Genetic and chemical methods of ascertaining aflatoxigenicity give discordant results: A Case of Aspergillus species from Eastern Kenya

Background: Aflatoxins (AFs) are poisonous compounds produced by species of fungi belonging to the genus Aspergillus mainly A. flavus and A. parasiticus. However, there are some members of these species that do not produce toxins and have since become of interest for use in Biological Control programs. The species that produce AFs are believed to have clusters of aflatoxin-associated genes that confer aflatoxigenicity. However, recent findings suggest that the presence of aflatoxin genes may not imply aflatoxin production capability. We therefore examined the extent to which the presence or absence of aflatoxin-associated genes (genetic) infers aflatoxigenicity using the Dichlorvos-Ammonia (chemical) method as reference.Results: We report a dissonance between the genetic and chemical methods of ascertaining aflatoxigenicity. Fungi (n = 314) that were morphologically identified as A. flavus and A. parasiticus were isolated from maize kernels and soil from Eastern Kenya. The fungi were further characterized to confirm their identities using a PCR-sequence analysis of the internal transcribed spacer (ITS) region of rDNA which confirmed them to be Aspergillus species. We selected and induced 16 isolates in YES media amended with Dichlorvos to produce AFs. Seven of the sixteen isolates were DM-AM positive (aflatoxigenic) but lacked at least one of the key aflatoxin-associated genes. Even more confounding, A. flavus isolate (1EM2606) had all aflatoxin-associated genes but was non-aflatoxigenic (DV-AM negative) while A. flavus (1EM1901) having all genes was aflatoxigenic (DV-AM positive). The genetic abundance of the AF-asscociated genes among the isolates was as follows: aflQ (100%), aflD (62.5%) followed by aflR (37.5%) and aflP (25%). Conclusion: We report that either method alone is limited in ascertaining aflatoxigenicity among Aspergillus section Flavi. The discordance between presence of aflatoxin-associated genes and aflatoxigenicity may be due to epigenetic factors that affect secondary metabolism, or even other signaling processes that alter toxin production that still remain poorly understood.

High resolution estimates of relative gene abundance with quantitative ratiometric regression PCR (qRR-PCR)

By leveraging characteristic fluorescence ratios of co-amplified gene targets, quantitative ratiometric regression PCR (qRR-PCR) enables high resolution estimates of relative genetic abundance.

Application of Sequence-Specific Labeled 16S rRNA Gene Oligonucleotide Probes for Genetic Profiling of Cyanobacterial Abundance and Diversity by Array Hybridization

ABSTRACT DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (Nostoc,Microcystis, and Planktothrix species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats.