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.