Dataset Information

Occurrence and toxigenic potential of Aspergillus section Flavi on wheat and sorghum silages in Uruguay.

ABSTRACT: Species belonging to Aspergillus section Flavi occur naturally in crops and can cause food spoilage and/or toxin production. The aim of this study was to determine the occurrence and diversity of the species of Aspergillus section Flavi found in wheat and sorghum at harvest time and during silage storage, and to evaluate the toxigenic potential of the isolates to determine the contamination risk of mycotoxins in grains. Strains from Aspergillus flavus and Aspergillus parasiticus were found based on multi-gene phylogenetic analyses. This is the first report on the presence of A. parasiticus in wheat from Uruguay. Of the 80 isolates Aspergillus section Flavi, 30% produced aflatoxins (AFs), mainly type B1, and 25% produced cyclopiazonic acid (CPA). Within the isolates from wheat samples, 35% were AFs producers and 27.5% were CPA producers. Among the Aspergillus section Flavi isolates from sorghum, 25% were AFs producers while 22.5% were CPA producers. This work contributes to the knowledge of the species in crops and helps define appropriate strategies for the prevention and control of contamination with AFs and CPA by Aspergillus section Flavi fungi.

SUBMITTER: Del Palacio A

PROVIDER: S-EPMC7448941 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Occurrence and toxigenic potential of Aspergillus section Flavi on wheat and sorghum silages in Uruguay.

Del Palacio Agustina A Pan Dinorah D

Mycology 20200427 2

Species belonging to Aspergillus section Flavi occur naturally in crops and can cause food spoilage and/or toxin production. The aim of this study was to determine the occurrence and diversity of the species of Aspergillus section Flavi found in wheat and sorghum at harvest time and during silage storage, and to evaluate the toxigenic potential of the isolates to determine the contamination risk of mycotoxins in grains. Strains from Aspergillus flavus and As ...[more]

PMID: 32923022

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Project description:Microfungal isolates were routinely identified depending on both macro and micro morphological characteristics, sometimes, some fungal isolates appeared to be similar and such cases caused severe confusion for mycologists during the preliminary identification. During our previous studies dealing with isolation of fungi for some biotechnological applications; two mystifying species Aspergillus flavus and Aspergillus oryzae showed similar cultural and macroscopic features. Therefore, the current study aimed to easily distinguish between these two species depending on simple approaches which are routinely followed by a large segment of researchers. Investigation of the macroscopic features was performed to check the fungal growth on four different media (PDA, MEA, YES, and CYA) followed by microscopic examination using an ordinary light microscope, and scanning electron microscope SEM. Also, screening of secondary metabolites for both strains was preliminarily identified to find out the difference between their metabolic profiles. Finally, ITS rDNA was involved to clarify the molecular differences along their partial sequence. Conclusively, the BLAST strategy confirmed the similarity of ITS rDNA segments of both fungal strains that supported our hypothesis. The color of the fungal growth is a very critical factor whereas it is extensively influenced by the type of cultivation media. Accordingly, the YES medium was an inspiring tool assisting in prompt differentiation during the culture investigation step whereas A. oryzae and A. flavus appeared significant mustard yellow and olive green respectively. During the microscopic examination, the CYA medium also had a robust effect on the formation of the conidial chain whereas the knit long chain was observed in A. oryzae while the conidia appeared scattered and not in a chain in the case of A. flavus. Likewise, both two strains possessed different metabolic profiles where A. oryzae is not an Afla toxin producer, unlike A. flavus.

Project description:For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade has relied nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome editing system that gave extremely high (>95%) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of the codon-optimized cas9 gene was driven by the A. nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by the A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortless. Additionally, multiple-gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated by a sequential approach. Cotransforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily applicable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis, and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (>90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. IMPORTANCE CRISPR/Cas9 genome editing systems have been developed for many aspergilli. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. They are also mostly strain specific or species specific. This developed system is highly efficient and allows knocking out multiple genes in A. flavus efficiently either by sequential transformation or by cotransformation of individual sgRNA vectors if desired. It is readily applicable to section Flavi species and aspergilli in other sections ("section" is a taxonomic rank between genus and species). This cross-Aspergillus section system is for wild-type isolates and does not require homologous donor DNAs to be added, NHEJ-deficient strains to be created, or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.

Dataset Information

Occurrence and toxigenic potential of Aspergillus section Flavi on wheat and sorghum silages in Uruguay.

Publications

Occurrence and toxigenic potential of <i>Aspergillus</i> section <i>Flavi</i> on wheat and sorghum silages in Uruguay.

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