Deleterious missense variants in the aflatoxin biosynthesis genes explain the low toxicity of Aspergillus flavus from infected rice.

Aspergillus flavus is one of the most natural contaminants of the improperly stored rice grains. It produces several secondary metabolites, like aflatoxins, which are well known hepatotoxic, hepatocarcinogenic and mutagenic agents. This study describes the in silico consequences of the missense mutations identified in several genes of aflatoxins biosynthesis in rice-contaminating A. flavus isolates. In the in vitro portion of the study, aflatoxins production profile was measured, and PCR-single strand-conformation polymorphism (SSCP)-sequencing method was used to genotype the studied genetic loci: aflP, aflM, aflR, PEP, and cob. Results showed aflatoxigenic potential in 79 out of 109 A. flavus isolates. Twenty-two missense and fifty-five synonymous mutations were found to be distributed variably on the studied loci. In the in silico portion of this study, several computations were utilized to predict the effect of each observed missense mutation on proteins structure, function, and stability. Seven mutations (O-methyl transferase: p.G256C; ver-1 dehydrogenase: p.K179 N and p.V183L; aspergillopepsin-1: p.P137L, p.S138F, p.G154C, and p.S158C) were found to be highly deleterious among the missense variants with damaging effects on their proteins' structure and function. In contrast to these detected variations in the aflatoxigenic loci, all missense mutations in the control non-aflatoxigenic cob gene were found to be neutral. These findings indicated that the observed mutations may reduce the concomitant biohazard of their biosynthesized aflatoxins. The current findings suggest that the naturally available variants may reduce or eliminates the dangerous consequences of aflatoxins upon ingesting the rice infected with A. flavus. To the best of our knowledge, this study is the first comprehensive report to analyze the missense mutations on the aflatoxin biosynthesis genes using in vitro and the state-of-art bio-computational tools.

A highly efficient electrophoretic method for discrimination between two Neoscytalidium species using a specific fungal internal transcribed spacer (ITS) fragment.

Neoscytalidium (or N.) dimidiatum and N. novaehollandiae are two aggressive plant pathogenic species that affect several agricultural crops. Early detection and identification of these fungi are of critical importance to bring about the effective minimization to the threat they pose to the infected plants. Herein, two species of Neoscytalidium were rapidly discriminated by utilizing the rRNA internal transcribed (ITS4-5.8S-ITS5) PCR primers. A total of 100 isolates of Neoscytalidium species, which were isolated from Iraqi canker-infected fig trees, were included in this study. Two discrete electrophoretic PCR bands were observed in Neoscytalidium isolates-A-variants were about 546 bp, while B-variants were about 993 bp in length. The comprehensive phylogenetic analysis of both DNA variants revealed that A-variants resided between N. novaehollandiae and N. hyalinum, while B-variants were closely related to N. dimidiatum. Furthermore, the highly specific re-constructed tree of both electrophoretic variants demonstrated that B-variants share a high similarity with N. novaehollandiae. Additionally, the secondary structures for both variants were predicted computationally to reveal the structural patterns that each variant follows. In conclusion, a small rRNA locus comprising 22 nucleotides that differs in the two variants is potentially responsible for this species-specific classification. The main divergence in the amplified loci led to the classification of these fungal variants into two main species, namely N. dimidiatum and N. novaehollandiae, demonstrating that the amplification by ITS4-ITS5 rRNA fragment is a beneficial strategy that can be employed for the assessment of Neoscytalidium diversity in the natural ecosystems.