Soil microbes are among the most abundant and diverse organisms on Earth but remain poorly characterized. New technologies have made possible to sequence the DNA of uncultivated microorganisms in soil and other complex ecosystems. Genome assembly is crucial for understanding their functional potential. Nanopore sequencing technologies allow to sequence long DNA fragments, optimizing production of metagenome-assembled genomes compared to short-read technology. Extracting DNA with a very high purity and high molecular weight is key to get the most out of this long read technologies. Here we present two extraction protocols to get DNA with high purity. First protocol is optimized to reach DNA quality suiting Nanopore shotgun metagenomics. It uses a non-toxic centrifugation gradient to separate bacterial cells from soil to extract DNA directly on cells. The median length of the acquired DNA sequences (N50) was 3 to 7 times greater than previously published in the literature, achieving an N50 of ∼14kb. The other, a modification of a commercially available MP Biomedical DNA extraction kit, yielded high-purity DNA for full-length 16S Oxford Nanopore metabarcoding, with an N50 of ∼8kb. The MP-based protocol achieves higher yields of ultra-pure DNA compared to the Nycodenz protocol, at the expense of shorter fragment lengths.

High-input agriculture involves low within-field crop genetic diversity, while plant diversity in natural ecosystems was shown to promote ecosystem functioning. Increasing intra-specific diversity in agroecosystems is a promising strategy to stabilize crop productivity and promote the associated diversity of soil biota. We investigated the effect of within-field diversity of bread wheat varieties on arbuscular mycorrhizal fungi (AMF) and two enzymatic activities involved in organic nitrogen and phosphorus mineralization. We set up a field experiment to test whether varietal or functional diversity modulate the abundance and diversity of AMF and the activity of leucine aminopeptidases and phosphatases in the root zone, considering the influence of root morphology. We used sixteen wheat varieties clustered into four groups according to previously measured traits. The abundance of AMF in roots was measured by qPCR, community composition was analyzed by Illumina metabarcoding on two AMF markers (SSU, LSU), and enzymatic activities were quantified by biochemical assays. Soil properties were the primary drivers of all response variables. Varietal diversity affected nitrogen-related soil enzymatic activities but not those related to phosphorus, with a significant increase of leucine-aminopeptidase activities with increasing varietal diversity. Wheat varietal and functional diversity marginally impacted the abundance of AMF, and functional diversity negatively affected AMF diversity on the SSU marker. Mean root traits modulated enzymatic activities, but not AMF communities. Increasing intra-specific crop diversity affects essential soil microbial processes, providing valuable insights for studying the relationship between plant diversity and soil microbiota in agroecosystems.

Within a landscape, multiple habitats exist for soil microbial communities. But how these habitats shape community composition requires an understanding of the way in which microbial diversity is impacted across a broad range of spatial scales. Mountain ecosystems are excellent systems to study microbial communities, because a multitude of climate and soil variables change within a relatively small distance. We investigated microbial community structure in bulk and rhizosphere soils beneath three plant species, Vaccinium myrtillus , Juniperus communis and Picea abies , that structure local plant communities along an elevation gradient in the French Alps. We examined the impact that climate, soil properties, plant diversity and plant root chemical and morphological traits had on microbial α‐ and β‐diversities. The most abundant bacterial phyla detected in both bulk and rhizosphere soils were Proteobacteria, Actinobacteria, Acidobacteria and Verrucomicrobia. Along the elevation gradient, bacterial phyla did not display a clear distribution pattern between bulk and rhizosphere soils. For fungi, dominant phyla were Ascomycota and Basidiomycota, and contrasting distribution patterns were found between bulk and rhizosphere soils. Overall, bacterial and fungal α‐diversity responded differently to elevation as well to soil compartments (bulk versus rhizosphere soil), revealing no significant patterns in bulk soil beneath any of the structuring plant species, but increasing in the rhizosphere compartment of P. abies just below the treeline. Changes in bacterial β‐diversity with elevation were related mostly to soil physical and chemical properties. Bacterial and fungal α‐diversity in rhizosphere communities were more related to plant species identity, vegetation diversity and belowground plant traits compared to soil properties, whilst the opposite was found for bulk soil. Our results highlight that environmental changes at the landscape scale (e.g. associated to elevation, soil properties or climate), impact significantly soil microbial communities, but vegetation refines communities at a local scale via the rhizosphere niche.

Abstract High-input agriculture has been associated with a drastic reduction of within-field crop genetic diversity, while plant (mostly functional) diversity in natural ecosystems has been shown to promote ecosystem functioning. Increasing intra-specific diversity in agroecosystems is a promising strategy to stabilize crop productivity and promote the associated diversity of fauna and microbiota. We investigated the effect of the within-field diversity of bread wheat varieties on arbuscular mycorrhizal fungi (AMF) and two enzymatic activities involved in organic nitrogen and phosphorus mineralization. A field experiment was designed to test whether the number of mixed wheat varieties in a plot, or their functional diversity (previously assessed), influence the abundance and diversity of AMF and the activity of leucine aminopeptidases and phosphatases in the root zone. The AMF abundance was measured by quantitative polymerase chain reaction, community composition was analyzed by Illumina metabarcoding on two AMF specific markers, and potential microbial activities were quantified by biochemical assays. Wheat traits related to root morphology and susceptibility to fungal diseases previously quantified for each variety were also used. Number of varieties significantly increased AMF abundance in roots, whereas functional cluster number did not, with no impact of root morphology. Functional cluster number influenced AMF diversity, though weakly and not linearly, responding most to binary mixtures. Both wheat variety and functional group number increased the potential leucine amino-peptidase activities in the root zone, while no effect was observed for phosphatase activities. Our results highlight that increasing crop intra-specific diversity triggered changes in key processes involved in nutrient acquisition.

Phytate is considered a poorly available plant P source but proved to be useful for particular soil bacteria strains. In soil-free conditions, it has been shown that bacteria locked up the mineralized phosphorus from phytate whereas bacterial grazers like nematodes were able to deliver P to plants. Here, we aimed to determine if the interactions between phytate-mineralizing bacteria, bacterial grazer nematodes, and mycorrhizal fungi could increase plant P acquisition from phytate in high P-adsorbing soils. Pinus pinaster was grown in a Cambisol supplemented with phytate. Plants, whether associated or not associated with the ectomycorrhizal fungus Hebeloma cylindrosporum, were either inoculated or not inoculated with the phytase-releasing bacteria Bacillus subtilis and the bacterial-feeding nematode Rhabditis sp. After 100 days, the dual inoculation of bacteria and nematodes significantly increased net plant P accumulation. We observed that, on average, mycorrhizal plants accumulated more P in their shoots than non-mycorrhizal plants. However, the highest plant P acquisition efficiency was found when the three soil organisms were present in the P. pinaster rhizosphere. We conclude that, in a highly inorganic P-fixing soil, plant P acquisition from phytate strongly depends on the grazing of phytate-mineralizing bacteria. Our results confirm the importance of the soil microbial loop to improve plant P nutrition from phytate, which should be considered a route to improve the utilization of this source of poorly available P by plants.

Background & Aims Better understanding of belowground interactions in agroforestry systems is crucial for the success of plant co-existence. Beyond root competition, associated arbuscular mycorrhizal (AM) fungi can also be involved in plant to plant interactions. Thus far, the contribution of each agroforestry component (trees, herbaceous vegetation beneath trees -HVbT- and crops) in the establishment and maintenance of AM communities is poorly documented, particularly in temperate areas. This study investigates the spatio-temporal dynamics of both roots and AM fungi in two alley-cropping sites located in southwestern France. Methods Over a one-year period, (i) root length density, production and distribution, (ii) AM activity (root mycorrhization rate and extra-radical hyphal production) and (iii) AM diversity (metabarcoding) were assessed at different distances from tree rows in two agroforestry systems. Results The mycorrhization rate and hyphal production increased at the interface between tree rows and cultivated alleys, showing a positive effect of the presence of a perennial system (tree and HVbT) and of plant diversity. Compared to HVbT, tree roots colonized farther into superficial layers of the cultivated alleys. However, due to higher root densities and well-established AM fungi observed throughout all the year, HVbT appeared to be more relevant in maintaining an active source of AM inoculum for newly developing crop roots in winter. Conclusion The spatial proximity of roots and common AM fungi provides new perspectives in deciphering the significance of arbuscular mycorrhizal communities in crop nutrition and yield in agroforestry systems.

Root exudation contributes to soil carbon allocation and also to microbial C and energy supply, which subsequently impacts soil aggregation around roots. Biologically-driven soil structural formation is an important driver of soil fertility. Plant genetic determinants of exudation and more generally of factors promoting rhizosphere soil aggregation are largely unknown. Here, we characterized rhizosphere aggregation in a panel of 86 pearl millet inbred lines using a ratio of root-adhering soil dry mass per root tissue dry mass (RAS/RT). This ratio showed significant variations between lines, with a roughly 2-fold amplitude between lowest and highest average values. For 9 lines with contrasting aggregation properties, we then compared the bacterial diversity and composition in root-adhering soil. Bacterial α-diversity metrics increased with the "RAS/RT ratio." Regarding taxonomic composition, the Rhizobiales were stimulated in lines showing high aggregation level whereas Bacillales were more abundant in lines with low ratio. 184 strains of cultivable exopolysaccharides-producing bacteria have been isolated from the rhizosphere of some lines, including members from Rhizobiales and Bacillales. However, at this stage, we could not find a correlation between abundance of EPS-producing species in bacterial communities and the ratio RAS/RT. These results illustrated the impact of cereals genetic trait variation on soil physical properties and microbial diversity. This opens the possibility of considering plant breeding to help management of soil carbon content and physical characteristics through carbon rhizodeposition in soil.