Phylogenetic distance-decay patterns are not explained by local community assembly processes in freshwater lake microbial communities.

While water and sediment microbial communities exhibit pronounced spatio-temporal patterns in freshwater lakes, the underlying drivers are yet poorly understood. Here, we evaluated the importance of spatial and temporal variation in abiotic environmental factors for bacterial and microeukaryotic community assembly and distance-decay relationships in water and sediment niches in Hongze Lake. By sampling across the whole lake during both Autumn and Spring sampling time points, we show that only bacterial sediment communities were governed by deterministic community assembly processes due to abiotic environmental drivers. Nevertheless, consistent distance-decay relationships were found with both bacterial and microeukaryotic communities, which were relatively stable with both sampling time points. Our results suggest that spatio-temporal variation in environmental factors was important in explaining mainly bacterial community assembly in the sediment, possibly due lesser disturbance. However, clear distance-decay patterns emerged also when the community assembly was stochastic. Together, these results suggest that abiotic environmental factors do not clearly drive the spatial structuring of lake microbial communities, highlighting the need to understand the role of other potential drivers, such as spatial heterogeneity and biotic species interactions.

Viral and Bacterial Communities Collaborate through Complementary Assembly Processes in Soil to Survive Organochlorine Contamination.

The ecological drivers that direct the assembly of viral and host bacterial communities are largely unknown, even though viral-encoded accessory genes help host bacteria survive in polluted environments. To understand the ecological mechanism(s) of viruses and hosts synergistically surviving under organochlorine pesticide (OCP) stress, we investigated the community assembly processes of viruses and bacteria at the taxon and functional gene levels in clean and OCP-contaminated soils in China using a combination of metagenomics/viromics and bioinformatics approaches. We observed a decreased richness of bacterial taxa and functional genes but an increased richness of viral taxa and auxiliary metabolic genes (AMGs) in OCP-contaminated soils (from 0 to 2,617.6 mg · kg-1). In OCP-contaminated soils, the assembly of bacterial taxa and genes was dominated by a deterministic process, of which the relative significance was 93.0% and 88.7%, respectively. In contrast, the assembly of viral taxa and AMGs was driven by a stochastic process, which contributed 83.1% and 69.2%, respectively. The virus-host prediction analysis, which indicated Siphoviridae was linked to 75.0% of bacterial phyla, and the higher migration rate of viral taxa and AMGs in OCP-contaminated soil suggested that viruses show promise for the dissemination of functional genes among bacterial communities. Taken together, the results of this study indicated that the stochastic assembly processes of viral taxa and AMGs facilitated bacterial resistance to OCP stress in soils. Moreover, our findings provide a novel avenue for understanding the synergistic interactions between viruses and bacteria from the perspective of microbial ecology, highlighting the significance of viruses in mediating bioremediation of contaminated soils. IMPORTANCE The interaction between viral communities and microbial hosts has been studied extensively, and the viral community affects host community metabolic function through AMGs. Microbial community assembly is the process by which species colonize and interact to establish and maintain communities. This is the first study that aimed to understand the assembly process of bacterial and viral communities under OCP stress. The findings of this study provide information about microbial community responses to OCP stress and reveal the collaborative interactions between viral and bacterial communities to resist pollutant stress. Thereby, we highlight the importance of viruses in soil bioremediation from the perspective of community assembly.

Rhizobium nodule diversity and composition are influenced by clover host selection and local growth conditions.

While shaping of plant microbiome composition through 'host filtering' is well documented in legume-rhizobium symbioses, it is less clear to what extent different varieties and genotypes of the same plant species differentially influence symbiont community diversity and composition. Here, we compared how clover host varieties and genotypes affect the structure of Rhizobium populations in root nodules under conventional field and controlled greenhouse conditions. We first grew four Trifolium repens (white clover) F2 crosses and one variety in a conventional field trial and compared differences in root nodule Rhizobium leguminosarum symbiovar trifolii (Rlt) genotype diversity using high-throughput amplicon sequencing of chromosomal housekeeping (rpoB and recA) genes and auxiliary plasmid-borne symbiosis genes (nodA and nodD). We found that Rlt nodule diversities significantly differed between clover crosses, potentially due to host filtering. However, variance in Rlt diversity largely overlapped between crosses and was also explained by the spatial distribution of plants in the field, indicative of the role of local environmental conditions for nodule diversity. To test the effect of host filtering, we conducted a controlled greenhouse trial with a diverse Rlt inoculum and several host genotypes. We found that different clover varieties and genotypes of the same variety selected for significantly different Rlt nodule communities and that the strength of host filtering (deviation from the initial Rhizobium inoculant composition) was positively correlated with the efficiency of symbiosis (rate of plant greenness colouration). Together, our results suggest that selection by host genotype and local growth conditions jointly influence white clover Rlt nodule diversity and community composition.

Global diversity and distribution of prophages are lineage-specific within the Ralstonia solanacearum species complex.

BACKGROUND: Ralstonia solanacearum species complex (RSSC) strains are destructive plant pathogenic bacteria and the causative agents of bacterial wilt disease, infecting over 200 plant species worldwide. In addition to chromosomal genes, their virulence is mediated by mobile genetic elements including integrated DNA of bacteriophages, i.e., prophages, which may carry fitness-associated auxiliary genes or modulate host gene expression. Although experimental studies have characterised several prophages that shape RSSC virulence, the global diversity, distribution, and wider functional gene content of RSSC prophages are unknown. In this study, prophages were identified in a diverse collection of 192 RSSC draft genome assemblies originating from six continents. RESULTS: Prophages were identified bioinformatically and their diversity investigated using genetic distance measures, gene content, GC, and total length. Prophage distributions were characterised using metadata on RSSC strain geographic origin and lineage classification (phylotypes), and their functional gene content was assessed by identifying putative prophage-encoded auxiliary genes. In total, 313 intact prophages were identified, forming ten genetically distinct clusters. These included six prophage clusters with similarity to the Inoviridae, Myoviridae, and Siphoviridae phage families, and four uncharacterised clusters, possibly representing novel, previously undescribed phages. The prophages had broad geographical distributions, being present across multiple continents. However, they were generally host phylogenetic lineage-specific, and overall, prophage diversity was proportional to the genetic diversity of their hosts. The prophages contained many auxiliary genes involved in metabolism and virulence of both phage and bacteria. CONCLUSIONS: Our results show that while RSSC prophages are highly diverse globally, they make lineage-specific contributions to the RSSC accessory genome, which could have resulted from shared coevolutionary history.

Herbicide Selection Promotes Antibiotic Resistance in Soil Microbiomes.

Herbicides are one of the most widely used chemicals in agriculture. While they are known to be harmful to nontarget organisms, the effects of herbicides on the composition and functioning of soil microbial communities remain unclear. Here we show that application of three widely used herbicides-glyphosate, glufosinate, and dicamba-increase the prevalence of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in soil microbiomes without clear changes in the abundance, diversity and composition of bacterial communities. Mechanistically, these results could be explained by a positive selection for more tolerant genotypes that acquired several mutations in previously well-characterized herbicide and ARGs. Moreover, herbicide exposure increased cell membrane permeability and conjugation frequency of multidrug resistance plasmids, promoting ARG movement between bacteria. A similar pattern was found in agricultural soils across 11 provinces in China, where herbicide application, and the levels of glyphosate residues in soils, were associated with increased ARG and MGE abundances relative to herbicide-free control sites. Together, our results show that herbicide application can enrich ARGs and MGEs by changing the genetic composition of soil microbiomes, potentially contributing to the global antimicrobial resistance problem in agricultural environments.

Meta-analysis of diazotrophic signatures across terrestrial ecosystems at the continental scale.

Biological nitrogen fixation performed by diazotrophs forms a cornerstone of Earth's terrestrial ecosystem productivity. However, the composition, diversity and distribution of soil diazotrophs are poorly understood across different soil ecosystems. Furthermore, the biological potential of the key diazotroph species in relation to key environmental parameters is unknown. To address this, we used meta-analysis approach to merge together 39 independent diazotroph amplicon sequencing (nifH gene) datasets consisting of 1988 independent soil samples. We then employed multiple statistical analyses and machine-learning approaches to compare diazotroph community differences and indicator species between terrestrial ecosystems on a global scale. The distribution, composition and structure of diazotroph communities varied across seven different terrestrial ecosystems, with community composition exhibiting an especially clear effect. The Cyanobacteria were the most abundant taxa in crust ecosystems (accounting for ~45% of diazotrophs), while other terrestrial ecosystems were dominated by Proteobacteria, including Alpha-, Beta- and Gamma-Proteobacteria (accounting for ~70% of diazotrophs). Farmland ecosystems harboured the highest and crust ecosystems the lowest alpha and phylogenetic diversities. Azospirillum zeae, Skermanella aerolata and four Bradyrhizobium species were identified as key indicator species of potential diazotroph activity. Overall, diazotroph abundances and distribution were affected by multiple environmental parameters, including soil pH, nitrogen, organic carbon, C:N ratio and annual mean precipitation and temperature. Together, our findings suggest that based on the relative abundance and diversity of nifH marker gene, diazotrophs have adapted to a range of environmental niches globally.

Genetic variation is associated with differences in facilitative and competitive interactions in the Rhizobium leguminosarum species complex.

Competitive and facilitative interactions influence bacterial community composition, diversity and functioning. However, the role of genetic diversity for determining interactions between coexisting strains of the same, or closely related, species remains poorly understood. Here, we investigated the type (facilitative/inhibitory) and potential underlying mechanisms of pairwise interactions between 24 genetically diverse bacterial strains belonging to three genospecies (gsA,C,E) of the Rhizobium leguminosarum species complex. Interactions were determined indirectly, based on secreted compounds in cell-free supernatants, and directly, as growth inhibition in cocultures. We found supernatants mediated both facilitative and inhibitory interactions that varied greatly between strains and genospecies. Overall, gsE strains indirectly suppressed growth of gsA strains, while their own growth was facilitated by other genospecies' supernatants. Similar genospecies-level patterns were observed in direct competition, where gsA showed the highest susceptibility and gsE the highest inhibition capacity. At the genetic level, increased gsA susceptibility was associated with a non-random distribution of quorum sensing and secondary metabolite genes across genospecies. Together, our results suggest that genetic variation is associated with facilitative and competitive interactions, which could be important ecological mechanisms explaining R. leguminosarum diversity.

Airborne and indigenous microbiomes co-drive the rebound of antibiotic resistome during compost storage.

Composting is widely used to reduce the abundance of antibiotic resistance genes (ARGs) in solid waste. While ARG dynamics have been extensively investigated during composting, the fate and abundance of residual ARGs during the storage remain unexplored. Here, we tested experimentally how ARG and mobile genetic element (MGE) abundances change during compost storage using metagenomics, quantitative PCR and direct culturing. We found that 43.8% of ARGs and 39.9% of MGEs quickly recovered already during the first week of storage. This rebound effect was mainly driven by the regrowth of indigenous, antibiotic-resistant bacteria that survived the composting. Bacterial transmission from the surrounding air had a much smaller effect, being most evident as MGE rebound during the later stages of storage. While hyperthermophilic composting was more efficient at reducing the relative abundance of ARGs and MGEs, relatively greater ARG rebound was observed during the storage of hyperthermophilic compost, exceeding the initial levels of untreated sewage sludge. Our study reveals that residual ARGs and MGEs left in the treated compost can quickly rebound during the storage via airborne introduction and regrowth of surviving bacteria, highlighting the need to develop better storage strategies to prevent the rebound of ARGs and MGEs after composting.

The impact of intra-specific diversity in the rhizobia-legume symbiosis.

Rhizobia - nitrogen-fixing, root-nodulating bacteria - play a critical role in both plant ecosystems and sustainable agriculture. Rhizobia form intracellular infections within legumes roots where they produce plant accessible nitrogen from atmospheric nitrogen and thus reduce the reliance on industrial inputs. The rhizobia-legume symbiosis is often treated as a pairwise relationship between single genotypes, both in research and in the production of rhizobial inoculants. However in nature individual plants are infected by a high diversity of rhizobia symbionts. How this diversity affects productivity within the symbiosis is unclear. Here, we use a powerful statistical approach to assess the impact of diversity within the Rhizobium leguminosarum - clover symbiosis using a biodiversity-ecosystem function framework. Statistically, we found no significant impact of rhizobium diversity. However this relationship was weakly positive - rather than negative - indicating that there is no significant cost to increasing inoculant diversity. Productivity was influenced by the identity of the strains within an inoculant; strains with the highest individual performance showed a significant positive contribution within mixed inoculants. Overall, inoculant effectiveness was best predicted by the individual performance of the best inoculant member, and only weakly predicted by the worst performing member. Collectively, our data suggest that the Rhizobium leguminosarum - clover symbiosis displays a weak diversity-function relationship, but that inoculant performance can be improved through the inclusion of high performing strains. Given the wide environmental dependence of rhizobial inoculant quality, multi-strain inoculants could be highly successful as they increase the likelihood of including a strain well adapted to local conditions across different environments.

Functional diversity increases the efficacy of phage combinations.

Phage therapy is a promising alternative to traditional antibiotics for treating bacterial infections. Such phage-based therapeutics typically contain multiple phages, but how the efficacy of phage combinations scales with phage richness, identity and functional traits is unclear. Here, we experimentally tested the efficacy of 827 unique phage combinations ranging in phage richness from one to 12 phages. The efficacy of phage combinations increased with phage richness. However, complementarity between functionally diverse phages allowed efficacy to be maximized at lower levels of phage richness in functionally diverse combinations. These findings suggest that phage functional diversity is the key property of effective phage combinations, enabling the design of simple but effective phage therapies that overcome the practical and regulatory hurdles that limit development of more diverse phage therapy cocktails.

Introduction of probiotic bacterial consortia promotes plant growth via impacts on the resident rhizosphere microbiome.

Plant growth depends on a range of functions provided by their associated rhizosphere microbiome, including nutrient mineralization, hormone co-regulation and pathogen suppression. Improving the ability of plant-associated microbiomes to deliver these functions is thus important for developing robust and sustainable crop production. However, it is yet unclear how beneficial effects of probiotic microbial inoculants can be optimized and how their effects are mediated. Here, we sought to enhance tomato plant growth by targeted introduction of probiotic bacterial consortia consisting of up to eight plant-associated Pseudomonas strains. We found that the effect of probiotic consortium inoculation was richness-dependent: consortia that contained more Pseudomonas strains reached higher densities in the tomato rhizosphere and had clearer beneficial effects on multiple plant growth characteristics. Crucially, these effects were best explained by changes in the resident community diversity, composition and increase in the relative abundance of initially rare taxa, instead of introduction of plant-beneficial traits into the existing community along with probiotic consortia. Together, our results suggest that beneficial effects of microbial introductions can be driven indirectly through effects on the diversity and composition of the resident plant rhizosphere microbiome.

Cross-resistance is modular in bacteria-phage interactions.

Phages shape the structure of natural bacterial communities and can be effective therapeutic agents. Bacterial resistance to phage infection, however, limits the usefulness of phage therapies and could destabilise community structures, especially if individual resistance mutations provide cross-resistance against multiple phages. We currently understand very little about the evolution of cross-resistance in bacteria-phage interactions. Here we show that the network structure of cross-resistance among spontaneous resistance mutants of Pseudomonas aeruginosa evolved against each of 27 phages is highly modular. The cross-resistance network contained both symmetric (reciprocal) and asymmetric (nonreciprocal) cross-resistance, forming two cross-resistance modules defined by high within- but low between-module cross-resistance. Mutations conferring cross-resistance within modules targeted either lipopolysaccharide or type IV pilus biosynthesis, suggesting that the modularity of cross-resistance was structured by distinct phage receptors. In contrast, between-module cross-resistance was provided by mutations affecting the alternative sigma factor, RpoN, which controls many lifestyle-associated functions, including motility, biofilm formation, and quorum sensing. Broader cross-resistance range was not associated with higher fitness costs or weaker resistance against the focal phage used to select resistance. However, mutations in rpoN, providing between-module cross-resistance, were associated with higher fitness costs than mutations associated with within-module cross-resistance, i.e., in genes encoding either lipopolysaccharide or type IV pilus biosynthesis. The observed structure of cross-resistance predicted both the frequency of resistance mutations and the ability of phage combinations to suppress bacterial growth. These findings suggest that the evolution of cross-resistance is common, is likely to play an important role in the dynamic structure of bacteria-phage communities, and could inform the design principles for phage therapy treatments.

Bacterial community richness shifts the balance between volatile organic compound-mediated microbe-pathogen and microbe-plant interactions.

Even though bacteria are important in determining plant growth and health via volatile organic compounds (VOCs), it is unclear how these beneficial effects emerge in multi-species microbiomes. Here we studied this using a model plant-bacteria system, where we manipulated bacterial community richness and composition and determined the subsequent effects on VOC production and VOC-mediated pathogen suppression and plant growth-promotion. We assembled VOC-producing bacterial communities in different richness levels ranging from one to 12 strains using three soil-dwelling bacterial genera (Bacillus, Paenibacillus and Pseudomonas) and investigated how the composition and richness of bacterial community affect the production and functioning of VOCs. We found that VOC production correlated positively with pathogen suppression and plant growth promotion and that all bacteria produced a diverse set of VOCs. However, while pathogen suppression was maximized at intermediate community richness levels when the relative amount and the number of VOCs were the highest, plant growth promotion was maximized at low richness levels and was only affected by the relative amount of plant growth-promoting VOCs. The contrasting effects of richness could be explained by differences in the amount and number of produced VOCs and by opposing effects of community productivity and evenness on pathogen suppression and plant-growth promotion along the richness gradient. Together, these results suggest that the number of interacting bacterial species and the structure of the rhizosphere microbiome drive the balance between VOC-mediated microbe-pathogen and microbe-plant interactions potentially affecting plant disease outcomes in natural and agricultural ecosystems.

Facilitation promotes invasions in plant-associated microbial communities.

While several studies have established a positive correlation between community diversity and invasion resistance, it is less clear how species interactions within resident communities shape this process. Here, we experimentally tested how antagonistic and facilitative pairwise interactions within resident model microbial communities predict invasion by the plant-pathogenic bacterium Ralstonia solanacearum. We found that facilitative resident community interactions promoted and antagonistic interactions suppressed invasions both in the lab and in the tomato plant rhizosphere. Crucially, pairwise interactions reliably explained observed invasion outcomes also in multispecies communities, and mechanistically, this was linked to direct inhibition of the invader by antagonistic communities (antibiosis), and to a lesser degree by resource competition between members of the resident community and the invader. Together, our findings suggest that the type and strength of pairwise interactions can reliably predict the outcome of invasions in more complex multispecies communities.

Resource stoichiometry shapes community invasion resistance via productivity-mediated species identity effects.

Diversity-invasion resistance relationships are often variable and sensitive to environmental conditions such as resource availability. Resource stoichiometry, the relative concentration of different elements in the environment, has been shown to have strong effects on the physiology and interactions between different species. Yet, its role for diversity-invasion resistance relationships is still poorly understood. Here, we explored how the ratio of nitrogen (N) and phosphorus affects the productivity and invasion resistance of constructed microbial communities by a plant pathogenic bacterium, Ralstonia solanacearum. We found that resource stoichiometry and species identity effects affected the invasion resistance of communities. Both high N concentration and resident community diversity constrained invasions, and two resident species, in particular, had strong negative effects on the relative density of the invader and the resident community productivity. While resource stoichiometry did not affect the mean productivity of the resident community, it favoured the growth of two species that strongly constrained invasions turning the slope of productivity-invasion resistance relationship more negative. Together our findings suggest that alterations in resource stoichiometry can change the community resistance to invasions by having disproportionate effects on species growth, potentially explaining changes in microbial community composition under eutrophication.

Mesorhizobium zhangyense sp. nov., isolated from wild Thermopsis lanceolate in northwestern China.

A Gram-stain-negative strain, 23-3-2T, was isolated from a nodule of Thermopsis lanceolate grown in Northwest China. Phylogenetic analysis of 16S rRNA gene sequence showed that the strain was closely related to Mesorhizobium camelthorni CCNWXJ 40-4T and M. alhagi CCNWXJ 12-2T having 98.0 and 97.9% similarities, respectively. Phylogenetic analysis based on the protein-coding genes atpD and glnA showed lower similarity with the same closely related species (94.5 and 89.9%, respectively), which suggest that 23-3-2T strain represents a distinctly delineated genospecies of the genus Mesorhizobium. The 23-3-2T strain grew at 20-37 °C temperature (optimum 28 °C) and 5.0-9.0 pH range (optimum pH 7.0). The cells contained Q-10 as the sole respiratory quinone and 18:1ω7c (24.56%) as the major cellular fatty acid. The DNA relatedness between the strain 23-3-2T and the two reference strains was 39-44%. Based on the phenotypic, chemotaxonomic and phylogenetic properties, strain 23-3-2T represents a novel species of the genus Mesorhizobium, for which the name Mesorhizobium zhangyense sp. nov. is proposed. The type strain is 23-3-2T (= CGMCC 1.15528T = NBRC 112337T). The respective DPD Taxon Number is TA00147.

Hyperthermophilic Composting Accelerates the Removal of Antibiotic Resistance Genes and Mobile Genetic Elements in Sewage Sludge.

Composting is an efficient way to convert organic waste into fertilizers. However, waste materials often contain large amounts of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) that can reduce the efficacy of antibiotic treatments when transmitted to humans. Because conventional composting often fails to remove these compounds, we evaluated if hyperthermophilic composting with elevated temperature is more efficient at removing ARGs and MGEs and explored the underlying mechanisms of ARG removal of the two composting methods. We found that hyperthermophilic composting removed ARGs and MGEs more efficiently than conventional composting (89% and 49%, respectively). Furthermore, the half-lives of ARGs and MGEs were lower in hyperthermophilic compositing compared to conventional composting (67% and 58%, respectively). More-efficient removal of ARGs and MGEs was associated with a higher reduction in bacterial abundance and diversity of potential ARG hosts. Partial least-squares path modeling suggested that reduction of MGEs played a key role in ARG removal in hyperthermophilic composting, while ARG reduction was mainly driven by changes in bacterial community composition under conventional composting. Together these results suggest that hyperthermophilic composting can significantly enhance the removal of ARGs and MGEs and that the mechanisms of ARG and MGE removal can depend on composting temperature.

Nocardioides astragali sp. nov., isolated from a nodule of wild Astragalus chrysopterus in northwestern China.

A Gram-positive, non-motile, rod-shaped bacterial strain, designated HH06T, was isolated from a nodule of Astragalus chrysopterus in northwestern China. Phylogenetic analysis of the 16S rRNA gene sequence showed that the strain is closely related to Nocardioides alpinus Cr7-14T and Nocardioides furvisabuli DSM 18445T with 98.5 and 98.1% similiarity, respectively. Growth was observed at 4-28 °C in R2A medium (optimum at 25 °C), at 10-30 °C in YMA and LB medium (optimum in both at 28 °C) and at pH 5.0-10.0 in R2A medium (optimum at pH 7.0-8.0). The cell wall peptidoglycan was found to contain LL-diaminopimelic acid as the principal diamino acid and MK-8(H4) was identified as the predominant menaquinone. The major polar lipids were identified as phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, two unidentified glycolipids and two unidentified polar lipids. The major fatty acids were identified as iso-C16:0 (32.8%) and C18:1 ω9c (15.1%). The DNA G+C content of strain HH06T was determined to be 71.4 mol%. Based on phenotypic, chemotaxonomic, phylogenetic properties and DNA-DNA relatedness, it is concluded that strain HH06T represents a novel species of the genus Nocardioides, for which the name Nocardioides astragali sp. nov. is proposed. The type strain is HH06T (= CGMCC 4.7327T = NBRC 112322T).

Resource availability modulates biodiversity-invasion relationships by altering competitive interactions.

Community diversity affects the survival of newly introduced species via resource competition. Competitive interactions can be modulated by resource availability and we hypothesized that this may alter biodiversity-invasion relationships. To study this, we assessed the growth of a bacterial invader, Ralstonia solanacearum, when introduced into communities comprised of one to five closely related resident species under different resource concentrations. The invader growth was then examined as a function of resident community richness, species composition and resource availability. We found that the relative density of the invader was reduced by increasing resident community richness and resource availability. Mechanistically, this could be explained by changes in the competitive interactions between the resident species and the invader along the resource availability gradient. At low resource availability, resident species with a high catabolic similarity with the invader efficiently reduced the invader relative density, while at high resource availability, fast-growing resident species became more important for the invader suppression. These results indicate that the relative importance of different resident community species can change dynamically along to resource availability gradient. Diverse communities could be thus more robust to invasions by providing a set of significant species that can take suppressive roles across different environments.