FlowPot axenic plant growth system for microbiota research

Abstract

The presence of resident microbiota on and inside plants is hypothesized to influence many phenotypic attributes of the host. Likewise, host factors and microbe-microbe interactions are believed to influence microbial community assembly. Rigorous testing of these hypotheses necessitates the ability to grow plants in the absence or presence of resident or defined microbiota. To enable such experiments, we developed the scalable and inexpensive FlowPot growth platform. FlowPots have a sterile peat substrate amenable to colonization by microbiota, and the platform supports growth of the model plant Arabidopsis thaliana in the absence or presence of soil-derived microbial communities. Mechanically, the FlowPot system is unique in that it allows for total-saturation of the sterile substrate by "flushing" with water and/or nutrient solution via an irrigation port. The irrigation port also facilitates passive drainage of the substrate, preventing root anoxia. Materials to construct an individual FlowPot total ~$2.A simple experiment with 12 FlowPots requires ~4.5 h of labor following peat and seed sterilization. Plants are grown on FlowPots within a standard tissue culture microbox after inoculation, thus the Flowpot system is modular and does not require a sterile growth chamber. Here, we provide a detailed assembly and microbiota inoculation protocol for the FlowPot system. Collectively, this standardized suite of tools and colonization protocols empowers the plant microbiome research community to conduct harmonized experiments to elucidate the rules microbial community assembly, the impact of microbiota on host phenotypes, and mechanisms by which host factors influence the structure and function of plant microbiota.3

Microbial Interkingdom Interactions in Roots Promote Arabidopsis Survival

“…Using microbes that were exclusively recovered from the roots of A. thaliana or close relatives grown in CAS, we assembled seven complex synthetic microbial communities consisting of 148 bacteria (labeled “B”), 34 fungi (labeled “F”), or eight oomycetes (labeled “O”), as well as all corresponding combinations of multi-kingdom microbial consortia (BO, BF, FO, BFO; Table S3 ). These microbes, selected based on their taxonomic diversity, were used to re-populate the gnotobiotic FlowPot system containing peat and vermiculite as sterile soil matrix onto which surface-sterilized A. thaliana Columbia-0 (Col-0) seeds were placed ( Kremer et al., 2018 ). Upon co-incubation of these microbial communities and the plant host for 4 weeks in this substrate, the filamentous eukaryotic microbes in the absence of bacterial root commensals (F, O, FO) had a strongly detrimental impact on plant growth (p < 0.05, Kruskal-Wallis with Dunn’s post hoc test) and their survival rate, whereas in combination with the bacterial community (BO, BF), plant growth was rescued to similar levels as in microbe-free (MF) control plants ( Figure 4 A).…”

“…FlowPots containing seeds were inoculated with half-strength Murashige and Skoog (MS) medium without sucrose (Sigma-Aldrich M5519, pH 5.7) with full-strength MES buffer (MES anhydrous, BioChemica). Combiness boxes containing Flowpots with plants ( Kremer et al., 2018 ) were incubated under short-day conditions at 21°C with light (10 hr) and at 19°C in the dark (14 hr).…”

Microbial interkingdom interactions in roots promote Arabidopsis survival

“…Using microbes that were exclusively 153 recovered from the roots of A. thaliana or close relatives grown in CAS soil, we assembled 154 seven complex synthetic microbial communities consisting of 148 bacteria (B), 34 fungi (F) 155 or nine oomycetes (O) as well as all corresponding combinations of multi-kingdom microbial 156 consortia (BO, BF, FO, BFO; Table S4). These microbes, selected based on their taxonomic 157 diversity, were used to re-populate the gnotobiotic FlowPot system containing peat and 158 vermiculite as sterile soil matrix onto which surface-sterilized A. thaliana Col-0 seeds were 159 placed (Kremer et al, 2018). Upon co-incubation of these microbial communities and the 160 plant host for four weeks in this substrate, the filamentous eukaryotic microbes in the absence 161 Figure 4C; Figure S10).…”

Litterbox—A gnotobiotic Zeolite-Clay System to Investigate Arabidopsis–Microbe Interactions

“…In future, it may be of use for rhizosphere studies since bacteria reproduced in the rhizosphere and the zeolite (Figure 4). However, other systems, such as the recently published FlowPot system, which allows rapid changes in media composition, might be advantageous for studies focussing on the rhizosphere [71]. For phyllosphere studies, the Litterbox system is the optimal choice.…”