Characterization of Aspergillus section Flavi associated with stored grains

Abstract

Increased frequencies of Aspergillus section Flavi and aflatoxins in cereal grains have been seen in recent years due to changes in climate circumstances, such as high temperatures and drought. To assess the microbiological risks of contamination, it is critical to have a reliable and accurate means of identifying the fungi. The main goal of this study was to characterize Aspergillus species from section Flavi obtained from twenty-three samples of barley and maize grains, gathered from different markets in Qena, Egypt, using morphological and molecular techniques. Twenty-three isolates were chosen, one isolate from each sample; they were identified as A. aflatoxiformans (4 isolates), A. flavus (18), and A. parasiticus (1). The existence of four aflatoxin biosynthesis genes was also investigated in relation to the strains' ability to produce total aflatoxins and aflatoxin B1, focusing on the regulatory gene aflR and the structural genes aflD and aflM. All strains producing aflatoxins were linked to the presence of aflR1 and/or aflR2, except two isolates that exhibited aflatoxins but from which aflR1 or aflR2 were not detected, which may be due to one or more missing or unstudied additional genes involved in aflatoxin production. AflD and aflM genes were amplified by 10 and 9 isolates, respectively. Five samples of barley and maize were contaminated by aflatoxins. Fifteen isolates were positive for producing total aflatoxins in the range of 0.1-240 ppm. Antagonistic activity of Trichoderma viride against A. flavus (F5) was assessed at 31.3%. Trichoderma reduced total aflatoxins in all treated seeds, particularly those subjected to Trichoderma formulation.

Keywords: Aflatoxins; Calmodulin gene; Flavi; Trichoderma.

Fig. 1

7 days old colonies on MEA at 25 °C: left to right 1–4: A. aflatoxiformans, 5: reverse colony of A. aflatoxiformans, 6–11: A. flavus group (1), 12–23: A. flavus group (2), 24: A. parasiticus, 25: the reverse of A. flavus colony

Fig. 2

Microscopic characteristics after a 7-day incubation period on MEA at 25 °C, Aspergillus aflatoxiformans (A), A. flavus (B, C), and A. parasiticus (D). From left to right: 1, 2, 4, 5, 7, 8, 10, and 11 at × 40 and 3, 6, 9, and 12 at × 100

Fig. 3

Principal component analysis (PCA), analysis of Aspergillus section Flavi based on the morphology, isolates inside the red circle was Aspergillus flavus groups; the green circle was A. aflatoxiformans, and the black circle was A. parasiticus

Fig. 4

Phylogenetic tree of Aspergillus section Flavi strains isolated from barley and maize based on calmodulin sequence data. The numbers above branches indicate bootstrap values that were constructed after a run of 1000 replications (isolates of this study were marked with stars)

Fig. 5

The growth inhibition effect of Trichoderma viride on Aspergillus flavus on Czapek Dox agar medium at 28 °C for 7 days