Effects of the Temperature and Limosilactobacillus fermentum Co-Inoculation on the Expression of AFB1-Synthesis Genes and the Level of Toxin Produced by Aspergillus flavus Zt41 in Corn Silage

Aflatoxin B1 (AFB1), a highly potent Group 1 human carcinogen produced by Aspergillus flavus (A. flavus), poses a significant contamination risk to corn silage, a threat that is further intensified by rising global temperatures. This study aimed to characterize the combined effects of temperature and co-inoculation with the lactic acid bacterium Limosilactobacillus fermentum (L. fermentum) on AFB1 production and the expression of key biosynthetic genes in A. flavus colonizing corn silage. Corn silage was incubated at 20 °C, 30 °C, and 37 °C with and without L. fermentum. Using qRT-PCR and HPLC, we found that elevated temperatures, particularly 37 °C, strongly induced the expression of the aflatoxin biosynthetic cluster, including the regulatory gene aflR and structural genes such as omtA and ordA. Co-inoculation with L. fermentum consistently reduced in the final AFB1 concentration by approximately 50–60% at all three temperatures. Molecular analysis revealed that this reduction was associated with transcriptional repression at 30 °C and 37 °C. L. fermentum consistently and markedly down-regulated the expression of aflR and all structural genes. A particularly pronounced suppression was observed for the late-pathway gene ordA at 30 °C. These findings provide molecular evidence supporting the incorporation of selected L. fermentum strains into silage inoculant formulations to mitigate the AFB1 risk under high-temperature conditions.