Abstract
Aspergillus flavus is the main producer of carcinogenic aflatoxins in agricultural commodities such as maize. This fungus occurs naturally on crops, and produces aflatoxins when environmental conditions are favorable. The aim of this study is to analyse the genetic variability among 109 A. flavus isolates previously recovered from maize sampled from a known aflatoxin-hotspot (Eastern region, Kenya) and the major maize-growing area in the Rift Valley (Kenya), and to determine their toxigenic potential. DNA analyses of internal transcribed spacer (ITS) regions of ribosomal DNA, partial β-tubulin gene (benA) and calmodulin gene (CaM) sequences were used. The strains were further analyzed for the presence of four aflatoxin-biosynthesis genes in relation to their capability to produce aflatoxins and other metabolites, targeting the regulatory gene aflR and the structural genes aflP, aflD, and aflQ. In addition, the metabolic profile of the fungal strains was unraveled using state-of-the-art LC-MS/MS instrumentation. The three gene-sequence data grouped the isolates into two major clades, A. minisclerotigenes and A. flavus. A. minisclerotigenes was most prevalent in Eastern Kenya, while A. flavus was common in both regions. A. parasiticus was represented by a single isolate collected from Rift Valley. Diversity existed within the A. flavus population, which formed several subclades. An inconsistency in identification of some isolates using the three markers was observed. The calmodulin gene sequences showed wider variation of polymorphisms. The aflatoxin production pattern was not consistent with the presence of aflatoxigenic genes, suggesting an inability of the primers to always detect the genes or presence of genetic mutations. Significant variation was observed in toxin profiles of the isolates. This is the first time that a profound metabolic profiling of A. flavus isolates was done in Kenya. Positive associations were evident for some metabolites, while for others no associations were found and for a few metabolite-pairs negative associations were seen. Additionally, the growth medium influenced the mycotoxin metabolite production. These results confirm the wide variation that exists among the group A. flavus and the need for more insight in clustering the group.
Highlights
The filamentous fungus Aspergillus flavus is a cosmopolitan soil-born saprophytic organism with opportunistic parasitic behaviors to plants, animals and humans
To verify whether there was a correlation between the distances between isolates based on their toxin profile produced on Czapek yeast extract agar (CYA) and the distance between isolates based on their toxin profile on yeast extract sucrose agar (YESA), a Mantel test with 1000 permutations was used (Figure 2)
A. minisclerotigenes was common in Eastern Kenya while only one isolate of the species was identified from the Rift Valley using the β-tubulin gene marker
Summary
The filamentous fungus Aspergillus flavus is a cosmopolitan soil-born saprophytic organism with opportunistic parasitic behaviors to plants, animals and humans. Variability in the A. flavus phenotype exists, for example, in sclerotia formation, culture characteristics, and ability to produce aflatoxins. Culture and molecular methods have been used to group isolates into aflatoxigenic and non-aflatoxigenic strains (Zarrin and Erfaninejad, 2016), and to investigate the variability in fungal species and subspecies (Geiser et al, 1998a,b, 2000; Frisvad et al, 2007; Jurjevicet al., 2015). Geiser et al (2000) subdivided A. flavus into Group 1 and Group 11 on the basis of sclerotia type and aflatoxin profile. Several authors have given evidence that A. flavus sensu lato may consist of several species (Geiser et al, 1998a,b, 2000; Pildain et al, 2008). The variation within the A. flavus isolates suggests the need for an extended taxonomic delineation for the benefit of the development of effective and safe biocontrol application technologies
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