Marcela Mendoza‐Suárez

-legume symbioses represents an environmentally friendly and inexpensive alternative to the use of chemical nitrogen fertilizers in legume crops. Rhizobial inoculants, applied frequently as biofertilizers, play an important role in sustainable agriculture. However, inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields. Strains with excellent performance under controlled conditions are typically selected as inoculants, but the rates of nodule occupancy compared to native strains are rarely investigated. Lack of persistence in the field after agricultural cycles, usually due to the transfer of symbiotic genes from the inoculant strain to naturalized populations, also limits the suitability of commercial inoculants. When rhizobial inoculants are based on native strains with a high nitrogen fixation ability, they often have superior performance in the field due to their genetic adaptations to the local environment. Therefore, knowledge from laboratory studies assessing competition and understanding how diverse strains of rhizobia behave, together with assays done under field conditions, may allow us to exploit the effectiveness of native populations selected as elite strains and to breed specific host cultivar-rhizobial strain combinations. Here, we review current knowledge at the molecular level on competition for nodulation and the advances in molecular tools for assessing competitiveness. We then describe ongoing approaches for inoculant development based on native strains and emphasize future perspectives and applications using a multidisciplinary approach to ensure optimal performance of both symbiotic partners.

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N 2 ) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N 2 fixation are independent traits, making their measurement extremely time-consuming with low experimental throughput. To transform the experimental assessment of rhizobial competitiveness and effectiveness, we have used synthetic biology to develop reporter plasmids that allow simultaneous high-throughput measurement of N 2 fixation in individual nodules using green fluorescent protein (GFP) and barcode strain identification (Plasmid ID) through next generation sequencing (NGS). In a proof-of-concept experiment using this technology in an agricultural soil, we simultaneously monitored 84 different Rhizobium leguminosarum strains, identifying a supercompetitive and highly effective rhizobial symbiont for peas. We also observed a remarkable frequency of nodule coinfection by rhizobia, with mixed occupancy identified in ∼20% of nodules, containing up to six different strains. Critically, this process can be adapted to multiple Rhizobium -legume symbioses, soil types, and environmental conditions to permit easy identification of optimal rhizobial inoculants for field testing to maximize agricultural yield.

gene-inducing flavonoids at sites where nodules developed subsequently. These bioreporters will be particularly helpful in understanding the dynamics of root exudation and the role of different molecules secreted into the rhizosphere.

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilising the Type IIS restriction enzyme Esp3I and have demonstrated its use for Golden Gate cloning in Escherichia coli. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. Using the principles outlined here, new modules for specific applications, e.g. origin of replication for plasmids in other bacteria, can easily be designed. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach. To demonstrate the potential of the system three example vectors were constructed and tested. Golden Gate level 1 vectors; pOGG024, with a broad-host range and high copy number was used for gene expression in laboratory-cultured Rhizobium leguminosarum, and pOGG026, with a broad-host range a lower copy number and excellent stability, even in the absence of antibiotic selection. The application of pOGG026 is demonstrated in environmental samples by bacterial gene expression in nitrogen-fixing nodules on pea plants roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 is a broad-host range, medium copy number, for which we demonstrate an application by constructing a dual reporter plasmid expressing green and red fluorescent proteins.