Molecular detection and biocontrol of Aflatoxigenic Fungi and revealed genetic diversity and driven evolution of Aspergillus flavus by Simple sequence repeat (SSR) marker

Aspergillus flavus is a major post-harvest pathogen responsible for significant yield losses and aflatoxin contamination in agricultural commodities, posing serious risks to food safety and public health. This study comprehensively investigated the morphological diversity, genetic structure, aflatoxin biosynthetic potential, and antifungal management of A. flavus isolates associated with maize (Zea mays L.) and chili (Capsicum annuum L.) collected from multiple districts of southwestern Bangladesh. A total of 38 isolates were characterized using combined microbiological, molecular, and bioassay-based approaches. Morphological assessment under four growth conditions revealed pronounced time- and environment- dependent phenotypic plasticity, with green morphotypes predominating under standard conditions and reduced pigmentation under restricted environments. Shannon’s Diversity Index indicated maximum morphological diversity in reverse colony views after 30 days of incubation, while statistical analyses confirmed no significant association between morphological and genetic variation, highlighting environmental influence on phenotypic traits. Genetic diversity was evaluated using 13 SSR markers, revealing high polymorphism and strong population differentiation (F_ST=0.630–0.981) with low gene flow between host-associated populations. STRUCTURE, UPGMA, and Neighbor-Joining analyses consistently supported two genetically distinct clusters corresponding to maize and chili hosts, with limited admixture and evidence of clonal lineages. Molecular screening of key aflatoxin biosynthetic genes (aflR, aflP, aflO, and tub1) demonstrated widespread aflatoxigenic potential, with aflR showing the highest prevalence (97.36%). While most maize isolates harbored all four genes, variability in aflP occurrence suggested genetic divergence among certain genotypes. In parallel, antifungal efficacy of five medicinal plant extracts (Azadirachta indica, Piper betle, Centella asiatica, Ocimum tenuiflorum, and Mentha spicata) was assessed against A. flavus. Azadirachta indica exhibited the strongest antifungal activity, surpassing the standard fungicide fluconazole at higher concentrations, and demonstrated a low minimum inhibitory concentration (MIC = 9.375 mg ml⁻¹). Other extracts showed moderate to satisfactory inhibition, supporting their potential as eco-friendly alternatives. Overall, this study demonstrates host-associated genetic structuring, high aflatoxigenic risk, and pronounced morphological plasticity in A. flavus populations of Bangladesh, while highlighting medicinal plant extracts as promising sustainable tools for managing post-harvest fungal contamination. 

• Research Project