Peptide nucleic acid (PNA) clamps offer a strategy to reduce the amplification of plant organellar 16S rRNA genes that often overwhelm 16S rRNA gene profiling of endophytic microbiota in plant tissues. Here, we evaluated the efficacy of universal plastid- (pPNA) and mitochondria-targeting (mPNA) clamps in root endosphere and rhizosphere samples of cabbage (Brassica rapa), pepper (Capsicum annuum), and soybean (Glycine max). Each PNA was added at 0.25 or 1.0 μM for endosphere samples and at 0.25 μM for rhizosphere samples. In the endosphere, the 1.0 μM PNA treatment reduced chloroplast- and mitochondrial-derived 16S rRNA gene reads and increased mean post-filter bacterial reads by approximately 29.7-fold in cabbage, 20.6-fold in pepper, and 23.0-fold in soybean relative to the corresponding no-PNA groups. Alpha-diversity indices showed non-significant but consistent trends toward higher richness and evenness under PNA treatment. However, reductions in chloroplast reads were host dependent, and persistent chloroplast signals in cabbage showed that the universal PNA clamps did not fully block all chloroplast and mitochondrial amplicon sequence variants. In contrast, in the rhizosphere, where host reads were already rare, PNA minimally affected read counts or diversity metrics. These results indicate that, under the controlled growth-chamber conditions tested, universal PNA clamps serve as a practical, effective default tool for root endosphere microbiome profiling in three plant species, while highlighting that clamp concentration and sequence may require host-specific optimization.
