Genetic and Toxigenic Variability within Aspergillus flavus Population Isolated from Maize in Two Diverse Environments in Kenya

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.

Development of an ELISA-Based Method for Testing Aflatoxigenicity and Aflatoxigenic Variability among Aspergillus species in Culture

“…This information is important considering the phenomenal growth of biological control programs targeting the reduction of aflatoxin contamination in food material not only in Africa but across the globe. However, to date, the study of aflatoxigenicity and aflatoxigenic variability among Aspergillus species relies on morphological [13] and chemical characteristics [8] as well as a complex combination of metabolic and molecular methods of assessment [5, 8]. To some extent, these different methods have returned an inconclusive verdict on aflatoxigenic variability and aflatoxigenicity among Aspergillus species.…”

“…In order to be successful, such biological control program based on fungi-fungi interaction must of essence first, correctly identify candidate non-aflatoxigenic strains of Aspergillus species; then accurately predict their behavior while interacting with aflatoxigenic fungi in field conditions. To identify candidate fungi for use in biological control formulations, researchers rely on diverse morphological, chemical [4], and to some extent a combination of molecular and metabolic methods to establish aflatoxigenicity [5]. The aforementioned methods currently in use enjoy some degree of success as well as inconsistencies in reporting non-aflatoxigenic fungi thus leaving a knowledge gap occasioned by the inconclusive nature of their findings.…”

Isolation of Aflatoxigenic Aspergillus flavus from Animal-Feed and Exploration of the Genetic Basis of Aflatoxin Biosynthesis

“…The isolated genomic DNA was used as a template to amplify ITS (Internal transcribed spacer) region by polymerase chain reaction using ITS1 (forward) and ITS4 (reverse) primers (Saleemi et al, 2012;Okoth et al, 2018). A 25μL reaction mixture containing; 2.5L (100ng) template DNA, 0.5μL dNTP mix (10mM), 2.5μL Pfu buffer with MgCl2 (10X), 0.5μL of each Forward and Reverse Primers (10 pmol/μL), 0.5μL Pfu polymerase (2.5U/μL) and 18μL double distilled deionized H2O was prepared.…”

“…Three purified ITS region amplicons were sent for sequencing to Macrogen (Korea) and compared to ITS nucleotide sequences (Kwak et al, 2013) Sequences were assembled in contigs by DNA Dragon Software (Version 1.6.0, SequentiX-Digital DNA Processing, Germany) and were submitted to the GenBank. Multiple sequence alignment of closely related type strains ITS sequences of validly named Aspergillus species retrieved from NCBI along with the sequences of current study was done using ClustalW and a phylogenetic tree was constructed by the Neighbour-Joining method using MEGA software (Version 7.0) with 1000 bootstrap value (Costa et al, 2011;Okoth et al, 2018).…”

“…During the recent past, PCR detection of aflatoxin RESEARCH ARTICLE biosynthesis gene expression or existence has been used as an analytical tool for the characterization of toxigenic fungi in particular feed ingredients (Miranda et al, 2019). Sequence variability and omissions in numerous regions of the aflatoxin biosynthetic pathway have been used to conclude the polyphyletic assemblage of toxigenic A. flavus species (Okoth et al, 2018).…”