structure of aflatoxin-producing fungal communities. Furthermore, though most species within Aspergillus section Flavi produce sclerotia, divergent sclerotial morphologies (small or S-type sclerotia vs. large or L-type sclerotia) and differences in types and quantities of aflatoxins produced suggest lineages are adapted to different life strategies. Temperature is a key parameter influencing pre- and post-harvest aflatoxin contamination of crops. We tested the hypothesis that species of aflatoxin-producing fungi that differ in sclerotial morphology will vary in competitive ability and that outcomes of competition and aflatoxin production will be modulated by temperature. Paired competition experiments between highly aflatoxigenic S-type species (A. aflatoxiformans and Lethal Aflatoxicosis Fungus) and L-type species (A. flavus L morphotype and A. parasiticus) were conducted on maize kernels at 25 and 30°C. Proportions of each isolate growing within and sporulating on kernels were measured using quantitative pyrosequencing. At 30°C, S-type fungi were more effective at host colonization compared to L-type isolates. Total aflatoxins and the proportion of B vs. G aflatoxins were greater at 30°C compared to 25°C. Sporulation by L-type isolates was reduced during competition with S-type fungi at 30°C, while relative quantities of conidia produced by S-type species either increased or did not change during competition. Results indicate that both species interactions and temperature can shape population structure of Aspergillus section Flavi, with warmer temperatures favoring growth and dispersal of highly toxigenic species with S-type sclerotia.

Consumption of illegal home-made alcohol in Malawi: A neglected public health threat.

This study assessed the ethanol and methanol contents of homemade spirit (Kachasu) sold in Blantyre, Malawi. The likelihood of ethanol and methanol toxicity, respectively, was determined through Monte Carlo simulations using reported Kachasu intake volumes of 21 consumers and the determined methanol and ethanol contents. Ethanol concentration, in samples from 20 different distillers, ranged from 11 to 55% v/v. Methanol was detected in 10 of the 20 samples (0.01-0.28% v/v). The likely mean ethanol intake of drinkers in Blantyre was found to be 214 ± 93 mL per day (90% CI, 68.9-373.4 mL), and mean methanol intake was 0.44 ± 0.37 mL (90% CI, 0.03-1.17 mL). The intake values translated to mean blood ethanol and methanol concentrations of 38 ± 16 mg/mL and 0.05 ± 0.04 mg/mL, respectively. Therefore, the risk of methanol toxicity was considered as negligible. However, there was a high risk of ethanol toxicity. Since production and selling of Kachasu are already illegal in Malawi, enforcement of regulations should be strengthened to reverse the current situation where Kachasu is being distilled and sold openly even within cities. Consumers should also be sensitized about the likely risks associated with consumption of Kachasu in Malawi so that they can make informed choices.