Examining the Effects of the Nitrogen Environment on Growth and N2-Fixation of Endophytic Herbaspirillum seropedicae in Maize Seedlings by Applying 11C Radiotracing

Spenser Waller; Stacy L Wilder; Michael J Schueller; Alexandra B Housh; Stephanie Scott; Mary Benoit; Avery Powell; Garren Powell; Richard A Ferrieri

doi:10.3390/microorganisms9081582

Examining the Effects of the Nitrogen Environment on Growth and N₂-Fixation of Endophytic Herbaspirillum seropedicae in Maize Seedlings by Applying ¹¹C Radiotracing

Microorganisms. 2021 Jul 25;9(8):1582. doi: 10.3390/microorganisms9081582.

Authors

Spenser Waller^{1

2}, Stacy L Wilder¹, Michael J Schueller^{1

3}, Alexandra B Housh^{1

3}, Stephanie Scott^{1

4}, Mary Benoit^{1

5}, Avery Powell^{1

2}, Garren Powell^{1

4}, Richard A Ferrieri^{1

3

5}

Affiliations

¹ Missouri Research Reactor Center, University of Missouri, Columbia, MO 65211, USA.
² School of Natural Resources, University of Missouri, Columbia, MO 65211, USA.
³ Chemistry Department, University of Missouri, Columbia, MO 65211, USA.
⁴ Biochemistry Department, University of Missouri, Columbia, MO 65211, USA.
⁵ Division of Plant Sciences, Interdisciplinary Plant Group, University of Missouri, Columbia, MO 65211, USA.

Abstract

Herbaspirillum seropedicae, as an endophyte and prolific root colonizer of numerous cereal crops, occupies an important ecological niche in agriculture because of its ability to promote plant growth and potentially improve crop yield. More importantly, there exists the untapped potential to harness its ability, as a diazotroph, to fix atmospheric N₂ as an alternative nitrogen resource to synthetic fertilizers. While mechanisms for plant growth promotion remain controversial, especially in cereal crops, one irrefutable fact is these microorganisms rely heavily on plant-borne carbon as their main energy source in support of their own growth and biological functions. Biological nitrogen fixation (BNF), a microbial function that is reliant on nitrogenase enzyme activity, is extremely sensitive to the localized nitrogen environment of the microorganism. However, whether internal root colonization can serve to shield the microorganisms and de-sensitize nitrogenase activity to changes in the soil nitrogen status remains unanswered. We used RAM10, a GFP-reporting strain of H. seropedicae, and administered radioactive ¹¹CO₂ tracer to intact 3-week-old maize leaves and followed ¹¹C-photosynthates to sites within intact roots where actively fluorescing microbial colonies assimilated the tracer. We examined the influence of administering either 1 mM or 10 mM nitrate during plant growth on microbial demands for plant-borne ¹¹C. Nitrogenase activity was also examined under the same growth conditions using the acetylene reduction assay. We found that plant growth under low nitrate resulted in higher nitrogenase activity as well as higher microbial demands for plant-borne carbon than plant growth under high nitrate. However, carbon availability was significantly diminished under low nitrate growth due to reduced host CO₂ fixation and reduced allocation of carbon resources to the roots. This response of the host caused significant inhibition of microbial growth. In summary, internal root colonization did little to shield these endophytic microorganisms from the nitrogen environment.

Keywords: Herbaspirillum seropedicae; carbon-11; endophytic rhizobacteria; green fluorescence reporting; maize roots; plant-borne carbon.

Abstract

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