Competition, Nodule Occupancy, and Persistence of Inoculant Strains: Key Factors in the Rhizobium-Legume Symbioses.

Biological nitrogen fixation by Rhizobium-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.

Optimizing Rhizobium-legume symbioses by simultaneous measurement of rhizobial competitiveness and N2 fixation in nodules.

Legumes tend to be nodulated by competitive rhizobia that do not maximize nitrogen (N2) fixation, resulting in suboptimal yields. Rhizobial nodulation competitiveness and effectiveness at N2 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 N2 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.

Speed breeding in growth chambers and glasshouses for crop breeding and model plant research.

'Speed breeding' (SB) shortens the breeding cycle and accelerates crop research through rapid generation advancement. SB can be carried out in numerous ways, one of which involves extending the duration of plants' daily exposure to light, combined with early seed harvest, to cycle quickly from seed to seed, thereby reducing the generation times for some long-day (LD) or day-neutral crops. In this protocol, we present glasshouse and growth chamber-based SB approaches with supporting data from experimentation with several crops. We describe the conditions that promote the rapid growth of bread wheat, durum wheat, barley, oat, various Brassica species, chickpea, pea, grass pea, quinoa and Brachypodium distachyon. Points of flexibility within the protocols are highlighted, including how plant density can be increased to efficiently scale up plant numbers for single-seed descent (SSD). In addition, instructions are provided on how to perform SB on a small scale in a benchtop growth cabinet, enabling optimization of parameters at a low cost.

A Bacterial Expression Vector Archive (BEVA) for Flexible Modular Assembly of Golden Gate-Compatible Vectors.

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing 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 (e.g., origin of replication for plasmids in other bacteria) can easily be designed for specific applications. 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. The Golden Gate level 1 vector pOGG024 (pBBR1-based broad-host range and medium copy number) was used for gene expression in laboratory-cultured Rhizobium leguminosarum. The Golden Gate level 1 vector pOGG026 (RK2-based broad-host range, lower copy number and stable in the absence of antibiotic selection) was used to demonstrate bacterial gene expression in nitrogen-fixing nodules on pea plant roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 (RK2-based broad-host range, medium copy number) was used to construct a dual reporter plasmid expressing green and red fluorescent proteins.

Bacterial Biosensors for in Vivo Spatiotemporal Mapping of Root Secretion.

Plants engineer the rhizosphere to their advantage by secreting various nutrients and secondary metabolites. Coupling transcriptomic and metabolomic analyses of the pea (Pisum sativum) rhizosphere, a suite of bioreporters has been developed in Rhizobium leguminosarum bv viciae strain 3841, and these detect metabolites secreted by roots in space and time. Fourteen bacterial lux fusion bioreporters, specific for sugars, polyols, amino acids, organic acids, or flavonoids, have been validated in vitro and in vivo. Using different bacterial mutants (nodC and nifH), the process of colonization and symbiosis has been analyzed, revealing compounds important in the different steps of the rhizobium-legume association. Dicarboxylates and sucrose are the main carbon sources within the nodules; in ineffective (nifH) nodules, particularly low levels of sucrose were observed, suggesting that plant sanctions affect carbon supply to nodules. In contrast, high myo-inositol levels were observed prior to nodule formation and also in nifH senescent nodules. Amino acid biosensors showed different patterns: a γ-aminobutyrate biosensor was active only inside nodules, whereas the phenylalanine bioreporter showed a high signal also in the rhizosphere. The bioreporters were further validated in vetch (Vicia hirsuta), producing similar results. In addition, vetch exhibited a local increase of nod 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.