Molecular and mycotoxigenic biodiversity of Aspergillus flavus isolated from Brazil nuts

The objective of this study was to carry out a transcription analysis of eight genes belonging to the aflatoxin (AF) and cyclopiazonic acid (CPA) biosynthesis pathway, and to detect aflatoxin B₁ (AFB₁) and CPA production in Aspergillus flavus strains isolated from Brazil nuts. Additionally, these genes were correlated with the different mycotoxigenic profiles of the same strains. Four previously identified A. flavus strains (ICB-01, ICB-151, ICB-161, and ICB-165) were grown on Brazil nut agar at 25°C for 10days. Mycotoxins were separated by high-performance liquid chromatography. Transcriptional analysis was performed by real-time RT-PCR using specific primers designed based on the conserved regions of two regulatory genes (aflR and aflS), three structural genes of the AFB₁ biosynthesis pathway (aflH, aflJ and aflP), and three structural genes of the CPA biosynthesis pathway (maoA, dmaT and pks-nrps). The expression of most genes in the A. flavus isolates varied according to the mycotoxin profile of each strain. The most expressed genes in the aflatoxigenic strain ICB-151 were aflJ (77.11%) and aflH (32.75%), while the CPA-producing strain ICB-161 mainly expressed dmaT (100%), maoA (63.72%), aflS (43.52%), and aflR (42.63%). The ICB-01 isolate was a producer of AFB₁ and CPA and the most expressed genes were aflS (47.79%), dmaT (42.77%), aflP (39.5%), and aflR (38.02%). ICB-198 did not produce any mycotoxin and exhibited lower expression of almost all genes analyzed. Furthermore, the ratio of aflS/aflR expression was correlated with the biosynthesis of AF and CPA in A. flavus strains producing exclusively AF or CPA or producing both AF and CPA. The ratio of aflS/aflR expression therefore seems to be related to the production of mycotoxins in Brazil nuts. Our results provide important data for the development of innovative and more cost-effective strategies to reduce and prevent AFB and CPA contamination in Brazil nuts.