Enabling sustainable growth of plants exploiting plant-microbe interactions

Background/Question/Methods:

Nitrogen (N) is often limited in non-leguminous plants, affecting plant growth and productivity. To alleviate this problem, large amounts of fertilizer is added, which has consequences of heavy energy demand and greenhouse gas emission during fertilizer production, and soil destabilization and hypertrophication from field run-offs during fertilizer application. It is well known that plant associated microbes can significantly influence nutrient availability, and diazotrophic endophytes are known to fix atmospheric N to ammonia in diverse crops. Our knowledge on a molecular level of how the plant recruits and maintains such beneficial bacteria from the surrounding soil and what are the metabolites exchanged between diazotrophic endophytes and host plants is very limited. Understanding the detailed mechanism of these processes is imperative, and could have significant impact on growing plant sustainably, eliminating fertilizers. Our research focuses on investigating interactions between plants and such endophytic diazotrophs, we present results from our investigation with endophytic diazotrophs associated with Tobacco (Nicotiana tabacum) and Switchgrass (Panicum virgatum).

Results/Conclusions:

We successfully isolated several endophytic strains from the roots and leaves of Tobacco and Switchgrass plants. N-fixation by the nitrogenase enzyme of these isolates was confirmed using the well established Acetylene Reduction Assay as well as by the detection of nifH genes. All the isolates readily utilized sugars and simple organic acids as Carbon source, and have a temperature optima of 25°C. To validate the N₂-fixing activity of isolated endophytic diazotrophs, and to localize the hot spots of N-fixation in-planta, aseptic Panicum virgatum seedlings were reinfected with one endophytic isolate, Raoultella strain R1Gly. Different sections of the seedling were examined by fluorescence imaging as well as phosphor imaging of short-lived [¹³N]N₂ radioactive gas. Further, in order to identify metabolites that potentially attract N-fixing bacteria to plants, diazotrophic isolate Azospirillum strain R1C was grown in root exudates collected from hydroponically grown plants, and analyzed for substrate uptake/release. Malate, glycolate, itaconate, citraconate were all consumed by strain R1C and oxalate, glyceric acid, methylmalonic acid were detected as being excreted by this strain. Detailed knowledge and understanding of such interactions between diazotrophic endophytes and host plant will allow us to design robust strategies to enhance plant biomass using microbial N-fixation and will ultimately allow for decreased dependency on fertilizers.