Effects of trait stacking on the maize phyllosphere microbiome: a shotgun metagenomic comparison

Current safety assessments for stacked-trait transgenic crops underrepresent the phyllosphere, a critical interface directly exposed to transgenic products, warranting strengthened risk evaluation. Here, we employed metagenomics to characterize the phyllosphere of three maize lines under herbicide-free conditions: wild-type (Corn.CK), herbicide-resistant single-trait (transgene of cp4-epsps, Corn.S), and insect- and herbicide-resistant stacked-trait (transgene of cry1Ab and cp4-epsps, Corn.D). Shannon diversity showed a consistent trend toward higher values in the cp4-epsps single-trait line than in the wild type (not significant after correction for multiple comparisons), whereas the stacked-trait line showed an intermediate increase. Transgenic events were associated with bidirectional taxonomic remodeling, characterized by the depletion of Enterobacterales and enrichment of environmentally adapted taxa. Functionally, transgenic lines displayed pervasive metabolic depletion across KEGG pathways and CAZyme categories, alongside reduced abundance of virulence-associated gene homologs (e.g., T6SS and Enterobactin) and lower antibiotic resistance gene diversity (wild-type > stacked-trait > single-trait), driven by the concomitant loss of Enterobacterales hosts. Notably, in most of the taxonomic, functional, virulence, and resistome dimensions examined, the stacked-trait line exhibited an intermediate microbiome profile between the wild-type and single-trait lines. These findings suggest that, for this genotype combination under these controlled conditions, trait stacking was not associated with microbiome perturbation greater than that observed for the single trait, and provide a preliminary, genotype- and stage-specific reference for phyllosphere biosafety assessment, pending field validation.