Unveiling Hidden Endophytes by Optimising Identification of Endophytic Bacterial Communities from Wild Grassland Plant Roots

Abstract

Endophytic bacteria are increasingly recognised for their roles in plant health through symbiosis. However, methodological challenges, such as inconsistent root sterilisation, inefficient microbial DNA extraction, and co-amplification of plant organellar DNA, limit accurate characterisation of these communities, especially in wild grassland plants and non model plant in general. To address this, we developed and tested a streamlined protocol for bacterial endophyte detection from wild grassland plant roots, encompassing surface sterilisation of roots, DNA extraction, clamping of plant internal mitochondrial and chloroplast DNA, and 16S rRNA amplicon sequencing. Our approach minimises plant DNA contamination and yields high-quality microbial profiles. The protocol is adaptable and specific to grassland plant species, offering a standardised foundation for endophyte studies in wild and non-model plants. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/706108v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@157df36org.highwire.dtl.DTLVardef@1ff645aorg.highwire.dtl.DTLVardef@1580ecorg.highwire.dtl.DTLVardef@1c31b89_HPS_FORMAT_FIGEXP M_FIG C_FIG (Haskins and Ajaz, 2026) https://BioRender.com/47gd2xr