Agricultural productivity relies on synthetic nitrogen fertilizers, yet half of that reactive nitrogen is lost to the environment. There is an urgent need for alternative nitrogen solutions to reduce the water pollution, ozone depletion, atmospheric particulate formation, and global greenhouse gas emissions associated with synthetic nitrogen fertilizer use. One such solution is biological nitrogen fixation (BNF), a component of the complex natural nitrogen cycle. BNF application to commercial agriculture is currently limited by fertilizer use and plant type. This paper describes the identification, development, and deployment of the first microbial product optimized using synthetic biology tools to enable BNF for corn (Zea mays) in fertilized fields, demonstrating the successful, safe commercialization of root-associated diazotrophs and realizing the potential of BNF to replace and reduce synthetic nitrogen fertilizer use in production agriculture. Derived from a wild nitrogen-fixing microbe isolated from agricultural soils, Klebsiella variicola 137-1036 (“Kv137-1036”) retains the capacity of the parent strain to colonize corn roots while increasing nitrogen fixation activity 122-fold in nitrogen-rich environments. This technical milestone was then commercialized in less than half of the time of a traditional biological product, with robust biosafety evaluations and product formulations contributing to consumer confidence and ease of use. Tested in multi-year, multi-site field trial experiments throughout the U.S. Corn Belt, fields grown with Kv137-1036 exhibited both higher yields (0.35 ± 0.092 t/ha ± SE or 5.2 ± 1.4 bushels/acre ± SE) and reduced within-field yield variance by 25% in 2018 and 8% in 2019 compared to fields fertilized with synthetic nitrogen fertilizers alone. These results demonstrate the capacity of a broad-acre BNF product to fix nitrogen for corn in field conditions with reliable agronomic benefits.

Genetically modified (GM) maize containing genes from the soil bacterium Bacillus thuringiensis (Bt) was cultivated on 29% of the total maize production area worldwide in 2009. Most studies to date compare Bt-maize varieties with their near isogenic lines; however, there is little information on the variability of conventional maize breeding lines and how the effects of Bt varieties are ranked within. In our study on the potential risks of Bt-maize varieties, we analyzed tissue quality and compared the effects of ten conventional and GM maize varieties on soil microbiological properties in a replicated climate chamber experiment. All maize varieties were cultivated twice in the same soil microcosm. Shoot yields and soluble C in leaf tissue of Bt varieties were higher than the ones of non-Bt. Soil dehydrogenase activity was reduced by 5% under Bt varieties compared to non-Bt, while most of the other soil microbial properties (soil microbial biomass, basal respiration) showed no significant differences between Bt and non-Bt varieties. The leaves and roots of one Bt variety were decomposed to a greater extent than the ones of its near isogenic line; the conventional breeding lines also showed higher values. Changes in crop and soil parameters were found when comparing the first and the second crops, but the effects of repeated cropping were the same for all tested varieties. For the studied parameters, the variation among nonBt-maize varieties was similar to the difference between Bt and non-Bt varieties.

Exploring natural diversity for biological nitrogen fixation in maize and its progenitors is a promising approach to reducing our dependence on synthetic fertilizer and enhancing the sustainability of our cropping systems. We have shown previously that maize accessions from the Sierra Mixe can support a nitrogen-fixing community in the mucilage produced by their abundant aerial roots and obtain a significant fraction of their nitrogen from the air through these associations. In this study, we demonstrate that mucilage production depends on root cap and border cells sensing water, as observed in underground roots. The diameter of aerial roots correlates with the volume of mucilage produced and the nitrogenase activity supported by each root. Young aerial roots produce more mucilage than older ones, probably due to their root cap’s integrity and their ability to produce border cells. Transcriptome analysis on aerial roots at two different growth stages before and after mucilage production confirmed the expression of genes involved in polysaccharide synthesis and degradation. Genes related to nitrogen uptake and assimilation were up-regulated upon water exposure. Altogether, our findings suggest that in addition to the number of nodes with aerial roots reported previously, the diameter of aerial roots and abundance of border cells, polysaccharide synthesis and degradation, and nitrogen uptake are critical factors to ensure efficient nitrogen fixation in maize aerial roots.

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