Molecular Identification and Characterisation of Aspergillus Flavus Isolates Originating from Serbian Wheat Grains

Abstract

During previous years, regarding the shifts in climate conditions in temperate region, such as occurrence of high temperatures and prolonged drought, increased occurrence frequencies of Aspergillus flavus and aflatoxins in cereal grains were recorded. A reliable and accurate identification of the fungi is of great importance for evaluating the microbiological risks of contamination. The essential point of the present investigation was molecular characterisation and identification of A. flavus isolates originating from common wheat and spelt grains collected after harvest during the period of three years (2015–2017) in Northern Serbia. A holistic approach that included PCR amplification of two DNA genomic regions and PCR-RFLP assay followed by fragment length analysis, provided complete and comprehensive characterisation of A. flavus isolated from wheat grains. The presented results indicate that there was no difference among the tested Aspergillus isolates on the molecular–genetic level. All 38 strains were identified as A. flavus by sequencing of combined ITS region and β-tubulin gene fragments (acc. no.: MH582473 to MH582510). PCR-RFLP method in combination with a Lab-on-a-chip (LoaC) electrophoresis can be successfully used to rapidly identify A. flavus isolates.

Molecular Characterization of Aspergillus flavus Strains Isolated from Animal Feeds

“…In previous studies, AFPA alone was used to identify A. flavus and A. parasiticus ( Abriba et al 2013 ; Fakruddin et al 2015 ; Hossain et al 2018 ; Krulj et al 2020 ). However, based only on the phenotypic characteristics, it was impossible to distinguish between the two closely related species A. flavus and A. parasiticus because of overlapping morphological features ( Rodrigues et al 2009 ).…”

First reported aflatoxigenic isolates of Aspergillus flavus in agroforestry systems in Buenaventura, Valle del Cauca-Colombia

“…In contrast, beta-tubulin (BenA) is a conserved gene that exhibits a high level of interspecific variability. Therefore, it is frequently used in phylogenetic studies of Aspergillus (Kim et al, 2020;Krulj et al, 2020). This gene allows for more precise identification of Aspergillus species, avoiding false positives caused by cross-reactions, which are limitations of ITS-based molecular identification (Kim et al, 2020).…”

PCR-RFLP method in combination with on-chip electrophoresis as a tool for determining of variability between important species of Aspergillus

“…Numerous molecular techniques have focused on Aspergillus species identification based on the partial or whole genome sequencing of different gene targets, including internal transcribed spacer (ITS) regions of rDNA and protein encoding genes such as calmodulin and β-tubulin (Wang et al, 2001;Samson et al, 2014;Chalupova, Raus, Sedlarova & Sebela, 2014;Nasri et al, 2015;Krulj et al, 2020). Samson and Varga (2009) recommend the use of at least two genomic sequences (e.g., ITS, calmodulin, β -tubulin, actin) for species description and identification through a multilocus approach.…”