Host-selective PMA-PCR enhances bacterial diversity recovery and lowers detection limits in plant microbiome profiling

Co-amplification of host DNA remains a significant obstacle in plant microbiome profiling, as universal taxonomic markers, such as the 16S rRNA gene, are also present in plant organellar genomes. While strategies like blocking primers can enrich bacterial reads, they rely on prior sequence knowledge and may introduce bias. We developed a sequence-independent approach that suppresses host DNA amplification by combining physical pretreatments that selectively compromise plant cell integrity with propidium monoazide (PMA) treatment, which binds exposed DNA and prevents its PCR amplification. We observed that various pretreatments-including cryopreservation and mechanical disruption enhanced plant cell susceptibility to PMA without affecting bacterial cells. The applied approach resulted in significantly increased proportions of bacterial reads and improved detection of bacterial diversity in downstream NGS. Compared to sequence-dependent strategies such as blocking primers, this method achieved comparable or superior performance in reducing host DNA interference-particularly in low-biomass samples. Our results establish cryopreservation-enhanced PMA-PCR as a robust, sequence-independent method for high-resolution plant microbiome profiling.