Soil microorganisms play important roles in mediating plant health, with beneficial bacteria promoting plant health by producing plant hormones, enhancing nutrient uptake and protecting against plant pathogens. Our knowledge of rhizosphere microbiology for the past 100+ years has been largely gained from laboratory culturing of microbes that associate with plant roots; however, recent molecular tools have revealed that the vast majority of soil microorganisms are not readily cultured under laboratory environments. Using cutting-edge metagenomic methods, the Liles lab has recently discovered novel bacterial phyla from soil sampled from the Cullars Rotation (circa 1911), the oldest, continuous soil fertility experiment in the Southern United States. Our research efforts, supported previously by AAES, NIH and NSF grants, have resulted in assembly of multiple bacterial genomes from bacteria affiliated with the "candidate phyla radiation" (CPR). Bacteria that are members of the CPR have been identified in groundwater and other environmental samples by other research groups using metagenomic methods, but CPR bacteria have not been previously characterized from soils. The novel CPR bacteria we have discovered from Cullars Rotation soil are ultra-small bacteria (< 0.45 mm), some of which are predicted to utilize bacterial peptidoglycan as a growth substrate. Despite their tiny physical and genome sizes, our data suggests that these novel soil CPR bacteria are numerous in soils and may have important microbial and/or plant associations that are completely unknown to science. There are many scientific questions regarding the contributions of CPR bacteria to soil microbial ecology and their interactions with microbial and plant hosts that we will address in this proposed research:Specific Objective #1: Determine how fertilizer application and soil pH affects soil CPR bacterial diversity in bulk soil and rhizosphere samples. Our discovery of soil CPR bacteria was from a plot of soil at the Cullars Rotation that has not received fertilizer application (NPK) for more than 100 years. We know that CPR bacteria are also present in Cullars Rotation soils that have received fertilizer application, but not how this has affected their abundance and diversity. We will explore CPR bacteria diversity within the 14 different plots at the Cullars Rotation, and in bulk soils and rhizosphere samples, using metagenomic methods.Specific Objective #2: Conduct a census of CPR bacteria within representative Alabama soils and use these data to develop CPR-specific molecular tools. We will sample representative agricultural soils from AL to identify the diversity of CPR bacterial taxa present in these diverse soil types. A metagenomic strategy will be used to assemble CPR bacterial genomes from each soil, and these genomes will be compared to each other in different soils to determine genomic variability and functional annotations. Using this larger dataset from multiple soils, we will identify CPR phylum-specific 16S rRNA gene sequences in order to target soil CPR bacteria using PCR or fluorescent in situ hybridization approaches.Specific Objective #3: Determine whether CPR bacteria have symbiotic associations with rhizosphere bacteria and/or plant roots. Based on their small genome size and lack of many metabolic capabilities, we hypothesize that soil CPR bacteria rely upon obligate symbiotic associations with microbial hosts, which explains why these CPR bacteria have never before been cultured under laboratory conditions. While isolation in pure culture may not be possible, we predict that co-culturing soil CPR bacteria with other microbes is possible. We will use our existing collection of plant growth-promoting Bacillus and other microbial species to identify specific strains that can promote co-culture of soil CPR bacteria. These co-cultures could enhance plant growth promotion and/or biocontrol activities, and be a useful model to study CPR-host associations.In summary, this research will accomplish multiple objectives: We will generate vast functional genomic information concerning novel CPR bacteria in agricultural soils, determine how agricultural management practices affect their abundance and diversity, develop molecular tools useful in studying soil CPR bacteria in future extramurally funded research, and study their associations with microbial and plant hosts. These studies will facilitate submission of multiple competitive extramural proposals.