Soil depth and geographic distance modulate bacterial ß-diversity in deep soil profiles throughout the U.S. Corn Belt.

Understanding how microbial communities are shaped across spatial dimensions is of fundamental importance in microbial ecology. However, most studies on soil biogeography have focused on the topsoil microbiome, while the factors driving the subsoil microbiome distribution are largely unknown. Here we used 16S rRNA amplicon sequencing to analyse the factors underlying the bacterial ß-diversity along vertical (0-240 cm of soil depth) and horizontal spatial dimensions (~500,000 km2 ) in the U.S. Corn Belt. With these data we tested whether the horizontal or vertical spatial variation had stronger impacts on the taxonomic (Bray-Curtis) and phylogenetic (weighted Unifrac) ß-diversity. Additionally, we assessed whether the distance-decay (horizontal dimension) was greater in the topsoil (0-30 cm) or subsoil (in each 30 cm layer from 30-240 cm) using Mantel tests. The influence of geographic distance versus edaphic variables on the bacterial communities from the different soil layers was also compared. Results indicated that the phylogenetic ß-diversity was impacted more by soil depth, while the taxonomic ß-diversity changed more between geographic locations. The distance-decay was lower in the topsoil than in all subsoil layers analysed. Moreover, some subsoil layers were influenced more by geographic distance than any edaphic variable, including pH. Although different factors affected the topsoil and subsoil biogeography, niche-based models explained the community assembly of all soil layers. This comprehensive study contributed to elucidating important aspects of soil bacterial biogeography including the major impact of soil depth on the phylogenetic ß-diversity, and the greater influence of geographic distance on subsoil than on topsoil bacterial communities in agroecosystems.

Corrigendum: Microbiome variation across populations of desert halophyte Zygophyllum qatarensis.

[This corrects the article DOI: 10.3389/fpls.2022.841217.].

Root exudate concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) affect maize rhizobacterial communities at specific developmental stages.

Root exudates shape the rhizosphere microbiome, but little is known about the specific compounds in root exudates that are important. Here, we investigated the impacts of the plant-synthesized phytohormones indole-3-acetic acid (IAA) and abscisic acid (ABA) exuded by roots on the maize rhizobacterial communities. To identify maize genotypes that differed in the root exudate concentrations of IAA and ABA, we screened hundreds of inbred lines using a semi-hydroponic system. Twelve genotypes with variable exudate concentrations of IAA and ABA were selected for a replicated field experiment. Bulk soil, rhizosphere, and root endosphere samples were collected at two vegetative and one reproductive maize developmental stage. IAA and ABA concentrations in rhizosphere samples were quantified by liquid chromatography-mass spectrometry. The bacterial communities were analyzed by V4 16S rRNA amplicon sequencing. Results indicated that IAA and ABA concentrations in root exudates significantly affected the rhizobacterial communities at specific developmental stages. ABA impacted the rhizosphere bacterial communities at later developmental stages, whereas IAA affected the rhizobacterial communities at the vegetative stages. This study contributed to our knowledge about the influence that specific root exudate compounds have on the rhizobiome composition, showing that the phytohormones IAA and ABA exuded by roots have a role in the plant-microbiome interactions.

Sugars and Jasmonic Acid Concentration in Root Exudates Affect Maize Rhizosphere Bacterial Communities.

Root exudates contribute to shaping the root-associated microbiomes, but it is unclear which of the many exudate compounds are important in this process. Here, we focused on understanding the influence of sugars and jasmonic acid (JA) concentrations in maize root exudates on the rhizobacterial communities. Twelve maize genotypes were identified with variable concentrations of sugars and JA based on a screening of 240 maize genotypes grown in a semihydroponic system. These twelve maize genotypes were grown in a replicated field experiment in which samples were collected at three maize developmental stages. The 16S rRNA gene (V4 region) was amplified and sequenced. Sugars and JA concentrations from rhizosphere soils were also quantified. The results indicated that the maize genotypic variability in sugars and JA concentration in root exudates, measured in the semihydroponic system, significantly affected the rhizosphere bacterial community composition at multiple stages plant development. In contrast, the root endosphere and bulk soil bacterial communities were only affected at specific growth stages. Sugars and JA concentration as quantified in rhizosphere soil samples confirmed that these two compounds affected the rhizobacterial communities at all developmental stages analyzed. The effects of specific sugars on the composition of the rhizobacterial communities were also measured, with larger effects of sucrose at earlier developmental stages and trehalose at later developmental stages. Our results indicate that JA and sugars are important root exudate compounds that influence the composition of the maize rhizobacterial communities. IMPORTANCE Roots secrete exudates that are important in interactions with soil microbes that promote plant growth and health. However, the exact chemical compounds in root exudates that participate in these interactions are not fully known. Here, we investigated whether sugars and the phytohormone jasmonic acid influence the composition of the rhizobacterial communities of maize, which is an important crop for food, feed, and energy. Our results revealed that both compounds contribute to the assemblage of rhizobacterial communities at different maize developmental stages. Knowledge about the specific compounds in root exudates that contribute to shape the rhizobiome will be important for future strategies to develop sustainable agricultural practices that are less dependent on agrochemicals.

Natural variation in root exudation of GABA and DIMBOA impacts the maize root endosphere and rhizosphere microbiomes.

Root exudates are important for shaping root-associated microbiomes. However, studies on a wider range of metabolites in exudates are required for a comprehensive understanding about their influence on microbial communities. We identified maize inbred lines that differ in exudate concentrations of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) and γ-aminobutyric acid (GABA) using a semi-hydroponic system. These lines were grown in the field to determine the changes in microbial diversity and gene expression due to varying concentrations of DIMBOA and GABA in exudates using 16S rRNA amplicon sequencing and metatranscriptomics. Results showed individual and interaction effects of DIMBOA and GABA on the rhizosphere and root endosphere ß-diversity, most strongly at the V10 growth stage. The main bacterial families affected by both compounds were Ktedonobacteraceae and Xanthomonadaceae. Higher concentrations of DIMBOA in exudates affected the rhizosphere metatranscriptome, enriching for metabolic pathways associated with plant disease. This study validated the use of natural variation within plant species as a powerful approach for understanding the role of root exudates on microbiome selection. We also showed that a semi-hydroponic system can be used to identify maize genotypes that differ in GABA and DIMBOA exudate concentrations under field conditions. The impact of GABA exudation on root-associated microbiomes is shown for the first time.

Microbiome Variation Across Populations of Desert Halophyte Zygophyllum qatarensis.

Microbial symbionts play a significant role in plant health and stress tolerance. However, few studies exist that address rare species of core-microbiome function during abiotic stress. In the current study, we compared the microbiome composition of succulent dwarf shrub halophyte Zygophyllum qatarensis Hadidi across desert populations. The results showed that rhizospheric and endosphere microbiome greatly varied due to soil texture (sandy and gravel). No specific bacterial amplicon sequence variants were observed in the core-microbiome of bulk soil and rhizosphere, however, bacterial genus Alcaligenes and fungal genus Acidea were abundantly distributed across root and shoot endospheres. We also analyzed major nutrients such as silicon (Si), magnesium, and calcium across different soil textures and Z. qatarensis populations. The results showed that the rhizosphere and root parts had significantly higher Si content than the bulk soil and shoot parts. The microbiome variation can be attributed to markedly higher Si - suggesting that selective microbes are contributing to the translocation of soluble Si to root. In conclusion, low core-microbiome species abundance might be due to the harsh growing conditions in the desert - making Z. qatarensis highly selective to associate with microbial communities. Utilizing rare microbial players from plant microbiomes may be vital for increasing crop stress tolerance and productivity during stresses.

Sweet Sorghum Genotypes Tolerant and Sensitive to Nitrogen Stress Select Distinct Root Endosphere and Rhizosphere Bacterial Communities.

The belowground microbiomes have many beneficial functions that assist plant growth, including nutrient cycling, acquisition and transport, as well as alleviation of stresses caused by nutrient limitations such as nitrogen (N). Here we analyzed the root endosphere, rhizosphere and soil bacterial communities of seven sweet sorghum genotypes differing in sensitivity to N-stress. Sorghum genotypes were grown in fields with no (low-N) or sufficient (high-N) N. The dry shoot weight ratio (low-N/high-N) was used to determine N-stress sensitivity. Our hypothesis was that genotypes tolerant and sensitive to N-stress select distinct bacterial communities. The endosphere and rhizosphere bacterial community structure were significantly different between the N-stress sensitive and tolerant genotypes in the high-N field, but not in the low-N field. However, significant changes in the relative abundance of specific bacterial taxa were observed in both fields. Streptomyces, a bacterial genus known to alleviate plant abiotic stresses, was enriched in the endosphere and rhizosphere of the tolerant genotypes in the low-N field. Our study indicates that sweet sorghum genotypes tolerant to N-stress select taxa that can potentially mitigate the N-stress, suggesting that the interactions between N-stress tolerant lines and the root-associated microbiome might be vital for coping with N-stress.

Alkaline soil pH affects bulk soil, rhizosphere and root endosphere microbiomes of plants growing in a Sandhills ecosystem.

Soil pH is a major factor shaping bulk soil microbial communities. However, it is unclear whether the belowground microbial habitats shaped by plants (e.g. rhizosphere and root endosphere) are also affected by soil pH. We investigated this question by comparing the microbial communities associated with plants growing in neutral and strongly alkaline soils in the Sandhills, which is the largest sand dune complex in the northern hemisphere. Bulk soil, rhizosphere and root endosphere DNA were extracted from multiple plant species and analyzed using 16S rRNA amplicon sequencing. Results showed that rhizosphere, root endosphere and bulk soil microbiomes were different in the contrasting soil pH ranges. The strongest impact of plant species on the belowground microbiomes was in alkaline soils, suggesting a greater selective effect under alkali stress. Evaluation of soil chemical components showed that in addition to soil pH, cation exchange capacity also had a strong impact on shaping bulk soil microbial communities. This study extends our knowledge regarding the importance of pH to microbial ecology showing that root endosphere and rhizosphere microbial communities were also influenced by this soil component, and highlights the important role that plants play particularly in shaping the belowground microbiomes in alkaline soils.

The Effects of Soil Depth on the Structure of Microbial Communities in Agricultural Soils in Iowa, USA.

This study investigated the differences in microbial community abundance, composition and diversity throughout the depth profiles in soils collected from corn and soybean fields in lowa, USA using 16S rRNA amplicon sequencing. The results revealed decreased richness and diversity in microbial communities at increasing soil depth. Soil microbial community composition differed due to crop type only in the top 60 cm and due to location only in the top 90 cm. While the relative abundance of most phyla decreased in deep soils, the relative abundance of the phylum Proteobacteria increased and dominated agricultural soils below the depth of 90 cm. Although soil depth was the most important factor shaping microbial communities, edaphic factors including soil organic matter, soil bulk density and the length of time that deep soils were saturated with water were all significant factors explaining the variation in soil microbial community composition. Soil organic matter showed the highest correlation with the exponential decrease in bacterial abundance with depth. A greater understanding of how soil depth influences the diversity and composition of soil microbial communities is vital for guiding sampling approaches in agricultural soils where plant roots extend beyond the upper soil profile. In the long term a greater knowledge of the influence of depth on microbial communities should contribute to new strategies that enhance the sustainability of soil which is a precious resource for food security.IMPORTANCE Determining how microbial properties change across different soils and within the soil depth profile, will be potentially beneficial to understanding the long-term processes that are involved in the health of agricultural ecosystems. Most literature on soil microbes has been restricted to the easily accessible surface soils. However, deep soils are important in soil formation, carbon sequestration, and in providing nutrients and water for plants. In the most productive agricultural systems in the USA where soybean and corn are grown, crop plant roots extend into the deeper regions of soils (> 100 cm), but little is known about the taxonomic diversity or the factors that shape deep soil microbial communities. The findings reported here highlight the importance of soil depth in shaping microbial communities, provide new information about edaphic factors that influence the deep soil communities and reveal more detailed information on taxa that exist in deep agricultural soils.

Genomic and metabolic differences between Pseudomonas putida populations inhabiting sugarcane rhizosphere or bulk soil.

Pseudomonas putida is one of 13 major groups of Pseudomonas spp. and contains numerous species occupying diverse niches and performing many functions such as plant growth promotion and bioremediation. Here we compared a set of 19 P. putida isolates obtained from sugarcane rhizosphere or bulk soil using a population genomics approach aiming to assess genomic and metabolic differences between populations from these habitats. Phylogenomics placed rhizosphere versus bulk soil strains in separate clades clustering with different type strains of the P. putida group. Multivariate analyses indicated that the rhizosphere and bulk soil isolates form distinct populations. Comparative genomics identified several genetic functions (GO-terms) significantly different between populations, including some exclusively present in the rhizosphere or bulk soil strains, such as D-galactonic acid catabolism and cellulose biosynthesis, respectively. The metabolic profiles of rhizosphere and bulk soil populations analyzed by Biolog Ecoplates also differ significantly, most notably by the higher oxidation of D-galactonic/D-galacturonic acid by the rhizosphere population. Accordingly, D-galactonate catabolism operon (dgo) was present in all rhizosphere isolates and absent in the bulk soil population. This study showed that sugarcane rhizosphere and bulk soil harbor different populations of P. putida and identified genes and functions potentially associated with their soil niches.

Evolution of the U.S. Biological Select Agent Rathayibacter toxicus.

Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species.IMPORTANCERathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicusR. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.

Genome variations between rhizosphere and bulk soil ecotypes of a Pseudomonas koreensis population.

Bulk soil and rhizosphere are soil compartments selecting different microbial communities. However, it is unknown whether this selection also can change the genome content of specific bacterial taxa, splitting a population in distinct ecotypes. To answer this question we compared the genome sequences of 53 isolates obtained from sugarcane rhizosphere (28) and bulk soil (25). These isolates were previously classified in the Pseudomonas koreensis subgroup of the P. fluorescens complex. Phylogenomics showed a trend of separation between bulk soil and rhizosphere isolates. Discriminant analysis of principal components (DAPC) identified differences in the accessory genome of rhizosphere and bulk soil sub-populations. We found significant changes in gene frequencies distinguishing rhizosphere from bulk soil ecotypes, for example, enrichment of phosphatases and xylose utilization (xut) genes, respectively. Phenotypic assays and deletion of xutA gene indicated that accumulation of xut genes in the bulk soil sub-population provided a higher growth capacity in a d-xylose medium, supporting the corresponding genomic differences. Despite the clear differences distinguishing the two ecotypes, all 53 isolates were classified in a single 16S rRNA gene OTU. Collectively, our results revealed that the gene pool and ecological behavior of a bacterial population can be different for ecotypes living in neighbouring soil habitats.

Tropical soils are a reservoir for fluorescent Pseudomonas spp. biodiversity.

Fluorescent Pseudomonas spp. are widely studied for their beneficial activities to plants. To explore the genetic diversity of Pseudomonas spp. in tropical regions, we collected 76 isolates from a Brazilian soil. Genomes were sequenced and compared to known strains, mostly collected from temperate regions. Phylogenetic analyses classified the isolates in the P. fluorescens (57) and P. putida (19) groups. Among the isolates in the P. fluorescens group, most (37) were classified in the P. koreensis subgroup and two in the P. jessenii subgroup. The remaining 18 isolates fell into two phylogenetic subclades distinct from currently recognized P. fluorescens subgroups, and probably represent new subgroups. Consistent with their phylogenetic distance from described subgroups, the genome sequences of strains in these subclades are asyntenous to the genome sequences of members of their neighbour subgroups. The tropical isolates have several functional genes also present in known fluorescent Pseudomonas spp. strains. However, members of the new subclades share exclusive genes not detected in other subgroups, pointing to the potential for novel functions. Additionally, we identified 12 potential new species among the 76 isolates from the tropical soil. The unexplored diversity found in the tropical soil is possibly related to biogeographical patterns.

Exploring the sheep rumen microbiome for carbohydrate-active enzymes.

The rumen is a complex ecosystem enriched for microorganisms able to degrade biomass during the animal's digestion process. The recovery of new enzymes from naturally evolved biomass-degrading microbial communities is a promising strategy to overcome the inefficient enzymatic plant destruction in industrial production of biofuels. In this context, this study aimed to describe the bacterial composition and functions in the sheep rumen microbiome, focusing on carbohydrate-active enzymes (CAE). Here, we used phylogenetic profiling analysis (inventory of 16S rRNA genes) combined with metagenomics to access the rumen microbiome of four sheep and explore its potential to identify fibrolytic enzymes. The bacterial community was dominated by Bacteroidetes and Firmicutes, followed by Proteobacteria. As observed for other ruminants, Prevotella was the dominant genus in the microbiome, comprising more than 30 % of the total bacterial community. Multivariate analysis of the phylogenetic profiling data and chemical parameters showed a positive correlation between the abundance of Prevotellaceae (Bacteroidetes phylum) and organic matter degradability. A negative correlation was observed between Succinivibrionaceae (Proteobacteria phylum) and methane production. An average of 2 % of the shotgun metagenomic reads was assigned to putative CAE when considering nine protein databases. In addition, assembled contigs allowed recognition of 67 putative partial CAE (NCBI-Refseq) representing 12 glycosyl hydrolase families (Pfam database). Overall, we identified a total of 28 lignocellulases, 22 amylases and 9 other putative CAE, showing the sheep rumen microbiome as a promising source of new fibrolytic enzymes.