DRIVERS OF EFFICIENT BELOWGROUND NITROGEN-FIXATION IN CALIFORNIA LEGUMES

The goals are to characterize the genetic and ecological mechanisms that drive rhizobial competitiveness to nodulate California crop legumes and to apply this knowledge to improve crop sustainability via biological nitrogen fixation.1. Method Development: A novel method to discover elite crop inoculum strains will be developed. The protocol, termed biogeographic genetic screening, is a method that could ultimately be patented.2. Biogeographic genetic screening will be conducted on crop-field rhizobial populations to develop a California legume microbiome strain library. Agronomic soil sources will be genetically surveyed to quantify relative abundance of rhizobial genotypes and their capacity to compete for nodulation on a panel of crop genotypes. Frequency distributions of rhizobia genotypes from the field samples will be quantified in a set of cultured nodules from a factorial soil-inoculation experiment to uncover competitive rhizobia compatible with multiple soils and host genotypes.3. Elite inoculum rhizobia will be developed that are superior in their capacity to establish on target legume hosts. These novel biofertilizer strains will be developed from the biogeographic genetic screen, and will provide a cheap and sustainable alternative to chemical nitrogen fertilization in California. Rhizobial populations are often characterized by a subset of genotypes that dominate the host plants at a local site and spread geographically to new host populations. Yet, little work has studied the drivers of these epidemic rhizobial genotypes, as has been the intense research focus for infectious diseases. To uncover epidemic drivers, it is critical to gather a geographically widespread sample of a microbial population, to genotype the isolates at multiple loci, and to investigate genotype-frequency distributions to uncover common strains and the conditions in which they flourish. Only by examining rhizobia genotype frequencies at a large spatial scale and with multiple host and soil types can we uncover the rhizobia that are best suited to compete for nodulation under diverse agronomic settings. A common toolbox that was developed to tackle the mechanistic bases of bacterial epidemics is applicable to our goals of biogeographic genetic screening. A typical protocol includes i) widespread screening and genotyping of the bacterial population to assess genotype frequencies in different ecological scenarios, ii) testing representative isolates in hosts to investigate infectiousness and host effects, and iii) comparing genome sequences and testing gene knockouts to identify molecular mechanisms of infectiousness. This three step approach has been successful in uncovering the ecological and molecular mechanisms that drive the epidemic spread of Escherichia coli (O157:H7), Pseudomonas aeruginosa (PAPI1), and Yersinia pestis (HPI). The genomic mechanisms that drive their epidemics - attributed to the acquisition of fitness-associated mobile elements - is of particular relevance here. These pathogens are all proteobacteria (like rhizobia), and share similarities in their modular genome architecture and lifestyle, in which they must thrive in the environment and must also colonize and infect specific hosts. Because rhizobial populations are most often characterized by an epidemic population structure, a similar approach will be initiated here to study drivers of rhizobial success under the agronomic conditions.