Periodic Reporting for period 1 - ROOTPHENOBIOME (INTERACTIONS OF ROOT PHENOTYPES AND ROOT MICROBIOME IN MAIZE UNDER NITROGEN LIMITING CONDITIONS)
Okres sprawozdawczy: 2020-02-01 do 2022-01-31
One of the most critical challenges in modern agriculture is the low nitrogen uptake in cereals. This lack of nutrient uptake efficiency causes environmental contamination and food insecurity globally. Sustainable cereal cultivation can be developed by integrating efficient combinations of root phenotypes and root microorganisms. For example, nitrogen uptake is enhanced in plants with root systems that are located where nitrogen is more abundant in the soil profile. Specifically, plants with deep roots have the capability to acquire more nitrogen in intensively managed agricultural soil because nitrogen-containing fertilizers are quickly mobilized to deep soil layers. The selection of plants with root phenotypes specifically targeted to improve resource acquisition could be a solution to improve plant yields reductions caused by inefficient nitrogen uptake. Also, the utilization of microorganisms to improve plant nutrient uptake under low nitrogen conditions is a promising strategy to help improve nitrogen availability in the root-soil interphase. The study of the root system as habitat for microorganisms is an important topic to be explored in the context of root phenotyping for sustainable agriculture. This project aimed to study the effects of root phenotypes on the composition and function of microorganisms that participate in the nitrogen cycle under low nitrogen conditions, with the ultimate goal of understanding root phenotypes as factor of variation of the root microbial communities important for nitrogen uptake.
This project focused on 3 main goals: 1. Describe the distribution of archaea and bacteria along the maize root systems under optimal and suboptimal nitrogen fertilization (WP1), 2. Study the associations of prokaryotes with specific root anatomical and architectural traits that are important under low-nitrogen conditions (WP2), 3. Explore the significance of microbial-root associations within the nitrogen cycle (WP3), 4. Explore mechanisms of microbe-plant associations using metabolic profiles (WP1, WP2, WP3).
In WP1 we characterized the bacteria and archaea present in bulk soil (soil lacking the influence of roots), rhizosphere (soil surrounding and being influenced by the roots) and endosphere (root tissue without soil) associated with maize grown in 30-litre columns (so-called mesocosms) via metabarcoding of 16S rRNA genes. These mesocosms, previously used in root phenomics, resemble the soil volume and depth available to a maize plant in the field. We evaluated the prokaryotic diversity at five points along the root system of a single maize genotype. In our experiment we combined two factors: N levels (high and low) and potting mixtures (one containing grassland soil and the other containing agricultural soil). Root anatomical and architectural traits, metabolic signatures of root tissue, and leaf metabolomes were measured. Prokaryotic communities differed by depths and root types regardless of the N level in both the rhizosphere and endosphere. Soil mixture had a significant effect on the leaf but not on root metabolomes. Leaf non-polar metabolite composition and polar metabolite compositions significantly differed between the N treatments. Across the whole experiment, significant taxon-metabolite associations were found for rhizosphere and endosphere. We are using multivariate statistical methods to find significant associations between root traits and microbial taxa under optimal, suboptimal severe low-N conditions.
In WP2 we used the protocol generated in WP1 for root and microbiome sampling to evaluate the interaction of root architecture and anatomy on prokaryotic associations. For this purpose, we used four closely-related maize genotypes that were previously phenotyped under similar experimental conditions, including low-N. We grew the plants under the factorial combination of nitrogen level and genotype and studied root microbiome, root anatomy and architecture, and roots and shoot metabolomics. Although the genotypes had contrasting root architecture the effect of genotypes on microbiome diversity was marginal. We continue to study individual root traits (such as root branching density, root vertical distribution, and number of axial roots) on the microbial communities. We are also working on the analyses of metabolic data that were produced at the end of the project. Root anatomy was phenotyped but the data are being analyzed. A MSc thesis was presented and approved, and a paper is in preparation.
In WP3 we aimed to measure the relative contribution of the root phenotype effects in comparison with microbiome effects using sterilized soil as a treatment in addition to the factors genotype and nitrogen level. However, soil sterilization demonstrated to be a very difficult task because microbial growth is recovered within days after opening the container where it has been sterilized, reaching microbial counts similar to non-sterilized soils. Therefore, we could not obtain microbe-free substrate under our experimental conditions. Consequently, we changed the scope of our research to study the soil legacy effect, as the microbial communities did change with the sterilization treatment. We are currently studying the effect of these tree factors on the microbial conditions, with a MSc thesis already presented and approved and a paper in preparation.
The results obtained in this project have been presented in several scientific conferences, invited lectures and seminars. The perspective and ideas underlying this project were recently published in scientific journal. Two funded proposals to continue with the phase on proof of concept of our ideas have been granted. Concepts and results have been shared in non-scientific events. Results obtained with this project provide evidence of the importance of integrating root architecture and anatomy as well as root metabolomes to better understand microbial root colonization under low-N conditions for a more sustainable agriculture.
In WP1 we characterized the bacteria and archaea present in bulk soil (soil lacking the influence of roots), rhizosphere (soil surrounding and being influenced by the roots) and endosphere (root tissue without soil) associated with maize grown in 30-litre columns (so-called mesocosms) via metabarcoding of 16S rRNA genes. These mesocosms, previously used in root phenomics, resemble the soil volume and depth available to a maize plant in the field. We evaluated the prokaryotic diversity at five points along the root system of a single maize genotype. In our experiment we combined two factors: N levels (high and low) and potting mixtures (one containing grassland soil and the other containing agricultural soil). Root anatomical and architectural traits, metabolic signatures of root tissue, and leaf metabolomes were measured. Prokaryotic communities differed by depths and root types regardless of the N level in both the rhizosphere and endosphere. Soil mixture had a significant effect on the leaf but not on root metabolomes. Leaf non-polar metabolite composition and polar metabolite compositions significantly differed between the N treatments. Across the whole experiment, significant taxon-metabolite associations were found for rhizosphere and endosphere. We are using multivariate statistical methods to find significant associations between root traits and microbial taxa under optimal, suboptimal severe low-N conditions.
In WP2 we used the protocol generated in WP1 for root and microbiome sampling to evaluate the interaction of root architecture and anatomy on prokaryotic associations. For this purpose, we used four closely-related maize genotypes that were previously phenotyped under similar experimental conditions, including low-N. We grew the plants under the factorial combination of nitrogen level and genotype and studied root microbiome, root anatomy and architecture, and roots and shoot metabolomics. Although the genotypes had contrasting root architecture the effect of genotypes on microbiome diversity was marginal. We continue to study individual root traits (such as root branching density, root vertical distribution, and number of axial roots) on the microbial communities. We are also working on the analyses of metabolic data that were produced at the end of the project. Root anatomy was phenotyped but the data are being analyzed. A MSc thesis was presented and approved, and a paper is in preparation.
In WP3 we aimed to measure the relative contribution of the root phenotype effects in comparison with microbiome effects using sterilized soil as a treatment in addition to the factors genotype and nitrogen level. However, soil sterilization demonstrated to be a very difficult task because microbial growth is recovered within days after opening the container where it has been sterilized, reaching microbial counts similar to non-sterilized soils. Therefore, we could not obtain microbe-free substrate under our experimental conditions. Consequently, we changed the scope of our research to study the soil legacy effect, as the microbial communities did change with the sterilization treatment. We are currently studying the effect of these tree factors on the microbial conditions, with a MSc thesis already presented and approved and a paper in preparation.
The results obtained in this project have been presented in several scientific conferences, invited lectures and seminars. The perspective and ideas underlying this project were recently published in scientific journal. Two funded proposals to continue with the phase on proof of concept of our ideas have been granted. Concepts and results have been shared in non-scientific events. Results obtained with this project provide evidence of the importance of integrating root architecture and anatomy as well as root metabolomes to better understand microbial root colonization under low-N conditions for a more sustainable agriculture.
The study of interactions between root microbiomes and root traits has been just recently considered in plant science research of model plants such as Arabidopsis. However, studies using crops are very scarce and usually not performed under abiotic stress conditions. The present project is unique and timely in considering not only roots or microbes, but the combination of the two partners to develop new technologies using plant and microbiome breeding.