Optimizing Biological Nitrogen Fixation on Sorghum by Manipulating Microbial Communities
Claire Palmer1* (firstname.lastname@example.org), Paulo Ivan Fernandes-Júnior1,2, April MacIntyre1,3, Sushmita Roy1, Wilfred Vermerris4, Ophelia Venturelli1, and Jean-Michel Ané1
1University of Wisconsin–Madison; 2Brazilian Agricultural Research Corporation (Embrapa); 3Valent BioSciences, Inc.; and 4University of Florida
As part of a multidisciplinary collaboration, this project aims to address key sustainability challenges facing the cultivation of sweet sorghum, an important biofuel crop. In particular, this project aims to decrease reliance on synthetic nitrogen fertilizers by improving biological nitrogen fixation, focusing on sorghum aerial roots as the main sites for diazotrophic activity. In tandem with collaborators investigating this trait from the plant perspective, researchers evaluate the bacterial communities associated with sorghum responsible for biological nitrogen fixation. The team plans to (1) isolate and characterize diverse bacterial strains from aerial root mucilage, (2) determine the bacterial interspecies interactions impacting biological nitrogen fixation, and (3) develop and test synthetic communities with robust biological nitrogen fixation.
Cultivation of the key biofuel crop, sorghum, relies heavily on using natural gas-intensive and environmentally damaging nitrogen fertilizers (Rütting, Aronsson, and Delin 2018). As an alternative, biological nitrogen fixation (BNF) through the activity of plant-associated diazotrophic bacteria has the potential to improve crop production sustainability and reduce environmental damage (Pankievicz et al. 2019). Demonstrating the potential of BNF in cereals, some indigenous corn landraces from Central America can obtain 29%–82% of their nitrogen from BNF in the low oxygen and sugar-rich mucilage secreted by aerial roots (Deynze et al. 2018). Several sorghum accessions also produce aerial roots and mucilage, but the diazotrophic activity of sorghum aerial root–associated communities has not been investigated. Following these observations, the team hypothesized that aerial root mucilage provides the ideal environment in which to improve BNF in sorghum.
The project uses a synthetic microbial community approach to investigate the bacterial interspecies interactions impacting BNF. Researchers drew upon a previous study that identified a seven-member community that stably and reproducibly assembled on corn roots and added five additional diazotrophic strains of interest, developing a 12-member nitrogen-fixing community which is referred to as PComm1 (Niu et al. 2017). Community BNF and composition in low and high species richness subcommunities of PComm1 is evaluated using acetylene reduction assays followed by 16S sequencing. All possible 1-, 2-, 11-, and 12-member communities are investigated. Preliminary results suggest that most interspecies interactions negatively impact nitrogen fixation, with less nitrogenase activity observed in communities with more members. This data set will be used to build computational models to further elucidate interspecies interactions impacting community growth and fixation, as well as design communities with enhanced BNF.
In addition to investigating community BNF in a simplified model system, researchers are also working to isolate and characterize bacteria from aerial root mucilage. Previously, the team isolated a collection of ~90 individual strains with a range of plant growth–promoting traits, such as auxin production, siderophore production, phosphate solubilization, and 1-aminocyclopropane-1-carboxylate (ACC) degradation. However, less than 10% of these isolates have nitrogen-fixing capabilities. Therefore, focus is currently on expanding the diversity of nitrogen-fixing strains in the project’s collection. A semisolid nitrogen-free medium approach was used to obtain new bacterial strains and increase the genetic variability of the mucilage-borne strain collection. Researchers obtained more than 320 new bacterial strains and used DNA fingerprinting to narrow down the collection to ~200 strains, excluding redundant profiles. In the next steps, the team will identify these strains and determine their diazotrophic ability, aiming to select new strains for future community manipulation and genetic engineering.
Deynze, A. V., et al. 2018. “Nitrogen Fixation in a Landrace of Maize Is Supported by a Mucilage-Associated Diazotrophic Microbiota,” PLOS Biology 16, e2006352.
Niu, B., et al. 2017. “Simplified and Representative Bacterial Community of Maize Roots,” Proceedings of the National Academy of Sciences of the United States of America 114, E2450–59.
Pankievicz, V. C. S., et al. 2019. “Are We There Yet? The Long Walk Towards the Development of Efficient Symbiotic Associations Between Nitrogen-Fixing Bacteria and Non-Leguminous Crops,” BMC Biology 17, 99.
Rütting, T., H. Aronsson, and S. Delin. 2018. “Efficient Use of Nitrogen in Agriculture,” Nutrient Cycling in Agroecosystems 110, 1–5.
The authors gratefully acknowledge funding from the U.S. Department of Energy Biological and Environmental Research (BER) Program grant no. DE-SC0021052.