Discussion
The increased focus on sustainable agricultural practices has increased the need for knowledge on the impact of land management on biodiversity of all living things from mammals to fungi and bacteria. For legume crops, a healthy soil microbiome, and especially the availability of nitrogen-fixing rhizobial symbionts capable of establishing an effective symbiosis, plays an important role in establishing a high-yielding agricultural practice.
Most studies to assess field bacterial diversity have focused onRhizobium isolates from Trifolium and Vicia nodules . Here, we present an in-depth study of clover field trials and organic fields where strains have previously been isolated , using the MAUI-seq method on nodule populations to investigate the reported level of diversity within the R. leguminosarum community found in white clover nodules when using a HTAS approach compared to a more traditional isolate-based approach.
It has been estimated that 99% or more of environmental prokaryotes cannot be cultured using classical methods in the laboratory. These obvious limitations are a challenge for many bacterial species, and HTAS is commonly used to investigate communities without the need for cultivation . R. leguminosarum is easily cultured from nodules in the laboratory, but it is slow growing and requires several rounds of single colony isolation before it can be separated from faster growing bacteria present in and around the nodule. Studying large numbers of nodules therefore requires both time and resources. HTAS offers the advantage that it allows for screening a large number of samples with many organisms per sample in a fast and efficient manner. We have adapted this method using MAUI-seq to study the intraspecies diversity of R. leguminosarum in nodules, to allow screening of large nodule populations from many sampling sites. While amplicon studies are ideal for sampling large numbers of samples (here, nodules), good quality sequences of the organism of choice are necessary to design specific, yet effective primers for any non-standard amplicon. The 196 sequenced Rlt isolates have formed the basis of the phylogenetic analysis and thereby the introgression analysis of this study. Here, we use amplicon sequencing to check that the isolates are representative of the nodule population as a whole.
There was good agreement between the two methods in estimating the allele frequency and genospecies composition of the nodule populations, though more rare alleles were observed in the MAUI-seq data probably due to the deeper sampling; 85 times more nodules were sampled using MAUI-seq compared to the isolate-based study. Many amplicon-based studies have assessed enrichment of many bacterial species in the rhizosphere or other soil compartments compared to bulk soil or between managements . Similarly, a modified version of MAUI-seq has been used to investigate the Rhizobium population in soil and how it differs from nodule populations from Vicia and Trifolium host plants of Rhizobium . Cultivating slow-growing bacteria from soil can be very difficult, and amplicon sequencing enables studies of populations in bulk soil that would otherwise have been unfeasible.
Using the broadly sampled set of nodule amplicons allowed us to make comparisons between nodule populations in soils under different managements and with different soil chemical and physical properties. Genetic differentiation for accessory genes is less correlated with distance than for core genes, perhaps reflecting differences in local adaptation. It seems likely that nodD and nodA are not adapted to local differences, but are adapted to the available symbiotic partner (here, white clover). The movement of symbiosis genes may be less restricted since they are located on conjugative plasmids . Mobility of accessory genes located on integrative conjugative elements (ICEs) has previously been hypothesised to lead to lower genetic differentiation over distance than core genes located on less mobile parts of the chromosome . In our case, the accessory symbiosis genes,nodA and nodD , are located on conjugative plasmids shown to cross genospecies boundaries, whereas there is little to no recombination between core genes . Isolation-by-distance has been shown to be more pronounced for the core genome for aquatic microbes, whereas the location of accessory genes on plasmids or on genomic islands renders them more mobile and more dispersed in the environment , which might also be true for bacterial populations in soil. In-depth analysis of allele frequencies in each sample is possible, due to the number of nodules sampled when using amplicon sequencing. Using only the isolate dataset, we would be limited to a few isolates per field/plot, and hence imprecise estimates of diversity.
Correlations between genetic differentiation and distance between populations are often confounded by differences in soil physical and chemical properties. We tested the correlations between soil chemical and physical properties, geospatial placement, and amplicon abundance. No significant correlations were observed between latitude/longitude and soil properties. The observed positive correlation between genetic differentiation and spatial separation is therefore unlikely to be due to soil chemical or physical properties. The UK site, which had a uniform and unique composition of only gsB, has the highest phosphorus (P) content. P content has been shown to be correlated with rhizobium population size in a previous study, but no genotyping was done . The high nutrient content might enhance the fitness difference between fast- and slow-growing Rlt strains , thereby driving the population differences between high and low P sites.
The level of nucleotide diversity (𝛑) observed within each nodule population sample was significantly higher for samples from fields under organic management than from fields used for clover breeding trials. This might reflect a lower diversity of Rlt in the soil at the clover breeding trial sites, possibly due to an increase in nitrogen application . The DK, F, and UK sites are very different in soil composition, making it less likely that the lower Rlt genetic diversity is due to a soil-management interaction than to a general effect of field management. The Rhizobium populations at the three sites have distinct sets of alleles, suggesting that the management does not select for a specific set of Rlt , but rather enriches already dominant or highly adapted strains, specific for each site. A more diverse collection of clover genotypes was grown at the DK, F, and UK sites than for DKO fields, so the reduction in diversity is unlikely to be an effect of clover genotype selection. The varied genospecies distribution of isolates from the DKO fields hinted at a higher diversity in these fields compared to the field trial sites, but the differences in sampling distance between DKO isolates (up to 200km) and isolates from individual field trials (<200m) impeded a detailed investigation. For the amplicon-based study, we collected 100 nodules from each field, which allowed us to treat each field as an individual data point in the diversity analysis. This revealed the striking differences in nucleotide diversity between fields under different management regimes.
A study of spatial variation of Rhizobium symbiotic performance used a field sampling layout to test the effect at different spatial scales . Similar study designs, with neighbouring fields managed in different ways, would be appropriate for a more in-depth assessment of the effect of management on Rhizobium populations while disentangling it from geographical and soil chemical variation. Our results, in combination with previous studies, provide an indication that there could be substantial effects of field management onRhizobium diversity and should motivate further studies on the effect of field management on soil microbial diversity at the level of individual species. We show that HTAS in the form of MAUI-seq on pooled nodules is an efficient method for estimating Rhizobium diversity in nodules, and a previous study has shown that the method can also be applied to soil samples . There was good agreement between the alleles detected by amplicon sequencing and those found in isolates cultured from nodules at the same sites, and MAUI-seq can provide more detailed estimates of allele frequencies without the need to culture and characterise large numbers of individual isolates.