Optimizing plant-microbe interactions for sustainable supply of nitrogen

Background/Question/Methods:

It is well known that plants are strongly affected by their associated microbes, as microbial activity directly influences nutrient availability, produces plant growth factors and confers disease resistance among others. N often is limited in plants, affecting plant growth and productivity. To alleviate this problem, large amounts of fertilizer is added which in turn has consequences of heavy energy demand and greenhouse gas emission during its production, and nutrient leaching and hypertrophication from field run-offs during its application. Diazotrophs are known to fix atmospheric N to ammonia in diverse plant species. Although there has been increasing evidence that the soil microbiome is orchestrated by root exudates our knowledge on a molecular level of how the plant recruits beneficial bacteria from the surrounding soil is still limited. Understanding the detailed mechanism of this process is imperative and could have direct implications in reduced use of fertilizers. As a sustainable alternative, our research focuses on investigating interactions between plants and such beneficial bacteria. Here, we present results from our investigation with microbes associated with Tobacco (Nicotiana tabacum) and Switchgrass (Panicum virgatum) that we were able to isolate and propagate in the lab.

Results/Conclusions

Several endophytic, root associated and rhizospheric bacterial isolates with representatives from Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes and Bacilli were obtained from the roots, leaves, rhizoplane and rhizosphere of these plants. We are investigating the physiology of these isolates – substrates transformed, requirements for growth and additionally, their ability to fix N₂ and to solubilize Phosphorus, another key element for plant growth. As demonstrated by PCR amplification of nifH, several of them are N₂-fixing bacteria, and the results were confirmed by measuring nitrogenase activity by using the standard acetylene-reduction assay. To validate the N₂-fixing activity of isolated endophytic diazotrophs in-planta, fluorescent-tagged strains were used to re-infect aseptic Panicum virgatum seedlings and examined by different imaging techniques. The tagged strains were imaged localized in the leaves. In addition, we investigated the metabolite profile of another isolated diazotroph closely related to Azospirillum lipoferum grown on tobacco root exudates. Preliminary results reveal the utilization of organic acids malate, glycolate, itaconate, citraconate from the exudates. An understanding of key metabolites exchanged and key pathways stimulated in both the plants and the N₂-fixing strains will allow us to design robust strategies to engineer diverse crops able to “fix” atmospheric N₂ and decreasing dependency on fertilizers.