P-345 - Developing a Gnotobiotic Tomato System for Controlled Phyllosphere Microbiome Studies

The establishment of gnotobiotic systems is essential for studying phyllosphere microbiome. Autoclaving soil, especially when done repeatedly, often inhibits plant growth due to accumulation of phytotoxic compounds. We hypothesized that activated charcoal (AC) and biochar (BC), that adsorb phytotoxic substances, could mitigate these effects and simplify post-autoclave processing. This study aimed to establish a gnotobiotic environment using AC and BC as amendments in sterile soil. Tomato seeds were surface sterilized and germinated on water agar before being transplanted into microboxes containing autoclaved soil (x3) amended with AC (0.1%, 1%, 2%), BC (0.2%, 0.6%, 1%), or soil washed with sterile water, along non-sterile controls. After 10 days, plant growth parameters (survival rate, height, and leaf count) were measured. AC 1%, AC 2%, and BC 0.6% outperformed other amendments. Additionally, plants were treated with a field microbial community, along non-sprayed controls. 16S amplicon sequencing results revealed that Lachnospirales and Rhodobacterales  were present in all samples, however, higher Shannon diversity was observed in the microbial community-treated plants. Furthermore, plants grown under gnotobiotic systems were sprayed with Pseudomonas syringae pv. tomato, and the pathogen successfully colonized the plants. Our findings suggest that growing plants in microboxes containing autoclaved soil amended with AC and BC provides a viable gnotobiotic system. This system holds valuable potential for advancing foliar disease research and microbial ecology studies.