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1. A microbiome engineering framework to evaluate rhizobial symbionts of legumes

   https://doi.org/10.1007/s11104-021-04892-2  

   Quides, Kenjiro W.; Atamian, Hagop S. (June 2021, Plant and Soil)

   null (Ed.)

   Abstract Background For well over a century, rhizobia have been recognized as effective biofertilizer options for legume crops. This has led to the widespread use of rhizobial inoculants in agricultural systems, but a recurring issue has emerged: applied rhizobia struggle to provide growth benefits to legume crops. This has largely been attributed to the presence of soil rhizobia and has been termed the ‘rhizobial competition problem.’ Scope Microbiome engineering has emerged as a methodology to circumvent the rhizobial competition problem by creating legume microbiomes that do not require exogenous rhizobia. However, we highlight an alternative implementation of microbiome engineering that focuses on untangling the complexities of the symbiosis that contribute to the rhizobial competition problem. We outline three approaches that use different starting inocula to test hypotheses to overcome the rhizobial competition problem. Conclusions The approaches we suggest are targeted at various stages of the legume-rhizobium symbiosis and will help us uncover underlying molecular mechanisms that contribute to the rhizobial competition problem. We conclude with an integrative perspective of these different approaches and suggest a path forward for future research on legumes and their complex microbiome. 

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2. Nitrogen enrichment alters selection on rhizobial genes

   https://doi.org/10.1101/2024.11.25.625319  

   Hill, Caleb A; McMullen, John G; Lennon, Jay T (November 2024, bioRxiv)

   1 Abstract Mutualisms evolve over time when individuals belonging to different species derive fitness benefits through the exchange of resources and services. Although prevalent in natural and managed ecosystems, mutualisms can be destabilized by environmental fluctuations that alter the costs and benefits of maintaining the symbiosis. In the rhizobia-legume mutualism, bacteria provide reduced nitrogen to the host plant in exchange for photosynthates that support bacterial metabolism. However, this relationship can be disrupted by the addition of external nitrogen sources to the soil, such as fertilizers. While the molecular mechanisms underpinning the rhizobia-legume symbiosis are well-characterized, the genome-wide fitness effects of nitrogen enrichment on symbiotic rhizobia are less clear. Here, we inoculated a randomly barcoded transposon-site sequencing (RB-TnSeq) library of the bacteriumEnsifer(Sinorhizobium)melilotiinto soils containing a host plant, alfalfa (Medicago sativa), under conditions of low and high nitrogen availability. Although plant performance remained robust to fertilization, nitrogen enrichment altered gene fitness for specific traits and functions in the rhizobial partner. Genes involved in carbohydrate metabolism showed increased fitness irrespective of soil nutrient content, whereas fitness gains in quorum-sensing genes were only observed in high-nitrogen environments. We also documented reductions in the fitness of nucleotide metabolism and cell-growth genes, while genes from oxidative phosphorylation and various amino-acid biosynthesis pathways were detrimental to fitness under elevated soil nitrogen, underscoring the complex trade-offs in rhizobial responses to nutrient enrichment. Our experimental functional genomics approach identified gene functions and pathways across allE. melilotireplicons that may be associated with the disruption of an agronomically important mutualism. 2ImportanceUnderstanding the evolutionary dynamics of the rhizobia-legume mutualism is important for elucidating how plant-soil-microbe interactions operate in natural and managed ecosystems. Legumes constitute a significant portion of global food production and generate 25% of all terrestrially fixed nitrogen. The application of chemical fertilizers can disrupt the mutualism by altering the selective pressures experienced by symbiotic rhizobia, potentially affecting gene fitness throughout the microbial genome and leading to the evolution of less productive or cooperative mutualists. To investigate how exogenous nitrogen inputs influence gene fitness during the complex rhizobial lifecycle, we used a barcoded genome-wide mutagenesis screen to quantify gene-level fitness across the rhizobial genome during symbiosis and identify metabolic functions affected by nitrogen enrichment. Our findings provide genomic insight into potential eco-evolutionary mechanisms by which symbioses are maintained or degraded over time in response to changing environmental conditions. 

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3. Lotus japonicus regulates root nodulation and nitrogen fixation dependent on the molecular form of nitrogen fertilizer

   https://doi.org/10.1007/s11104-022-05762-1  

   Ortiz-Barbosa, G. S.; Torres-Martínez, L.; Rothschild, J.; Sachs, J. L. (November 2022, Plant and Soil)

   Purpose Legumes form root nodules to gain fixed nitrogen from rhizobia and can also access nitrogen in soil. Data suggest that plants might discriminate among these sources to optimize growth, but recogni- tion of symbiotically fixed nitrogen and its regulation remain poorly understood. Methods A greenhouse inoculation study manipu- lated the molecular form and concentration of nitro- gen available using two Lotus japonicus genotypes and the nitrogen-fixing symbiont, Mesorhizobium loti. Plants were supplied with sole organic and inorganic nitrogen sources to simulate forms that plants might receive from symbiotic nitrogen fixation or from the soil. Host benefit from and regulation of symbiosis was investigated by quantifying symbiotic trait varia- tion and isotopic analysis of nitrogen fixation. Results Host growth varied in response to fertili- zation with alanine, aspartic acid, ammonium, and nitrate, suggesting differences in catabolism effi- ciency. Net benefits of nodulation were reduced or eliminated under all forms of extrinsic fertilization. However, even when symbiosis imposed significant costs, hosts did not reduce investment into nodulation or nitrogen fixation when exposed to aspartic acid, unlike with other nitrogen sources. Conclusions L. japonicus can adaptively down- regulate investment into symbiosis in the presence of some but not all nitrogen sources. Failure to down- regulate any aspect of symbiosis in the presence of aspartic acid suggests that it might be jamming the main signal used by L. japonicus to detect nitrogen fixation. 

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4. Pangenome Evolution Reconciles Robustness and Instability of Rhizobial Symbiosis

   https://doi.org/10.1128/mbio.00074-22  

   Weisberg, Alexandra J.; Rahman, Arafat; Backus, Dakota; Tyavanagimatt, Parinita; Chang, Jeff H.; Sachs, Joel L. (June 2022, mBio)

   Cooper, Vaughn S. (Ed.)

   ABSTRACT Root nodulating rhizobia are nearly ubiquitous in soils and provide the critical service of nitrogen fixation to thousands of legume species, including staple crops. However, the magnitude of fixed nitrogen provided to hosts varies markedly among rhizobia strains, despite host legumes having mechanisms to selectively reward beneficial strains and to punish ones that do not fix sufficient nitrogen. Variation in the services of microbial mutualists is considered paradoxical given host mechanisms to select beneficial genotypes. Moreover, the recurrent evolution of non-fixing symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. Here, we deconstructed hundreds of genome sequences from genotypically and phenotypically diverse Bradyrhizobium strains and revealed mechanisms that generate variation in symbiotic nitrogen fixation. We show that this trait is conferred by a modular system consisting of many extremely large integrative conjugative elements and few conjugative plasmids. Their transmissibility and propensity to reshuffle genes generate new combinations that lead to uncooperative genotypes and make individual partnerships unstable. We also demonstrate that these same properties extend beneficial associations to diverse host species and transfer symbiotic capacity among diverse strains. Hence, symbiotic nitrogen fixation is underpinned by modularity, which engenders flexibility, a feature that reconciles evolutionary robustness and instability. These results provide new insights into mechanisms driving the evolution of mobile genetic elements. Moreover, they yield a new predictive model on the evolution of rhizobial symbioses, one that informs on the health of organisms and ecosystems that are hosts to symbionts and that helps resolve the long-standing paradox. IMPORTANCE Genetic variation is fundamental to evolution yet is paradoxical in symbiosis. Symbionts exhibit extensive variation in the magnitude of services they provide despite hosts having mechanisms to select and increase the abundance of beneficial genotypes. Additionally, evolution of uncooperative symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. We analyzed genome sequences of Bradyrhizobium, bacteria that in symbioses with legume hosts, fix nitrogen, a nutrient essential for ecosystems. We show that genes for symbiotic nitrogen fixation are within elements that can move between bacteria and reshuffle gene combinations that change host range and quality of symbiosis services. Consequently, nitrogen fixation is evolutionarily unstable for individual partnerships, but is evolutionarily stable for legume- Bradyrhizobium symbioses in general. We developed a holistic model of symbiosis evolution that reconciles robustness and instability of symbiosis and informs on applications of rhizobia in agricultural settings. 

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5. Quantitative effects of soil salinity on the symbiosis of wild lima bean (Phaseolus lunatus L.) and Bradyrhizobium in Costa Rica

   https://doi.org/DOI:10.5073/JABFQ.2018.091.039  

   Ballhorn, D.J.; Wolfe, E.R.; Tyler, Jess; Ronan, W.; Sands-Gouner, S; Shaw, C..; Balkan, M.A. (January 2018, Journal of applied botany and food quality)

   Global climate change and local anthropogenic activities are increasing soil salinization with permanent negative effects on agricultural and ecosystem productivity. While salt stress is known to affect plant performance, its effects on the association with key microbial plant symbionts, such as legume-associated nitrogen-fixing rhizobia, are less understood. In this field study conducted in Costa Rica (Puntarenas), we used sympatrically-occurring wild lima bean (Phaseolus lunatus L.) and Bradyrhizobium to quantify biomass production of unfertilized rhizobial (R+) and fertilized rhizobia-free (R-) plants at different levels of experimentally manipulated salinity in native soil. In response to salt stress, nodulation was significantly reduced even at slightly increased salt levels. Plants growing at soil salinity levels of 2, 4, 6, and 8 mS/cm showed a mean reduction of nodules by 60.22, 76.52, 83.98, and 92.5% compared to the controls. Similarly, we also observed a significant decline in plant biomass at elevated salinity. However, biomass accumulation of R- plants was significantly less impacted compared to R+ plants, suggesting that the plant-microbe symbiosis is more salt-sensitive than the plant host itself. We suggest that the search for more salt-tolerant, crop plant-compatible rhizobial strains may provide a sustainable approach to maintain agricultural productivity on low to moderately saline soils. 

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