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Abstract Soil microbial growth, a vital biogeochemical process, governs both the accrual and loss of soil carbon. Here, we investigate the biogeography of soil microbiome potential growth rates and show that microbiomes in resource-rich (high organic matter and nutrients) and acid-neutral soils from cold and humid regions exhibit high potential growth. Conversely, in resource-poor, dry, hot, and hypersaline soils, soil microbiomes display lower potential growth rates, suggesting trade-offs between growth and resource acquisition or stress tolerance. In addition, the potential growth rates of soil microbiomes positively correlates with genome size and the number of ribosomal RNA operons but negatively correlates with optimum temperature, biomass carbon-to-phosphorus and nitrogen-to-phosphorus ratios. The spatial variation of microbial potential growth rates aligns with several macroecological theories. These findings not only enhance our understanding of microbial adaptation to diverse environments but also aid in realistically parameterizing microbial physiology in soil carbon cycling models.

Abstract Understanding the large-scale pattern of soil microbial carbon use efficiency (CUE) and its temperature sensitivity (CUET) is critical for understanding soil carbon–climate feedback. We used the 18O-H2O tracer method to quantify CUE and CUET along a north-south forest transect. Climate was the primary factor that affected CUE and CUET, predominantly through direct pathways, then by altering soil properties, carbon fractions, microbial structure and functions. Negative CUET (CUE decreases with measuring temperature) in cold forests (mean annual temperature lower than 10 °C) and positive CUET (CUE increases with measuring temperature) in warm forests (mean annual temperature greater than 10 °C) suggest that microbial CUE optimally operates at their adapted temperature. Overall, the plasticity of microbial CUE and its temperature sensitivity alter the feedback of soil carbon to climate warming; that is, a climate-adaptive microbial community has the capacity to reduce carbon loss from soil matrices under corresponding favorable climate conditions.

Abstract Soil organic carbon (SOC) persistence is predominantly governed by mineral protection, consequently, soil mineral-associated (MAOC) and particulate organic carbon (POC) turnovers have different impacts on the vulnerability of SOC to climate change. Here, we generate the global MAOC and POC maps using 8341 observations and then infer the turnover times of MAOC and POC by a data-model integration approach. Global MAOC and POC storages are $${975}_{964}^{987}$$ 975 964 987 Pg C (mean with 5% and 95% quantiles) and $${330}_{323}^{337}$$ 330 323 337 Pg C, while global mean MAOC and POC turnover times are $${129}_{45}^{383}$$ 129 45 383 yr and $${23}_{5}^{82}$$ 23 5 82 yr in the top meter, respectively. Climate warming-induced acceleration of MAOC and POC decomposition is greater in subsoil than that in topsoil. Overall, the global atlas of MAOC and POC turnover, together with the global distributions of MAOC and POC stocks, provide a benchmark for Earth system models to diagnose SOC-climate change feedback.

Abstract Agriculture contributes to a decline in local species diversity and to above- and below-ground biotic homogenization. Here, we conduct a continental survey using 1185 soil samples and compare microbial communities from natural ecosystems (forest, grassland, and wetland) with converted agricultural land. We combine our continental survey results with a global meta-analysis of available sequencing data that cover more than 2400 samples across six continents. Our combined results demonstrate that land conversion to agricultural land results in taxonomic and functional homogenization of soil bacteria, mainly driven by the increase in the geographic ranges of taxa in croplands. We find that 20% of phylotypes are decreased and 23% are increased by land conversion, with croplands enriched in Chloroflexi, Gemmatimonadota, Planctomycetota, Myxcoccota and Latescibacterota. Although there is no significant difference in functional composition between natural ecosystems and agricultural land, functional genes involved in nitrogen fixation, phosphorus mineralization and transportation are depleted in cropland. Our results provide a global insight into the consequences of land-use change on soil microbial taxonomic and functional diversity.

Abstract Background The severity and frequency of drought are expected to increase substantially in the coming century and dramatically reduce crop yields. Manipulation of rhizosphere microbiomes is an emerging strategy for mitigating drought stress in agroecosystems. However, little is known about the mechanisms underlying how drought-resistant plant recruitment of specific rhizosphere fungi enhances drought adaptation of drought-sensitive wheats. Here, we investigated microbial community assembly features and functional profiles of rhizosphere microbiomes related to drought-resistant and drought-sensitive wheats by amplicon and shotgun metagenome sequencing techniques. We then established evident linkages between root morphology traits and putative keystone taxa based on microbial inoculation experiments. Furthermore, root RNA sequencing and RT-qPCR were employed to explore the mechanisms how rhizosphere microbes modify plant response traits to drought stresses. Results Our results indicated that host plant signature, plant niche compartment, and planting site jointly contribute to the variation of soil microbiome assembly and functional adaptation, with a relatively greater effect of host plant signature observed for the rhizosphere fungi community. Importantly, drought-resistant wheat (Yunhan 618) possessed more diverse bacterial and fungal taxa than that of the drought-sensitive wheat (Chinese Spring), particularly for specific fungal species. In terms of microbial interkingdom association networks, the drought-resistant variety possessed more complex microbial networks. Metagenomics analyses further suggested that the enriched rhizosphere microbiomes belonging to the drought-resistant cultivar had a higher investment in energy metabolism, particularly in carbon cycling, that shaped their distinctive drought tolerance via the mediation of drought-induced feedback functional pathways. Furthermore, we observed that host plant signature drives the differentiation in the ecological role of the cultivable fungal species Mortierella alpine (M. alpina) and Epicoccum nigrum (E. nigrum). The successful colonization of M. alpina on the root surface enhanced the resistance of wheats in response to drought stresses via activation of drought-responsive genes (e.g., CIPK9 and PP2C30). Notably, we found that lateral roots and root hairs were significantly suppressed by co-colonization of a drought-enriched fungus (M. alpina) and a drought-depleted fungus (E. nigrum). Conclusions Collectively, our findings revealed host genotypes profoundly influence rhizosphere microbiome assembly and functional adaptation, as well as it provides evidence that drought-resistant plant recruitment of specific rhizosphere fungi enhances drought tolerance of drought-sensitive wheats. These findings significantly underpin our understanding of the complex feedbacks between plants and microbes during drought, and lay a foundation for steering “beneficial keystone biome” to develop more resilient and productive crops under climate change. Video Abstract

Abstract Background The growth of alfalfa (Medicago sativa L.) is significantly hampered by drought and nutrient deficiencies. The identification of root architectural and anatomical characteristics holds paramount importance for the development of alfalfa genotypes with enhanced adaptation to adverse environmental conditions. In this study, we employed a visual rhizobox system to investigate the variability in root system architecture (including root depth, root length, root tips number, etc.), anatomical features (such as cortical traits, total stele area, number and area of vessel, etc.), as well as nitrogen and phosphorus uptake across 53 alfalfa genotypes during the seedling stage. Results Out of the 42 traits measured, 21 root traits, along with nitrogen (N) and phosphorus (P) uptake, displayed higher coefficients of variation (CVs ≥ 0.25) among the tested genotypes. Local root morphological and anatomical traits exhibited more significant variation than global root traits. Twenty-three traits with CVs ≥ 0.25 constituted to six principal components (eigenvalues > 1), collectively accounting for 88.0% of the overall genotypic variation. Traits such as total root length, number of root tips, maximal root depth, and others exhibited positive correlations with shoot dry mass and root dry mass. Additionally, total stele area and xylem vessel area showed positive correlations with N and P uptake. Conclusions These root traits, which have demonstrated associations with biomass and nutrient uptake, may be considered for the breeding of alfalfa genotypes that possess efficient resource absorption and increased adaptability to abiotic stress, following validation during the entire growth period in the field.

An increasing number of anthropogenic pressures can have negative effects on biodiversity and ecosystem functioning. However, our understanding of how soil microbial communities and functions in response to multiple global change factors (GCFs) is still incomplete, particularly in less frequently disturbed subsoils. In this study, we examined the impact of different levels of GCFs (0–9) on soil functions and bacterial communities in both topsoils (0–20 cm) and subsoils (20–40 cm) of an agricultural ecosystem, and characterized the intrinsic factors influencing community resistance based on microbial life history strategy. Our experimental results showed a decline in soil multifunctionality, bacterial diversity, and community resistance as the number of GCFs increased, with a more drastic reduction in community resistance of subsoils. Specifically, we observed a significantly positive relationship between the oligotroph/copiotroph ratio and community resistance in subsoils, which was also verified by the negative correlation between 16S rRNA operon (rrn) copy number and community resistance. Structural equation modeling further revealed the direct effects of community resistance in promoting the ecosystem functioning, regardless of top- and subsoils. Therefore, these results suggested that subsoils may recruit more oligotrophic microbes to enhance their originally weaker community resistance under multiple GCFs, which was essential for maintaining sustainable agroecological functions and services. Overall, our study represents a significant advance in linking microbial life history strategy to the resistance of belowground microbial community and functionality.

Abstract Background Allelopathy is closely associated with rhizosphere biological processes, and rhizosphere microbial communities are essential for plant development. However, our understanding of rhizobacterial communities under influence of allelochemicals in licorice remains limited. In the present study, the responses and effects of rhizobacterial communities on licorice allelopathy were investigated using a combination of multi-omics sequencing and pot experiments, under allelochemical addition and rhizobacterial inoculation treatments. Results Here, we demonstrated that exogenous glycyrrhizin inhibits licorice development, and reshapes and enriches specific rhizobacteria and corresponding functions related to glycyrrhizin degradation. Moreover, the Novosphingobium genus accounted for a relatively high proportion of the enriched taxa and appeared in metagenomic assembly genomes. We further characterized the different capacities of single and synthetic inoculants to degrade glycyrrhizin and elucidated their distinct potency for alleviating licorice allelopathy. Notably, the single replenished N (Novosphingobium resinovorum) inoculant had the greatest allelopathy alleviation effects in licorice seedlings. Conclusions Altogether, the findings highlight that exogenous glycyrrhizin simulates the allelopathic autotoxicity effects of licorice, and indigenous single rhizobacteria had greater effects than synthetic inoculants in protecting licorice growth from allelopathy. The results of the present study enhance our understanding of rhizobacterial community dynamics during licorice allelopathy, with potential implications for resolving continuous cropping obstacle in medicinal plant agriculture using rhizobacterial biofertilizers. Video Abstract

ABSTRACT As the most common approach for the restoration of degraded grasslands, the use of grass-legume mixtures has long been recognized for its role in increasing aboveground biomass and resisting grassland degradation. However, whether the legumes in these mixtures can help neighboring plants resist the decline in biomass caused by the loss of soil microbial diversity remains a question worthy of investigation. To address this, we employed a dilution method to create a gradient of decreasing microbial diversity in soil and utilized full-factorial combinations of legumes and two grasses to investigate the crucial role of legumes in the mixture. The results showed that compared to monoculture, the mixture of Medicago sativa L. and Elymus dahuricus Turcz. enhanced the biomass of grass species under conditions of soil microbial diversity loss. We then discovered that a significantly enriched Pseudomonas (ASV53), in the grass-legume mixtures under conditions of microbial diversity loss, was positively correlated with plant biomass and nitrogen-fixing (nifH) gene abundance, implying that it could be a keystone species. In addition, the grass-legume mixture increased the deterministic processes of microbial community enrichment in the root zone soil by enhancing the process of homogeneous selection. Functional predictions revealed that grass-legume mixtures increased the potential abundance of N-related and phototrophy-related microbial communities in the root zone soil. This study provides an important insight into the mechanism underlying the role of legumes in increasing and maintaining grass biomass despite soil microbial diversity loss.IMPORTANCEGrass-legume mixtures are a common practice for establishing artificial grasslands, directly or indirectly contributing to the improvement of yield. In addition, this method helps maintain soil and plant health by reducing the use of chemical fertilizers. The impact of grass-legume mixtures on yield and its underlying microbial mechanisms have been a focus of scientific investigation. However, the benefits of mixtures in the context of soil microbial diversity loss remain a problem worthy of exploration. In this study, we examined different aboveground and belowground diversity combinations to elucidate the mechanisms by which grass-legume mixtures help maintain stable yields in the face of diversity loss. We identified the significantly enriched Pseudomonas genus microbial ASV53, which was gathered through homogeneous selection and served as a keystone in the co-occurrence network. ASV53 showed a strong positive correlation with biomass and the abundance of nitrogen-fixing genes. These findings provide a new theoretical foundation for utilizing grass-legume mixtures to enhance grass yields and address the challenges posed by diversity loss.

Abstract Background The rhizosphere microbiome, which is shaped by host genotypes, root exudates, and plant domestication, is crucial for sustaining agricultural plant growth. Despite its importance, how plant domestication builds up specific rhizosphere microbiomes and metabolic functions, as well as the importance of these affected rhizobiomes and relevant root exudates in maintaining plant growth, is not well understood. Here, we firstly investigated the rhizosphere bacterial and fungal communities of domestication and wild accessions of tetraploid wheat using amplicon sequencing (16S and ITS) after 9 years of domestication process at the main production sites in China. We then explored the ecological roles of root exudation in shaping rhizosphere microbiome functions by integrating metagenomics and metabolic genomics approaches. Furthermore, we established evident linkages between root morphology traits and keystone taxa based on microbial culture and plant inoculation experiments. Results Our results suggested that plant rhizosphere microbiomes were co-shaped by both host genotypes and domestication status. The wheat genomes contributed more variation in the microbial diversity and composition of rhizosphere bacterial communities than fungal communities, whereas plant domestication status exerted much stronger influences on the fungal communities. In terms of microbial interkingdom association networks, domestication destabilized microbial network and depleted the abundance of keystone fungal taxa. Moreover, we found that domestication shifted the rhizosphere microbiome from slow growing and fungi dominated to fast growing and bacteria dominated, thereby resulting in a shift from fungi-dominated membership with enrichment of carbon fixation genes to bacteria-dominated membership with enrichment of carbon degradation genes. Metagenomics analyses further indicated that wild cultivars of wheat possess higher microbial function diversity than domesticated cultivars. Notably, we found that wild cultivar is able to harness rhizosphere microorganism carrying N transformation (i.e., nitrification, denitrification) and P mineralization pathway, whereas rhizobiomes carrying inorganic N fixation, organic N ammonification, and inorganic P solubilization genes are recruited by the releasing of root exudates from domesticated wheat. More importantly, our metabolite-wide association study indicated that the contrasting functional roles of root exudates and the harnessed keystone microbial taxa with different nutrient acquisition strategies jointly determined the aboveground plant phenotypes. Furthermore, we observed that although domesticated and wild wheats recruited distinct microbial taxa and relevant functions, domestication-induced recruitment of keystone taxa led to a consistent growth regulation of root regardless of wheat domestication status. Conclusions Our results indicate that plant domestication profoundly influences rhizosphere microbiome assembly and metabolic functions and provide evidence that host plants are able to harness a differentiated ecological role of root-associated keystone microbiomes through the release of root exudates to sustain belowground multi-nutrient cycles and plant growth. These findings provide valuable insights into the mechanisms underlying plant-microbiome interactions and how to harness the rhizosphere microbiome for crop improvement in sustainable agriculture. Video Abstract

Abstract Predicting the distribution patterns of soil microbial communities requires consideration of more environmental drivers. The effects of soil micronutrients on composition of microbial communities are largely unknown despite micronutrients closely relating to soil fertility and plant communities. Here we used data from 228 agricultural fields to identify the importance of micronutrients (iron, zinc, copper and manganese) in shaping structure of soil microbial communities (bacteria, fungi and protist) along latitudinal gradient over 3400 km, across diverse edaphic conditions and climatic gradients. We found that micronutrients explained more variations in the structure of microbial communities than macronutrients in maize soils. Moreover, micronutrients, particularly iron and copper, explained a unique percentage of the variation in structure of microbial communities in maize soils even after controlling for climate, soil physicochemical properties and macronutrients, but these effects were stronger for fungi and protist than for bacteria. The ability of micronutrients to predict the structure of soil microbial communities declined greatly in paddy soils. Machine learning approach showed that the addition of micronutrients substantially increased the predictive power by 9–17% in predicting the structure of soil microbial communities with up to 69–78% accuracy. These results highlighted the considerable contributions of soil micronutrients to microbial community structure, and advocated that soil micronutrients should be considered when predicting the structure of microbial communities in a changing world.

Abstract Background Plant health and growth are negatively affected by pathogen invasion; however, plants can dynamically modulate their rhizosphere microbiome and adapt to such biotic stresses. Although plant-recruited protective microbes can be assembled into synthetic communities for application in the control of plant disease, rhizosphere microbial communities commonly contain some taxa at low abundance. The roles of low-abundance microbes in synthetic communities remain unclear; it is also unclear whether all the microbes enriched by plants can enhance host adaptation to the environment. Here, we assembled a synthetic community with a disease resistance function based on differential analysis of root-associated bacterial community composition. We further simplified the synthetic community and investigated the roles of low-abundance bacteria in the control of Astragalus mongholicus root rot disease by a simple synthetic community. Results Fusarium oxysporum infection reduced bacterial Shannon diversity and significantly affected the bacterial community composition in the rhizosphere and roots of Astragalus mongholicus. Under fungal pathogen challenge, Astragalus mongholicus recruited some beneficial bacteria such as Stenotrophomonas, Achromobacter, Pseudomonas, and Flavobacterium to the rhizosphere and roots. We constructed a disease-resistant bacterial community containing 10 high- and three low-abundance bacteria enriched in diseased roots. After the joint selection of plants and pathogens, the complex synthetic community was further simplified into a four-species community composed of three high-abundance bacteria (Stenotrophomonas sp., Rhizobium sp., Ochrobactrum sp.) and one low-abundance bacterium (Advenella sp.). Notably, a simple community containing these four strains and a thirteen-species community had similar effects on the control root rot disease. Furthermore, the simple community protected plants via a synergistic effect of highly abundant bacteria inhibiting fungal pathogen growth and less abundant bacteria activating plant-induced systemic resistance. Conclusions Our findings suggest that bacteria with low abundance play an important role in synthetic communities and that only a few bacterial taxa enriched in diseased roots are associated with disease resistance. Therefore, the construction and simplification of synthetic communities found in the present study could be a strategy employed by plants to adapt to environmental stress. Video abstract

Similar to pathogenic bacteria, rhizobia can inject effector proteins into host cells directly to promote infection via the type III secretion system (T3SS). Nodulation outer protein P (NopP), a specific T3SS effector of rhizobia, plays different roles in the establishment of multiple rhizobia-legume symbiotic systems. Mesorhizobium amorphae CCNWGS0123 (GS0123), which infects Robinia pseudoacacia specifically, secretes several T3SS effectors, including NopP. Here, we demonstrate that NopP is secreted through T3SS-I of GS0123 during the early stages of infection, and its deficiency decreases nodule nitrogenase activity of R. pseudoacacia nodules. A trafficking protein particle complex subunit 13–like protein (TRAPPC13) has been identified as a NopP target protein in R. pseudoacacia roots by screening a yeast two-hybrid library. The physical interaction between NopP and TRAPPC13 is verified by bimolecular fluorescence complementation and coimmunoprecipitation assays. In addition, subcellular localization analysis reveals that both NopP and its target, TRAPPC13, are colocalized on the plasma membrane. Compared with GS0123-inoculated R. pseudoacacia roots, some genes associated with cell wall remodeling and plant innate immunity down-regulated in ΔnopP-inoculated roots at 36 h postinoculation. The results suggest that NopP in M. amorphae CCNWGS0123 acts in multiple processes in R. pseudoacacia during the early stages of infection, and TRAPPC13 could participate in the process as a NopP target.

Dodders (Cuscuta chinensis) are rootless and holoparasitic herbs that can infect a variety of host plants, including the vitally important economic and bioenergy crop soybean (Glycine max). Although dodder parasitism severely affects the physiology of host plants, little is known about its effects on fungal communities and root secondary metabolites in hosts. In this study, variations in root-associated fungal communities and root metabolites of soybean under different parasitism conditions were investigated using ITS rRNA gene sequencing and UPLC–MS/MS metabolome detection technologies. The results showed that dodder parasitism significantly altered the composition and diversity of the fungal communities in the rhizosphere and endosphere of soybean. The relative abundance of the potential pathogenic fungus Alternaria significantly increased in the root endosphere of dodder-parasitized soybean. Furthermore, correlation analysis indicated that the fungal community in the root endosphere was susceptible to soil factors under dodder parasitism. Meanwhile, the content of soil total nitrogen was significantly and positively correlated with the relative abundance of Alternaria in the rhizosphere and endosphere of soybean. Metabolomic analysis indicated that dodder parasitism altered the accumulation of flavonoids in soybean roots, with significant upregulation of the contents of kaempferol and its downstream derivatives under different parasitism conditions. Taken together, this study highlighted the important role of dodder parasitism in shaping the fungal communities and secondary metabolites associated with soybean roots, providing new insights into the mechanisms of multiple interactions among dodder, soybean, microbial communities and the soil environment.

ABSTRACT The relative functional importance of rare and abundant species in driving relationships between biodiversity and ecosystem functions (BEF) remains unknown. Here, we investigated the functional roles of rare and abundant species diversity (multitrophic soil organism groups) on multifunctionality derived from 16 ecosystem functions in 228 agricultural fields relating to soil and crop health. The results revealed that the diversity of rare species, rather than of abundant species, was positively related to multifunctionality. Abundant taxa tended to maintain a larger number of functions than rare taxa, while rare subcommunity contributed more phylotypes supporting to the single ecosystem functions. Community assembly processes were closely related to the ecosystem functional performance of soil biodiversity, only observed in rare subcommunity. Higher relative contributions of stochastic assembly processes promoted the positive effects of diversity of rare taxa on multifunctionality, while reducing their diversity and multifunctionality overall. Our results highlight the importance of rare species for ecosystem multifunctionality and elucidate the linkage between ecological assembly processes and BEF relationships. IMPORTANCE The relative functional importance of rare and abundant species in driving relationships between biodiversity and ecosystem functions remains unknown. Here, we highlighted the importance of rare species for ecosystem multifunctionality. In addition, community assembly processes were closely related to the ecosystem functional performance of soil biodiversity in rare subcommunity. Stochastic assembly processes promoted the positive effects of diversity of rare taxa on multifunctionality, while reducing their diversity and multifunctionality overall. This study expands current understanding of the mechanisms underpinning the relationships between biodiversity and ecosystem functions and suggests that stochastic community assembly enhances BEF relationships.

Flowering stage of soybean is an important agronomic trait, which is important for soybean yield, quality and adaptability, and is the external expression of integrating external environmental factors and endogenous signals of the plant itself. Cropping system can change soil properties and fertility, which in turn determine plant growth and yield. The microbial community is the key regulator of plant health and production performance. Currently, there is limited understanding of the effects of cropping systems on microbial community composition, ecological processes controlling community assembly in different soil-plant continuum compartments of soybean. Here, we hope to clarify the structure and assembly process of different soybean compartments bacterial community at flowering stage through our work. The results showed that intercropping decreased the species diversity of rhizosphere and phyllosphere, and phylloaphere microbes mainly came from rhizosphere. FAPROTAX function prediction showed that indicator species sensitive to intercropping and crop rotation were involved in nitrogen/phosphorus cycle and degradation process, respectively. In addition, compared to the continuous cropping, intercropping increased the stochastic assembly processes of bacterial communities in plant-associated compartments, while crop rotation increased the complexity and stability of the rhizosphere network and the deterministic assembly process. Our study highlights the importance of intercropping and crop rotation, as well as rhizosphere and phyllosphere compartments for future crop management and sustainable agricultural regulation of crop microbial communities.

Abstract Background Phaseolus vulgaris (common bean) microsymbionts belonging to the bacterial genera Rhizobium, Bradyrhizobium, and Ensifer (Sinorhizobium) have been isolated across the globe. Individual symbiosis genes (e.g., nodC) of these rhizobia can be different within each genus and among distinct genera. Little information is available about the symbiotic structure of indigenous Rhizobium strains nodulating introduced bean plants or the emergence of a symbiotic ability to associate with bean plants in Bradyrhizobium and Ensifer strains. Here, we sequenced the genomes of 29 representative bean microsymbionts (21 Rhizobium, four Ensifer, and four Bradyrhizobium) and compared them with closely related reference strains to estimate the origins of symbiosis genes among these Chinese bean microsymbionts. Results Comparative genomics demonstrated horizontal gene transfer exclusively at the plasmid level, leading to expanded diversity of bean-nodulating Rhizobium strains. Analysis of vertically transferred genes uncovered 191 (out of the 2654) single-copy core genes with phylogenies strictly consistent with the taxonomic status of bacterial species, but none were found on symbiosis plasmids. A common symbiotic region was wholly conserved within the Rhizobium genus yet different from those of the other two genera. A single strain of Ensifer and two Bradyrhizobium strains shared similar gene content with soybean microsymbionts in both chromosomes and symbiotic regions. Conclusions The 19 native bean Rhizobium microsymbionts were assigned to four defined species and six putative novel species. The symbiosis genes of R. phaseoli, R. sophoriradicis, and R. esperanzae strains that originated from Mexican bean-nodulating strains were possibly introduced alongside bean seeds. R. anhuiense strains displayed distinct host ranges, indicating transition into bean microsymbionts. Among the six putative novel species exclusive to China, horizontal transfer of symbiosis genes suggested symbiosis with other indigenous legumes and loss of originally symbiotic regions or non-symbionts before the introduction of common bean into China. Genome data for Ensifer and Bradyrhizobium strains indicated symbiotic compatibility between microsymbionts of common bean and other hosts such as soybean.

Abstract Background Excessive application of chemical fertilizer has exerted a great threat to soil quality and the environment. The inoculation of plants with plant-growth-promoting rhizobacteria (PGPR) has emerged as a great prospect for ecosystem recovery. The aim of this work to isolate PGPRs and highlights the effect of bacterial inoculants on available N/P/K content in soil and on the growth of wheat under conditions of reduced fertilizer application. Results Thirty-nine PGPRs were isolated and tested for their growth-promoting potential. Thirteen isolates had nitrogen fixation ability, of which N9 (Azotobacter chroococcum) had the highest acetylene reduction activity of 156.26 nmol/gh. Eleven isolates had efficient phosphate solubilizing ability, of which P5 (Klebsiella variicola) released the most available phosphorus in liquid medium (231.68 mg/L). Fifteen isolates had efficient potassium solubilizing ability, of which K13 (Rhizobium larrymoorei) released the most available potassium in liquid medium (224.66 mg/L). In culture medium supplemented with tryptophan, P9 (Klebsiella pneumoniae) produced the greatest amount of IAA. Inoculation with the bacterial combination K14 + 176 + P9 + N8 + P5 increased the alkali-hydrolysed nitrogen, available phosphorus and available potassium in the soil by 49.46, 99.51 and 19.38%, respectively, and enhanced the N, P, and K content of wheat by 97.7, 96.4 and 42.1%, respectively. Moreover, reducing fertilizer application by 25% did not decrease the available nitrogen, phosphorus, and potassium in the soil and N/P/K content, plant height, and dry weight of wheat. Conclusions The bacterial combination K14 + 176 + P9 + N8 + P5 is superior candidates for biofertilizers that may reduce chemical fertilizer application without influencing the normal growth of wheat.

Abstract Revealing the roles of biotic factors in driving community assembly, which is crucial for the understanding of biodiversity and ecosystem functions, is a fundamental but infrequently investigated subject in microbial ecology. Here, combining a cross‐biome observational study with an experimental microcosm study, we provided evidence to reveal the major roles of biotic factors (i.e., soil fungi and cross‐kingdom species associations) in determining soil bacterial biogeography and community assembly in complex terrestrial ecosystems of the arid regions of northwest China. The results showed that the soil fungal richness mediates the balance of assembly processes of bacterial communities, and stochastic assembly processes decreased with increasing fungal richness. Our results further suggest that the predicted increase in aridity conditions due to climate change will reduce bacterial α‐diversity, particularly in desert soils and subsurface layer, and induce more negative species associations. Together, our study represents a significant advance in linking soil fungi to the mechanisms underlying bacterial biogeographic patterns and community assembly in arid ecosystems under climate aridity and land‐use change scenarios.

ABSTRACT Aridity, which is increasing worldwide due to climate change, affects the biodiversity and functions of dryland ecosystems. Whether aridification leads to gradual (or abrupt) and systemic (or specific) changes in the biogeography of abundant and rare microbial species is largely unknown. Here, we investigated stress-adaptive changes (aridity-driven, ranging from 0.65 to 0.94) and biogeographic patterns of abundant and rare bacterial communities in different habitats, including agricultural field, forest, wetland, grassland, and desert, in desert oasis transition zones in northern China. We observed abrupt changes at the breakpoint of aridity values (0.92), characterized by diversity (α-diversity and β-diversity), species coexistence, community assembly processes, and phylogenetic niche conservatism. Specifically, when aridity was 0.92, the systemic responses of abundant and rare taxa changed dramatically in a consistent direction, such that both subcommunities rapidly tended to have a more deterministic assembly, species coexistence, and stronger phylogenetic niche conservatism with increasing aridity. In addition, the change rates of abundant taxa were higher than those of rare taxa, indicating the more sensitive responses of abundant taxa along aridity variation. This finding has important implications for understanding the impact of aridity on the structure and function of abundant and rare soil taxa and how diversity maintenance is associated with soil microbiota responding to global change. The abrupt threshold of soil bacteria found can be used for buffering and for building effective adaptation and mitigation measures aimed at maintaining the capacity of drylands for basic ecosystem functioning. IMPORTANCE Aridity, which is increasing worldwide due to climate change, affects the biodiversity and functions of dryland ecosystems. We provided the first statistical evidence for abrupt changes of species coexistence, ecological processes, and niche conservation of abundant and rare soil bacteria triggered by diversity to abrupt increases in aridity. The abrupt threshold of soil bacterial community response to aridity is spatially heterogeneous at the local scale and should be specified according to local conditions for buffering and for building effective adaptation and mitigation measures aimed at maintaining the capacity of drylands for basic ecosystem functioning.

Efficient screening method is the prerequisite for getting plant growth-promoting (PGP) rhizobacteria (PGPR) which may play an important role in sustainable agriculture from the natural environment. Many current traditional preliminary screening criteria based on knowledge of PGP mechanisms do not always work well due to complex plant–microbe interactions and may lead to the low screening efficiency. More new screening criteria should be evaluated to establish a more effective screening system. However, the studies focused on this issue were not enough, and few new screening criteria had been proposed. The aim of this study was to analyze the correlation between the metabolic phenotypes of rhizobacterial isolates and their PGP ability. The feasibility of using these phenotypes as preliminary screening criteria for PGPR was also evaluated. Twenty-one rhizobacterial isolates were screened for their PGP ability, traditional PGP traits, and multiple metabolic phenotypes that are not directly related to PGP mechanisms, but are possibly related to rhizosphere colonization. Correlations between the PGP traits or metabolic phenotypes and increases in plant agronomic parameters were analyzed to find the indicators that are most closely related to PGP ability. The utilization of 11 nutrient substrates commonly found in root exudates, such as D-salicin, β-methyl-D-glucoside, and D-cellobiose, was significantly positively correlated with the PGP ability of the rhizobacterial isolates. The utilization of one amino acid and two organic acids, namely L-aspartic acid, α-keto-glutaric acid, and formic acid, was negatively correlated with PGP ability. There were no significant correlations between four PGP traits tested in this study and the PGP ability. The ability of rhizobacterial isolates to metabolize nutrient substrates that are identical or similar to root exudate components may act as better criteria than PGP traits for the primary screening of PGPR, because rhizosphere colonization is a prerequisite for PGPR to affect plants.

Root diameter and rooting depth lead to morphological and architectural heterogeneity of plant roots; however, little is known about their effects on root-associated microbial communities. Bacterial community assembly was explored across 156 samples from three rhizocompartments (the rhizosphere, rhizoplane, and endosphere) for different diameters (0.0–0.5 mm, 0.5–1.0 mm, 1.0–2.0 mm, and>2.0 mm) and depths (0–5 cm, 5–10 cm, 10–15 cm, and 15–20 cm) of soybean [Glycine max (L.) Merrill] root systems. The microbial communities of all samples were analyzed using amplicon sequencing of bacterial 16S rRNA genes. The results showed that root diameter significantly affected the rhizosphere and endosphere bacterial communities, while rooting depth significantly influenced the rhizosphere and rhizoplane bacterial communities. The bacterial alpha diversity decreased with increasing root diameter in all three rhizocompartments, and the diversity increased with increasing rooting depth only in the rhizoplane. Clearly, the hierarchical enrichment process of the bacterial community showed a change from the rhizosphere to the rhizoplane to the endosphere, and the bacterial enrichment was higher in thinner or deeper roots (except for the roots at a depth of 15–20 cm). Network analysis indicated that thinner or deeper roots led to higher bacterial network complexity. The core and keystone taxa associated with the specific root diameter class and rooting depth class harbored specific adaptation or selection strategies. Root diameter and rooting depth together affected the root-associated bacterial assembly and network complexity in the root system. Linking root traits to microbiota may enhance our understanding of plant root-microbe interactions and their role in developing environmentally resilient root ecosystems.

Legume plants are pioneer species in desert ecosystem that can be used to reestablish degraded ecosystem functions and soil properties, and reshape underground microbial communities. However, the distribution in soil nutrients and bacterial communities after introducing leguminous shrubs have not been well described. Here, we quantified the variations in soil nutrients and bacterial communities in soil profiles (0–100 cm) across stand ages (5-year, 8-year, 15-year) and severe desertification (SD), and further investigated the bacterial contributions to soil nutrients during stand development. The total phosphorus (TP), total nitrogen (TN) and organic carbon (OC) contents significantly increased, whereas the available nitrogen (AN) and available phosphorus (AP) contents showed a downward trend across stand ages. Bacterial diversity increased after introducing leguminous shrubs. Vertical spatial variation of bacterial communities attenuated during stand development. Bacterial beta-diversity, specific microbial phyla (i.e., Proteobacteria and Firmicutes) and nitrification process were strongly associated with vertical spatial variation in soil nutrients. Combined analyses revealed that leguminous shrubs can ameliorate soil OC, TN and TP with increasing legumes growth time, but could still exhibit no facilitating effect on the improvement of soil available N and P even for legumes aged 15 years. Improving the bioavailability of nutrient elements and microbial function diversity could be targeted for further studies in arid barren sandy.

As the main consumers of bacteria and fungi in farmed soils, protists remain poorly understood. The aim of this study was to explore protist community assembly and ecological roles in soybean fields. Here, we investigated differences in protist communities using high-throughput sequencing and their inferred potential interactions with bacteria and fungi between the bulk soil and rhizosphere compartments of three soybean cultivars collected from six ecological regions in China. Distinct protist community structures characterized the bulk soil and rhizosphere of soybean plants. A significantly higher relative abundance of phagotrophs was observed in the rhizosphere (25.1%) than in the bulk soil (11.3%). Spatial location (R2 = 0.37–0.51) explained more of the variation in protist community structures of soybean fields than either the compartment (R2 = 0.08–0.09) or cultivar type (R2 = 0.02–0.03). The rhizosphere protist network (76 nodes and 414 edges) was smaller and less complex than the bulk soil network (147 nodes and 880 edges), indicating a smaller potential of niche overlap and interactions in the rhizosphere due to the increased resources in the rhizosphere. Furthermore, more inferred potential predator-prey interactions occur in the rhizosphere. We conclude that protists have a crucial ecological role to play as an integral part of microbial co-occurrence networks in soybean fields.

Astragalus membranaceus (Fisch.) Bge. var. mongholicus, which is used in traditional Chinese medicine, contains several bioactive ingredients. The root-associated microbial communities play a crucial role in the production of secondary metabolites in plants. However, the correlation of root-associated bacteria and fungi with the bioactive ingredients production in A. mongholicus has not been elucidated. This study aimed to examine the changes in soil properties, root bioactive ingredients, and microbial communities in different cultivation years. The root-associated bacterial and fungal composition was analyzed using high-throughput sequencing. The correlation between root-associated bacteria and fungi, soil properties, and six major bioactive ingredients were examined using multivariate correlation analysis. Results showed that soil properties and bioactive ingredients were distinct across different cultivation years. The composition of the rhizosphere microbiome was different from that of the root endosphere microbiome. The bacterial community structure was affected by the cultivation year and exhibited a time-decay pattern. Soil properties affected the fungal community composition. It was found that 18 root-associated bacterial operational taxonomic units (OTUs) and four fungal OTUs were positively and negatively correlated with bioactive ingredient content, respectively. The abundance of Stenotrophomonas in the rhizosphere was positively correlated with astragaloside content. Phyllobacterium and Inquilinus in the endosphere were positively correlated with the calycosin content. In summary, this study provided a new opportunity and theoretical reference for improving the production and quality of in A. mongholicus, which thus increase the pharmacological value of A. mongholicus.

ABSTRACT Biodiversity is important for supporting ecosystem functioning. To evaluate the factors contributing to the strength of microbial diversity-function relationships in complex terrestrial ecosystems, we conducted a soil survey over different habitats, including an agricultural field, forest, wetland, grassland, and desert. Soil microbial multidiversity was estimated by the combination of bacterial and fungal diversity. Soil ecosystem functions were evaluated using a multinutrient cycling index (MNC) in relation to carbon, nitrate, phosphorus, and potassium cycling. Significant positive relationships between soil multidiversity and multinutrient cycling were observed in all habitats, except the grassland and desert. Specifically, community compositions showed stronger correlations with multinutrient cycling than α-diversity, indicating the crucial role of microbial community composition differences on soil nutrient cycling. Importantly, we revealed that changes in both the neutral processes (Sloan neutral modeling) and the proportion of negative bacterial-fungal associations were linked to the magnitude and direction of the diversity-MNC relationships. The habitats less governed by neutral processes and dominated by negative bacterial-fungal associations exhibited stronger negative microbial α-diversity–MNC relationships. Our findings suggested that the balance between positive and negative bacterial-fungal associations was connected to the link between soil biodiversity and ecosystem function in complex terrestrial ecosystems. This study elucidates the potential factors influencing diversity-function relationships, thereby enabling future studies to forecast the effects of belowground biodiversity on ecosystem function. IMPORTANCE The relationships between soil biodiversity and ecosystem functions are an important yet poorly understood topic in microbial ecology. This study presents an exploratory effort to gain predictive understanding of the factors driving the relationships between microbial diversity and potential soil nutrient cycling in complex terrestrial ecosystems. Our structural equation modeling and random forest analysis revealed that the balance between positive and negative bacterial-fungal associations was clearly linked to the strength of the relationships between soil microbial diversity and multiple nutrients cycling across different habitats. This study revealed the potential factors underpinning diversity-function relationships in terrestrial ecosystems and thus helps us to manage soil microbial communities for better provisioning of key ecosystem services.

Abstract Background Soil microbiomes play an important role in the services and functioning of terrestrial ecosystems. However, little is known of their vertical responses to restoration process and their contributions to soil nutrient cycling in the subsurface profiles. Here, we investigated the community assembly of soil bacteria, archaea, and fungi along vertical (i.e., soil depths of 0–300 cm) and horizontal (i.e., distance from trees of 30–90 cm) profiles in a chronosequence of reforestation sites that represent over 30 years of restoration. Results In the superficial layers (0–80 cm), bacterial and fungal diversity decreased, whereas archaeal diversity increased with increasing soil depth. As reforestation proceeded over time, the vertical spatial variation in bacterial communities decreased, while that in archaeal and fungal communities increased. Vertical distributions of the soil microbiomes were more related to the variation in soil properties, while their horizontal distributions may be driven by a gradient effect of roots extending from the tree. Bacterial and archaeal beta-diversity were strongly related to multi-nutrient cycling in the soil, respectively, playing major roles in deep and superficial layers. Conclusions Taken together, these results reveal a new perspective on the vertical and horizontal spatial variation in soil microbiomes at the fine scale of single trees. Distinct response patterns underpinned the contributions of soil bacteria, archaea, and fungi as a function of subsurface nutrient cycling during the reforestation of ex-arable land.

Pathogens require iron for their metabolism and virulence. Here the authors identify an iron acquisition system inPseudomonas aeruginosainvolving a protein secreted by a type VI secretion system, the PQS signalling compound and siderophore receptors.

Sinorhizobium meliloti CCNWSX0020, isolated from root nodules of Medicago lupulina growing in gold mine tailings in the northwest of China, displayed multiple heavy metal resistance and growth promotion of M. lupulina. In our previous work, the expression level of dmeR and dmeF genes were induced by Cu2+ through comparative transcriptome approach. Based on protein analysis, the dmeF encoded for a protein which showed a 37% similarity to the cation transporter DmeF of Cupriavidus metallidurans, whereas dmeR encoded transcriptional regulator which was highly homologous with DmeR belonging to RcnR/CsoR family metal-responsive transcriptional regulator. In addition to copper, quantitative real-time PCR analysis showed that dmeR and dmeF were also induced by nickel and cobalt. To investigate the functions of dmeR and dmeF in S. meliloti CCNWSX0020, the dmeR and dmeF deletion mutants were constructed. The dmeF mutant was more sensitive to Co2 + and Ni2 + than the wild type strain. Pot experiments were carried out to determine whether the growth of M. lupulina was affected when the dmeF gene was knocked out in the presence of nickel or cobalt. Results indicated that the nodule number of the host plant inoculated with the dmeF deletion mutant was significantly less than the S. meliloti CCNWSX0020 wild-type in the presence of Co2 + or Ni2 +. However, when standardized by nodule fresh weight, the nitrogenase activities of nodules infected by the dmeF deletion mutant was similar to nitrogenase activity of the wild type nodule.

Recent horizontal gene transfer (HGT) is crucial for enabling microbes to rapidly adapt to their novel environments without relying upon rare beneficial mutations that arise spontaneously. For several years now, computational approaches have been developed to detect HGT, but they typically lack the sensitivity and ability to detect recent HGT events. Here we introduce a novel strategy, named RecentHGT. The number of genes undergoing recent HGT between two bacterial genomes was estimated by a new algorithm derived from the expectation-maximization algorithm and is based on the theoretical sequence-similarity distribution of orthologous genes. We tested the proposed strategy by applying it to a set of 10 Rhizobium genomes, and detected several large-scale recent HGT events. We also found that our strategy was more sensitive than other available HGT detection methods. These HGT events were mainly mediated by symbiotic plasmids. Our new strategy can provide clear evidence of recent HGT events and thus it brings us closer to the goal of detecting these potentially adaptive evolution processes in rhizobia as well as pathogens.

Mesorhizobium alhagi CCNWXJ12-2T, isolated from root nodules of the desert plant Alhagi sparsifolia, contains two type III secretion systems (T3SSs). T3SSs are specialized machinery with wide distribution in bacteria that inject effector proteins into target cells. Our previous study showed that the expression of M. alhagi T3SS1 is upregulated in high-salt conditions. Here, phylogenetic analysis of T3SS1 using the core protein RhcU suggested that T3SS1 belongs to the α-Rhc II subgroup of the Rhc T3SS family. To elaborate the function of M. alhagi CCNWXJ12-2T T3SS1 in stress adaption, two T3SS1 mutants (ΔrhcQ and ΔMA29250) were constructed and analyzed. β-galactosidase transcriptional fusion assays showed that activity of the promoter of T3SS1 was induced by salts. Mutant ΔrhcQ was more sensitive to NaCl and LiCl than the wild-type, but ΔMA29250 was not. Both mutants were more sensitive to KCl than the wild-type. The intracellular Na+ concentration in ΔrhcQ in high-NaCl conditions (0.4 M) increased by 37% compared to that of the wild-type strain, while the Na+ concentration in ΔMA29250 increased by 13%. The K+ concentration in both mutants increased by 16% compared to the wild-type in high-KCl conditions (0.3 M). Strain ΔrhcQ showed decreased survival compared to the wild-type after treatment with H2O2, while the survival rate of ΔMA29250 was almost the same as that of the wild-type. Antioxidant enzyme activities in ΔrhcQ were lower than those in the wild-type strain, but this was not the case for ΔMA29250. Our data elucidate the beneficial effects of T3SS1 in the adaption of M. alhagi CCNWXJ12-2T to stress.

Rhizobium leguminosarum bv. viciae, which nodulates pea and vetch, makes a mixture of secreted nodulation signals (Nod factors) carrying either a C18:4 or a C18:1 N-linked acyl chain. Mutation of nodE blocks the formation of the C18:4 acyl chain, and nodE mutants, which produce only C18:1-containing Nod factors, are less efficient at nodulating pea. However, there is significant natural variation in the levels of nodulation of different pea cultivars by a nodE mutant of R. leguminosarum bv. viciae. Using recombinant inbred lines from two pea cultivars, one which nodulated relatively well and one very poorly by the nodE mutant, we mapped the nodE-dependent nodulation phenotype to a locus on pea linkage group I. This was close to Sym37 and PsK1, predicted to encode LysM-domain Nod-factor receptor-like proteins; the Sym2 locus that confers Nod-factor-specific nodulation is also in this region. We confirmed the map location using an introgression line carrying this region. Our data indicate that the nodE-dependent nodulation is not determined by the Sym2 locus. We identified several pea lines that are nodulated very poorly by the R. leguminosarum bv. viciae nodE mutant, sequenced the DNA of the predicted LysM-receptor domains of Sym37 and PsK1, and compared the sequences with those derived from pea cultivars that were relatively well nodulated by the nodE mutant. This revealed that one haplotype (encoding six conserved polymorphisms) of Sym37 is associated with very poor nodulation by the nodE mutant. There was no such correlation with polymorphisms at the PsK1 locus. We conclude that the natural variation in nodE-dependent nodulation in pea is most probably determined by the Sym37 haplotype.

Type VI secretion systems (T6SSs) are widespread multi-component machineries that translocate effectors into either eukaryotic or prokaryotic cells, for virulence or for interbacterial competition. Herein, we report that the T6SS-4 from Yersinia pseudotuberculosis displays an unexpected function in the transportation of Zn2+ to combat diverse stresses and host immunity. Environmental insults such as oxidative stress induce the expression of T6SS-4 via OxyR, the transcriptional factor that also regulates many oxidative response genes. Zinc transportation is achieved by T6SS-4-mediated translocation of a novel Zn2+-binding protein substrate YezP (YPK_3549), which has the capacity to rescue the sensitivity to oxidative stress exhibited by T6SS-4 mutants when added to extracellular milieu. Disruption of the classic zinc transporter ZnuABC together with T6SS-4 or yezP results in mutants that almost completely lost virulence against mice, further highlighting the importance of T6SS-4 in resistance to host immunity. These results assigned an unconventional role to T6SSs, which will lay the foundation for studying novel mechanisms of metal ion uptake by bacteria and the role of this process in their resistance to host immunity and survival in harmful environments.

Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km2) scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI) and the geometric mean of enzyme activities (GME). At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality.

BACKGROUND: Legume-rhizobium symbiosis is a complex process that is regulated in the host plant cell through gene expression network. Many nodulin genes that are upregulated during different stages of nodulation have been identified in leguminous herbs. However, no nodulin genes in woody legume trees, such as black locust (Robinia pseudoacacia), have yet been reported. METHODOLOGY/PRINCIPAL FINDINGS: To identify the nodulin genes involved in R. pseudoacacia-Mesorhizobium amorphae CCNWGS0123 symbiosis, a suppressive subtractive hybridization approach was applied to reveal profiling of differentially expressed genes and two subtracted cDNA libraries each containing 600 clones were constructed. Then, 114 unigenes were identified from forward SSH library by differential screening and the putative functions of these translational products were classified into 13 categories. With a particular interest in regulatory genes, twenty-one upregulated genes encoding potential regulatory proteins were selected based on the result of reverse transcription-polymerase chain reaction (RT-PCR) analysis. They included nine putative transcription genes, eight putative post-translational regulator genes and four membrane protein genes. The expression patterns of these genes were further analyzed by quantitative RT-PCR at different stages of nodule development. CONCLUSIONS: The data presented here offer the first insights into the molecular foundation underlying R. pseudoacacia-M. amorphae symbiosis. A number of regulatory genes screened in the present study revealed a high level of regulatory complexity (transcriptional, post-transcriptional, translational and post-translational) that is likely essential to develop symbiosis. In addition, the possible roles of these genes in black locust nodulation are discussed.

Message RNA (mRNA) carries a large number of local secondary structures, with structural stability to participate in the regulations of gene expression. A worthy question is how the local structural stability is maintained under the constraint that multiple selective pressures are imposed on mRNA local regions. Here, we performed the first genome-wide study of natural selection operating on high structural stability regions (HSRs) of mRNAs in Escherichia coli. We found that HSR tends to adjust the folded conformation to reduce the harm of mutations, showing a high level of mutational robustness. Moreover, guanine preference in HSR was observed, supporting the hypothesis that the selective constraint for high structural stability may partly account for the high percentage of G content in Escherichia coli genome. Notably, we found a substantially reduced synonymous substitution rate in HSRs compared with that in their adjacent regions. Surprisingly and interestingly, the non-key sites in HSRs, which have slight effect on structural stability, have synonymous substitution rate equivalent to background regions. To explain this result, we identified compensatory mutations in HSRs based on structural stability, and found that a considerable number of synonymous mutations occur to restore the structural stability decreased heavily by the mutations on key sites. Overall, these results suggest a significant role of local structural stability as a selective force operating on mRNA, which furthers our understanding of the constraints imposed on protein-coding RNAs.