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.