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.
