Research Article: Inferring the presence of aflatoxin-producing Aspergillus flavus strains using RNA sequencing and electronic probes as a transcriptomic screening tool

Date Published: October 16, 2018

Publisher: Public Library of Science

Author(s): Andres S. Espindola, William Schneider, Kitty F. Cardwell, Yisel Carrillo, Peter R. Hoyt, Stephen M. Marek, Hassan A. Melouk, Carla D. Garzon, Richard A. Wilson.


E-probe Diagnostic for Nucleic acid Analysis (EDNA) is a bioinformatic tool originally developed to detect plant pathogens in metagenomic databases. However, enhancements made to EDNA increased its capacity to conduct hypothesis directed detection of specific gene targets present in transcriptomic databases. To target specific pathogenicity factors used by the pathogen to infect its host or other targets of interest, e-probes need to be developed for transcripts related to that function. In this study, EDNA transcriptomics (EDNAtran) was developed to detect the expression of genes related to aflatoxin production at the transcriptomic level. E-probes were designed from genes up-regulated during A. flavus aflatoxin production. EDNAtran detected gene transcripts related to aflatoxin production in a transcriptomic database from corn, where aflatoxin was produced. The results were significantly different from e-probes being used in the transcriptomic database where aflatoxin was not produced (atoxigenic AF36 strain and toxigenic AF70 in Potato Dextrose Broth).

Partial Text

Maize [1], peanuts [2], tree nuts, dried spices [3] and cottonseed [4] are crops that can be infected during the pre-harvest, post-harvest and/or storage period with Aspergillus flavus Link. This fungus produces polyketide secondary metabolites named aflatoxins. Among the four known aflatoxins (B1, B2, G1, G2), B1 has been of special interest to food biosecurity due to its toxicity and potent carcinogenic properties [5]. A. flavus is a ubiquitous saprophytic ascomycete fungus grouped in the Aspergillus section Flavi, species with aflatoxin-producing strains including A. flavus, A. parasiticus and A. nomius [6,7].

EDNA has previously been proven to successfully detect a variety of plant pathogens from raw metagenomic databases [26,28,40]. DNA as the main source of identification has always been the gold standard for detecting organisms in a sample, although viability is not assessed. Therefore, the question about “dead or alive” is left undetermined unless the organism is isolated and cultured, or, transcriptome analysis is used as a complementary detection tool or, complementary molecular viability analysis is included [41]. Here we have been able to use e-probes to associate the production of a secondary metabolite with gene regulation. The amount of aflatoxin B1 measured under these experimental conditions when AF70 was inoculated in ground corn and PDB confirms that aflatoxin is produced and that EDNA transcriptomics can infer such production rapidly and without any assembly or read mapping to reference genomes. Using the same approach, AF36 showed trace amounts of aflatoxin in the ELISA test which are attributable to the LOD of the matrixes and possible cross contamination during the incubation period. Yet, EDNA transcriptomics also determined that aflatoxigenic genes were downregulated in AF36 in all experimental conditions.




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