Introduction
The interplay of plants and microorganisms in the soil has a multitude of beneficial functions in natural ecosystems, including protection against pathogens and abiotic stress such as drought, uptake of nutrients like phosphate and nitrogen, and growth promotion .
Understanding the microbial variability within and between fields, and which factors influence the number or diversity of microbes, is necessary to understand how to best optimise or work with the microbiome. The increase in genetic diversity over distance for microorganisms has been shown at scales ranging from metres to 100 kilometres . This effect can be explained by sampling a wide range of conditions or by isolation-by-distance, first described in aquaticSulfolobus , and since then verified in multiple species .
Biological nitrogen fixation (BN)F occurs as a result of a mutualistic symbiosis between legumes and soil bacteria, commonly known as rhizobia. Rhizobia are harboured in specialised root structures, known as nodules. To confidently establish the level of diversity within nodule populations, the most common assessment method uses cultured bacteria isolated from nodules . Isolate-based approaches rely on the culturability of the microbes and become very labour intensive if the desired number of isolates per site is high, though they have the advantage that isolates are available for evaluation as potential inoculants. For soil microbiome diversity studies, where many of the organisms cannot be cultured using traditional methods, high throughput amplicon sequencing (HTAS) is used to amplify sequences that distinguish microbial communities at different levels of resolution from environmental DNA samples in a cultivation-independent manner . This method can be adapted for Rhizobium nodule or soil populations using multiplexed amplicons with unique molecular identifiers (MAUI-seq), as has been shown in recent publications . How well diversity estimates from traditional isolate-based approaches compare to HTAS in evaluating the rhizobial diversity has not been explored in detail.
A number of studies have addressed the influence of land management on the soil microbiome by comparison with undisturbed soils such as native tropical forests and permanent grasslands . Land management was found to have an impact on the alpha-diversity of fungal and bacterial microbial communities. Managed land shifted the balance towards more abundant fungal communities at the expense of bacterial diversity. Application of nitrogen was shown to reduce alpha-diversity of protist, bacterial, and fungal soil communities in maize fields, with possible detrimental effects on the soil microbiome . Standard agricultural practices involve applying large amounts of N, both as synthetic and organic fertiliser, to ensure the yield and quality of the crop , with possible detrimental effects on microbiome diversity.
Defining the way in which land management influences the soil microbiome is important when moving towards more sustainable agricultural practices. For organic farmers, where nitrogen input is limited, BNF is essential in fertilising the soil and providing high-yield crops with high protein content. The health and biodiversity of the soil microbiome can influence the yield of legumes through this symbiosis by affecting the available genotypes of rhizobia . The changes to the soil microbiome by management and fertiliser application could affect the nitrogen (N) availability in agricultural settings by limiting legume nodulation efficiency due to decreased abundance of rhizobia and other nodule-associated bacteria .
For some legumes grown in non-native soils, an effective symbiont is not naturally present. The solution to this issue has been to inoculate fields with the appropriate rhizobial symbiont for the legume crop. However, these inoculum rhizobia can be outcompeted by native rhizobia before the end of the growth season, even in soils with low levels of native rhizobia . Therefore, when growing legumes in native soils with a high concentration of rhizobia, where inoculation is an even less effective tool than in low-rhizobia soils, it is important to maintain a rich diversity of highly adapted microbes in the soil to ensure that an appropriate, effective symbiont partner is present for the legume crop of choice .
White clover (Trifolium repens L.) is an important agricultural crop in temperate climates used by both conventional and organic farmers primarily to improve forage quality by raising protein content in perennial grass pastures. Its symbiotic partner, Rhizobium leguminosarum (Rl ), is a species complex comprising at least seven genetically distinct genospecies (gs) with limited gene flow between them . Rl can nodulate several legume species, and its specificity is determined by a group of symbiosis genes located on mobile plasmids. The population of Rl capable of establishing a symbiosis with clovers is the symbiovar trifolii (Rlt ). Recently, methods investigating microbial intraspecies diversity in environmental samples have been developed. MAUI-seq, which is the method used in this study, relies on multiplexed amplicons tagged with unique molecular identifiers (UMIs) . UMIs allow filtering of erroneous reads (chimaeras and polymerase errors) using a ratio of how often a sequence is observed as a primary UMI sequence (the most abundant sequence tagged with a given UMI) or a secondary sequence (a less abundant sequence for a given UMI).
We have previously characterised a set of genomes from 196 Rltisolates from pink white-clover nodules from three clover field trial sites in northern Europe, and 50 organic fields from Jutland, Denmark . These 196 genomes were distributed throughout five of the seven known genospecies in Rl , with the majority belonging to gsC. Here, theRlt nodule populations in the same fields were characterised using the MAUI-seq method by amplifying two core genes (rpoB andrecA ) and two accessory genes (nodA and nodD ) important in establishing symbiosis. We compare HTAS with the traditional isolate-based approach in evaluating the intraspeciesRlt diversity in white-clover nodule populations in field trials and organic fields, to investigate the allelic diversity at each site in greater depth.
While isolates potentially provide the full genome information and allow assessment of whole genome differences between strains, sample sizes are necessarily limited. In recent studies, the numbers of isolates ranged from 73 to 212 ( n=73; n=86; n=196; n=210; n=212). Our previous study characterised isolates from 196 nodules in detail and facilitated in-depth population genomics analysis and the discovery of movement of symbiosis genes between genospecies on a promiscuous plasmid . Using MAUI-seq, we were able to process and study the nodule population on a much larger scale, obtaining sequencing data of amplicons from 17,000 nodules.
We found that isolate-based and MAUI-seq diversity assessment were similar in terms of genospecies abundance and that all highly abundant sequences overlapped. MAUI-seq identified more rare alleles for all amplicons except recA . We concluded that the diversity observed was robustly determined by both methods, and a small set of chromosomal core genes and plasmid-borne accessory nod genes were significantly correlated with differences in soil clay and silt content. Core genes were affected by isolation by distance to a greater extent than plasmid-borne symbiosis genes in a set of samples from organic fields in Jutland. When comparing genetic diversity in nodule populations from fields under different management, samples from organic fields had significantly higher genetic diversity than fields used for conventional clover breeding trials, indicating that biodiversity of clover symbionts is affected by field management.