Gene-Specific Effects of Three Cry Transgenes on Rhizosphere Microbiota in Catalpa bungei

Catalpa bungei, a fast-growing timber tree, is threatened by the lepidopteran pest Omphisa plagialis. Previous studies in our laboratory successfully generated transgenic C. bungei lines overexpressing Cry genes (Cry1Ab, Cry2A, and Cry9-2) that exhibited resistance to O. plagialis, but their potential impact on soil bacterial communities remains unclear. In this study, we analyzed nine transgenic C. bungei lines (three independent lines for each Cry gene) to characterize their rhizosphere bacterial communities using high-throughput sequencing of the 16S ribosomal DNA (rDNA) V4–V5 regions. A total of 628 amplicon sequence variants (ASVs) were shared among all transgenic and wild-type (WT) lines, forming a stable core microbiome dominated by Proteobacteria, Bacteroidota, Acidobacteriota, and Actinobacteriota. Alpha diversity showed no significant differences, while beta diversity revealed minor but distinct compositional shifts. Cry1Ab lines exhibited higher abundances of fast-growing taxa, particularly Proteobacteria and Bacteroidota; Cry2A lines displayed intermediate profiles, whereas Cry9-2 lines were nearly indistinguishable from WT communities. Linear discriminant analysis of the effect size revealed significant enrichment of taxa such as Burkholderiaceae and Ralstonia in the Cry1Ab rhizosphere, in contrast to the higher abundance of Chloroflexi in the WT. Functional predictions indicated consistent metabolic pathways across all treatments, suggesting strong ecological redundancy. This study demonstrates minimal impact on rhizosphere microbial communities in transgenic C. bungei plants. The Cry9-2 construct exhibited superior environmental stability, whereas the Cry1Ab construct caused only slight but ecologically acceptable shifts. These findings support the ecological safety of Bt-transgenic C. bungei and identify Cry9-2 as a particularly favorable candidate for forestry applications. This comparative evaluation of three Cry genes in a tree species provides a framework for future gene-specific biosafety assessments in woody plants.