Periodic Reporting for period 1 - FITTER (Friends with benefits – the role of endophytic bacteria in legume nodules containing nitrogen-fixing rhizobia)
Berichtszeitraum: 2023-09-01 bis 2025-08-31
Modern agriculture faces the critical challenge of feeding a growing global population while protecting the environment and maintaining healthy soils for future generations. Conventional farming relies heavily on synthetic fertilizers, which cause water pollution and greenhouse gas emissions. Legumes (soybeans, clover, lucern, peas, beans), are important crops in agriculture due to their ability of biological nitrogen fixation.
Biological nitrogen fixation is the conversion of atmospheric nitrogen into ammonia, a form of nitrogen that plants need for development and growth. Legumes form a mutualistic relationship with symbiotic bacteria, the rhizobia, which are housed in root nodules. Within those nodules, rhizobia use specialized enzymes to fix nitrogen, while the plant provides carbohydrates and a protected environment within the nodule. This relationship reduces the need for synthetic nitrogen fertilizers, making legumes essential for sustainable farming.
Recent research reveals that root nodules harbour not only rhizobia but also diverse non-rhizobial endophytes (NRE), such as bacteria or fungi that may play important roles in plant health, stress tolerance and nutrient cycling. This raises intriguing questions about how these microbial communities contribute to the overall success of legumes and their ability to fix nitrogen. This project addressed this gap by investigating the diversity and functional roles of non-rhizobial endophytes in root nodules, shedding light on their contributions to legume health and productivity.
Objectives of this Marie Skłodowska Curie Action (MSCA) were first to characterize the microbial communities within legume root nodules, with a particular focus on non-rhizobial bacteria (NRE). By employing advanced sequencing technologies, such as 16S rRNA gene sequencing and metagenomics we uncovered the diversity and composition of nodule bacterial communities of legume, clover and soybean grown in the field. Additionally, we investigated the rhizosphere, which is the soil surrounding plant roots, to understand the role of the rhizosphere microbiome as a source of NRE colonizing nodules.
The second objective focused on uncovering the functional roles of the nodule bacterial community. Using a metagenomics approach, we aimed to identify the genes and metabolic pathways within nodule-associated bacteria. Additionally, we created a culture collection of rhizobia and NRE for future research.
Finally, the third objective was to understand how environmental factors influence the diversity and functional roles of NAB within legume nodules. Factors such as soil type, nutrient availability, climate conditions, and agricultural practices are known to shape microbial communities in the rhizosphere, but their impact on nodule-associated bacteria remains poorly understood.
Biological nitrogen fixation is the conversion of atmospheric nitrogen into ammonia, a form of nitrogen that plants need for development and growth. Legumes form a mutualistic relationship with symbiotic bacteria, the rhizobia, which are housed in root nodules. Within those nodules, rhizobia use specialized enzymes to fix nitrogen, while the plant provides carbohydrates and a protected environment within the nodule. This relationship reduces the need for synthetic nitrogen fertilizers, making legumes essential for sustainable farming.
Recent research reveals that root nodules harbour not only rhizobia but also diverse non-rhizobial endophytes (NRE), such as bacteria or fungi that may play important roles in plant health, stress tolerance and nutrient cycling. This raises intriguing questions about how these microbial communities contribute to the overall success of legumes and their ability to fix nitrogen. This project addressed this gap by investigating the diversity and functional roles of non-rhizobial endophytes in root nodules, shedding light on their contributions to legume health and productivity.
Objectives of this Marie Skłodowska Curie Action (MSCA) were first to characterize the microbial communities within legume root nodules, with a particular focus on non-rhizobial bacteria (NRE). By employing advanced sequencing technologies, such as 16S rRNA gene sequencing and metagenomics we uncovered the diversity and composition of nodule bacterial communities of legume, clover and soybean grown in the field. Additionally, we investigated the rhizosphere, which is the soil surrounding plant roots, to understand the role of the rhizosphere microbiome as a source of NRE colonizing nodules.
The second objective focused on uncovering the functional roles of the nodule bacterial community. Using a metagenomics approach, we aimed to identify the genes and metabolic pathways within nodule-associated bacteria. Additionally, we created a culture collection of rhizobia and NRE for future research.
Finally, the third objective was to understand how environmental factors influence the diversity and functional roles of NAB within legume nodules. Factors such as soil type, nutrient availability, climate conditions, and agricultural practices are known to shape microbial communities in the rhizosphere, but their impact on nodule-associated bacteria remains poorly understood.
In the initiation phase of the project, local farmers were contacted and a network of local farmers willing to participate in the study was established. During 2024, we conducted field sampling in Vienna and Lower Austria at organic and conventional fields growing soybean, clover or lucern (WP1). Each field was sampled three times, collecting soil and root systems, in spring (during active growth phase), summer (during flowering) and fall. In total, we processed 140 root systems in the laboratory by washing roots, cutting off nodules, followed by surface sterilisation to ensure we captured only the bacterial community within nodules (WP2). A total of 109 soil samples were also sent to the University of Natural Resources and Life Sciences (BOKU) in Vienna for detailed soil analysis (WP6). After DNA extraction of nodule and soil samples (WP2), two complementary research methods were used: First, we used traditional cultivation techniques, isolating individual bacteria from root nodules, growing them in the laboratory and making stocks for future use (WP3). This culture collection allows us to further study specific bacterial strains in detail in the future and we can identify potential candidates for future agricultural applications. Second, we used different DNA sequencing technologies for soil and nodule samples. First we specifically targeted marker genes that act like molecular barcodes for different microorganisms (bacteria or fungi) to investigate diversity in nodule and soil communities (WP4). These results were used to decide on further sequencing approaches to look at the functional profiles of the nodule bacterial community (WP5), where the total DNA of a given sample was sequenced.
Objective 1: The composition of nodule bacterial communities was distinct between the three legume types (soybean, lucern and red clover), and we found significant shifts in community profiles between the sampling timepoints and for the different agricultural practices (conventional vs. organic). Bacterial richness in nodules increased from spring to summer and decrease again towards fall, suggesting that at the peak of nitrogen fixing ability, bacterial richness is the highest. Whereas for soil, bacterial richness remained the same throughout the sampling period.
Objective 2: Nodules were dominated by their rhizobial partners with red clover nodules containing an average of 80% Rhizobium sp. or Ensifer sp. and lucern 87% Sinorhizobium sp. This correlates with what has been reported in previous studies. However, soybean nodules were almost solely inhabited by Bradyrhizobium sp. (99.4%), making them the least diverse communities regardless of agricultural practice.
The most common NRE were Pseudarthrobacter sp., Peribacillus, Bacillus, Glycomyces and Peribacillus found in >95% of samples, for which we were able to obtain representative genomes for detailed genetic analysis. Another achievement is the collection of 120 bacterial isolates from nodules of all three legume species, including isolates for the different rhizobial species (Sinorhizobium meliloti, Rhizobium leguminosarum and Bradyrhizobium japonicum), as well as representatives of the most common non-rhizobial endophtyes we found in the nodules (Bacillus sp., Peribacillus sp., Priestia sp. and many more). This valuable resource can be used for plant experiments, as well as detailed studies of their genomes in the future.
Objective 3: Detailed rhizosphere soil analysis showed that potassium and phosphate levels in the soil followed seasonal patterns with an increase towards fall for clover, but not for soybean and lucern. The pH of the soils remained constant for lucern and clover, whereas there was a small, but significant change in pH from 6.7. to 6.9 for soybean.
Further, we collaborated with researchers from the National Institute for Agriculture, Food and Environment in Toulouse (France) on a soybean project, where we investigated the effect of irrigation on the nodules communities of different soybean cultivars. We found that nodules were solely occupied by up to five different strains of Bradyrhizobium sp, but found no evidence of NRE in those samples.
Objective 1: The composition of nodule bacterial communities was distinct between the three legume types (soybean, lucern and red clover), and we found significant shifts in community profiles between the sampling timepoints and for the different agricultural practices (conventional vs. organic). Bacterial richness in nodules increased from spring to summer and decrease again towards fall, suggesting that at the peak of nitrogen fixing ability, bacterial richness is the highest. Whereas for soil, bacterial richness remained the same throughout the sampling period.
Objective 2: Nodules were dominated by their rhizobial partners with red clover nodules containing an average of 80% Rhizobium sp. or Ensifer sp. and lucern 87% Sinorhizobium sp. This correlates with what has been reported in previous studies. However, soybean nodules were almost solely inhabited by Bradyrhizobium sp. (99.4%), making them the least diverse communities regardless of agricultural practice.
The most common NRE were Pseudarthrobacter sp., Peribacillus, Bacillus, Glycomyces and Peribacillus found in >95% of samples, for which we were able to obtain representative genomes for detailed genetic analysis. Another achievement is the collection of 120 bacterial isolates from nodules of all three legume species, including isolates for the different rhizobial species (Sinorhizobium meliloti, Rhizobium leguminosarum and Bradyrhizobium japonicum), as well as representatives of the most common non-rhizobial endophtyes we found in the nodules (Bacillus sp., Peribacillus sp., Priestia sp. and many more). This valuable resource can be used for plant experiments, as well as detailed studies of their genomes in the future.
Objective 3: Detailed rhizosphere soil analysis showed that potassium and phosphate levels in the soil followed seasonal patterns with an increase towards fall for clover, but not for soybean and lucern. The pH of the soils remained constant for lucern and clover, whereas there was a small, but significant change in pH from 6.7. to 6.9 for soybean.
Further, we collaborated with researchers from the National Institute for Agriculture, Food and Environment in Toulouse (France) on a soybean project, where we investigated the effect of irrigation on the nodules communities of different soybean cultivars. We found that nodules were solely occupied by up to five different strains of Bradyrhizobium sp, but found no evidence of NRE in those samples.
Due to the high percentage of rhizobia in samples, we employed adaptive sequencing, a cutting-edge Oxford Nanopore technology that identifies and selects DNA sequences in real-time. This approach dramatically improved sequencing efficiency by discarding abundant rhizobial DNA and enriching for rare NRE sequences, allowing us to obtain high-quality whole genomes of the most common NRE (Bacillus, Peribacillus) that would otherwise be obscured.
By integrating cutting-edge sequencing technologies, functional analyses, and environmental studies, this research provides a deeper understanding of the diversity, function, and ecological drivers of NRE in legume nodules. This knowledge enables practical agricultural applications: NRE that enhance nitrogen fixation or plant growth can be incorporated into bacterial inoculants to boost crop performance while reducing reliance on synthetic fertilizers.
Ultimately, this project advances sustainable agriculture by revealing how invisible microbial partnerships support legume productivity. This is a crucial step toward harnessing these natural systems for global food security.
By integrating cutting-edge sequencing technologies, functional analyses, and environmental studies, this research provides a deeper understanding of the diversity, function, and ecological drivers of NRE in legume nodules. This knowledge enables practical agricultural applications: NRE that enhance nitrogen fixation or plant growth can be incorporated into bacterial inoculants to boost crop performance while reducing reliance on synthetic fertilizers.
Ultimately, this project advances sustainable agriculture by revealing how invisible microbial partnerships support legume productivity. This is a crucial step toward harnessing these natural systems for global food security.