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

Marcela A. Mendoza-Suárez; Barney A. Geddes; Carmen Sánchez-Cañizares; Ricardo H. Ramírez-González; Charlotte Kirchhelle; Beatriz Jorrin; Philip S. Poole

doi:10.1073/pnas.1921225117

New Research In

Edited by Éva Kondorosi, Hungarian Academy of Sciences, Biological Research Centre, Szeged, Hungary, and approved March 12, 2020 (received for review December 3, 2019)

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Significance

Increasing legume use in agriculture is a key requirement for the sustainable intensification of global farming systems. Maximizing the yield of legumes requires matching of the plant to elite rhizobia that are both competitive for nodulation and capable of high rates of nitrogen fixation. We have developed a high-throughput method that allows identification of elite strains at the single nodule level with the potential to revolutionize the search for elite indigenous rhizobia.

Abstract

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.

Footnotes

↵¹To whom correspondence may be addressed. Email: philip.poole{at}plants.ox.ac.uk.

Author contributions: M.A.M.-S., B.A.G., C.S.-C., and P.S.P. designed research; M.A.M.-S., R.H.R.-G., and C.K. performed research; M.A.M.-S. contributed new reagents/analytic tools; M.A.M.-S., B.A.G., C.S.-C., R.H.R.-G., C.K., B.J., and P.S.P. analyzed data; and M.A.M.-S., B.A.G., C.S.-C., and P.S.P. wrote the paper.
The authors declare no competing interest.
This article is a PNAS Direct Submission.
Data deposition: Raw reads for this study have been deposited in the European Nucleotide Archive (ENA) at European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI) under accession no. PRJEB37539.
This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1921225117/-/DCSupplemental.

Published under the PNAS license.

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