Rhizosphere-Associated Anammox Bacterial Diversity and Abundance of Nitrogen Cycle-Related Functional Genes of Emergent Macrophytes in Eutrophic Wetlands.

Rhizospheric microbial community of emergent macrophytes plays an important role in nitrogen removal, especially in the eutrophic wetlands. The objective of this study was to identify the differences in anammox bacterial community composition among different emergent macrophytes and investigate revealed the the main factors affecting on the composition, diversity, and abundance of anammox bacterial community. Results showed that the composition, diversity, and abundance of the anammox community were significantly different between the vegetated sediments of three emergent macrophytes and unvegetated sediment. The composition of the anammox bacterial community was different in the vegetated sediments of different emergent macrophytes. Also, the abundance of nitrogen cycle-related functional genes in the vegetated sediments was found to be higher than that in the unvegetated sediment. Canonical correspondence analysis (CCA) and structural equation models analysis (SEM) showed that salinity and pH were the main environmental factors influencing the composition and diversity of the anammox bacterial community and NO2--N indirectly affected anammox bacterial community diversity by affecting TOC. nirK-type denitrifying bacteria abundance had significant effects on the bacterial community composition, diversity, and abundance of anammox bacteria. The community composition of anammox bacteria varies with emergent macrophyte species. The rhizosphere of emergent macrophytes provides a favorable environment and promotes the growth of nitrogen cycling-related microorganisms that likely accelerate nitrogen removal in eutrophic wetlands.

Tardiphaga alba sp. nov., a heavy-metal-tolerant bacterium isolated from garden soil.

A previously undescribed, heavy-metal-tolerant, motile, Gram-negative bacterium, designated strain SK50-23T, was characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SK50-23T was closely related to Tardiphaga robiniae LMG 26467T and the non-phototrophic 'Rhodopseudomonas boonkerdii' NS23T (98.1 and 97.3 % 16S rRNA gene sequence similarity, respectively). Strain SK50-23T possessed a circular genome of 5.86 Mb, with a DNA G+C content of 61.9 mol%. Digital DNA-DNA hybridization showed 20.8-21.6 % similarity between strain SK50-23T and related species. In addition, the whole-genome average nucleotide identity values between strain SK50-23T and related species ranged from 75.1 to 83.5 %. The major cellular fatty acid identified in strain SK50-23T was C18 : 1ω7c, and the main isoprenoid quinone present was ubiquinone Q-10. Strain SK50-23T could be assigned to the genus Tardiphaga with the species name Tardiphaga alba sp. nov. based on morphological, chemotaxonomic and genome-based taxonomic characteristics, and 16S rRNA gene-based phylogenetic characteristics. The type strain of the proposed novel species is SK50-23T (=NBRC 108825T=CGMCC No. 1.12037T).

[Characteristics and Dominant Influencing Factors of the Fungal Community Structure in Soils Co-contaminated with Rare Earth Elements and Heavy Metals].

Rare earth elements (REEs) have been listed as emerging pollutants and are often enriched together in soils with heavy metals (HMs), which results in ecological crises. The ecological effects caused by REEs have been attracting increasing amounts of attention, but most studies neglect the synergistic effect of REEs and HMs. The soil fungal community plays an important role in maintaining ecosystem functions, and understanding the fungal community structure and its dominant influencing factors in the co-contaminated soils will help to develop soil remediation strategies that could reduce or remedy the impacts of human production activities on the environment. Currently, the effects of long-term contamination of REEs and HMs on the soil fungal communities remain unclear. The Baotou rare earth tailings dam (Inner Mongolia, China) was used as the area of study, and soil samples co-contaminated with REEs and HMs were collected. Illumina high-throughput sequencing with ITS1 gene amplicons was used to analyze the fungal community diversity and structural characteristics. The results showed that the heterogeneity of soil environmental variables determined the distribution of fungal communities in a small area and constituted its own unique ecological niche in the co-contaminated environment. The fungal community richness and diversity in the co-contaminated soils were significantly lower than those in the uncontaminated soils, and the composition of the fungal community was significantly different. The results of a random forest (RF) analysis showed that TN was the most important factor that affected the fungal community richness and diversity, followed by REEs, Zn, and AK. The results of a canonical correspondence analysis (CCA) showed that Zn was the most important factor that affected the fungal community structure. A variation partitioning analysis (VPA) was performed to quantify the relative contributions of different environmental variables on the changes in fungal community structure, and the analytical results showed that all the detected environmental variables could explain 93.3% of the variation in soil fungal community. The combined effect of soil physicochemical properties and pollution factors (REEs and HMs) accounted for 58.5% of the total variation, and their contribution alone accounted for 13.5% and 21%, respectively. The effects of these pollution factors on the fungal communities were slightly higher than those of the soil physicochemical properties. The synergistic contributions of REEs and HMs were 40.1%, and their individual effects were 21.8% and 17.9%, respectively. Therefore, the soil physicochemical properties, REEs, and HMs regulated the fungal community structure and composition in concert. The synergistic contributions of REEs and HMs were greater than their individual effects, and these results suggest that it is necessary to further strengthen the risk control of the co-contamination of REEs and HMs in the soil environment.

Paenibacillus sedimenti sp. nov., isolated from freshwater wetland sediment.

A Gram-stain-positive, motile, rod-shaped, facultatively anaerobic bacterium, designated strain WST5T, isolated from sediment was characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain WST5T was most closely related to Paenibacillus aestuarii CJ25T (96.8 % similarity). The genome size of the WST5T was 6.5 Mb, contained 4500 predicted protein-coding genes, and had a DNA G+C content of 46.6%. The values of whole-genome average nucleotide identity analysis and digital DNA-DNA hybridization between strain WST5T and its closely related type strains were less than 76 and 25.6 %, respectively. The predominant cellular fatty acids (>10 %) were anteiso-C15 : 0 and C16 : 1 ω5c and the main menaquinone was MK-7. The major polar lipids were identified as diphospholidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminophospholipids. Based on the results of phenotypic, genotypic, chemotaxonomic and phylogenetic analyses, strain WST5T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus sedimentum sp. nov. is proposed. The type strain is WST5T (=NBRC 115194 T=CGMCC 1.18706T).

Methanotrophy Alleviates Nitrogen Constraint of Carbon Turnover by Rice Root-Associated Microbiomes.

The bioavailability of nitrogen constrains primary productivity, and ecosystem stoichiometry implies stimulation of N2 fixation in association with carbon sequestration in hotspots such as paddy soils. In this study, we show that N2 fixation was triggered by methane oxidation and the methanotrophs serve as microbial engines driving the turnover of carbon and nitrogen in rice roots. 15N2-stable isotope probing showed that N2-fixing activity was stimulated 160-fold by CH4 oxidation from 0.27 to 43.3 µmol N g-1 dry weight root biomass, and approximately 42.5% of the fixed N existed in the form of 15N-NH4 + through microbial mineralization. Nitrate amendment almost completely abolished N2 fixation. Ecophysiology flux measurement indicated that methane oxidation-induced N2 fixation contributed only 1.9% of total nitrogen, whereas methanotrophy-primed mineralization accounted for 21.7% of total nitrogen to facilitate root carbon turnover. DNA-based stable isotope probing further indicated that gammaproteobacterial Methylomonas-like methanotrophs dominated N2 fixation in CH4-consuming roots, whereas nitrate addition resulted in the shift of the active population to alphaproteobacterial Methylocystis-like methanotrophs. Co-occurring pattern analysis of active microbial community further suggested that a number of keystone taxa could have played a major role in nitrogen acquisition through root decomposition and N2 fixation to facilitate nutrient cycling while maintaining soil productivity. This study thus highlights the importance of root-associated methanotrophs as both biofilters of greenhouse gas methane and microbial engines of bioavailable nitrogen for rice growth.

Claroideoglomus etunicatum affects the structural and functional genes of the rhizosphere microbial community to help maize resist Cd and La stresses.

Arbuscular mycorrhizal fungi (AMF) and plant rhizosphere microbes reportedly enhance plant tolerance to abiotic stresses and promote plant growth in contaminated soils. The co-contamination of soil by heavy metals (e.g., Cd) and rare earth elements (e.g., La) represents a severe environmental problem. Although the influence of AMF in the phytoremediation of contaminated soils is well documented, the underlying interactive mechanisms between AMF and rhizosphere microbes are still unclear. We conducted a greenhouse pot experiment to evaluate the effects of AMF (Claroideoglomus etunicatum) on maize growth, nutrient and metal uptake, rhizosphere microbial community, and functional genes in soils with separate and combined applications of Cd and La. The purpose of this experiment was to explore the mechanism of AMF affecting plant growth and metal uptake via interactions with rhizosphere microbes. We found that C. etunicatum (i) significantly enhanced plant nutritional level and biomass and decreased metal concentration in the co-contaminated soil; (ii) significantly altered the structure of maize rhizosphere bacterial and fungal communities; (iii) strongly enriched the abundance of carbohydrate metabolism genes, ammonia and nitrate production genes, IAA (indole-3-acetic acid) and ACC deaminase (1-aminocyclopropane-1-carboxylate) genes, and slightly altered the abundance of P-related functional genes; (iv) regulated the abundance of microbial quorum sensing system and metal membrane transporter genes, thereby improving the stability and adaptability of the rhizosphere microbial community. This study provides evidence of AMF improving plant growth and resistance to Cd and La stresses by regulating plant rhizosphere microbial communities and aids our understanding of the underlying mechanisms.

In Vivo Evidence of Single 13C and 15N Isotope-Labeled Methanotrophic Nitrogen-Fixing Bacterial Cells in Rice Roots.

Methane-oxidizing bacteria (methanotrophs) play an ecological role in methane and nitrogen fluxes because they are capable of nitrogen fixation and methane oxidation, as indicated by genomic and cultivation-dependent studies. However, the chemical relationships between methanotrophy and diazotrophy and aerobic and anaerobic reactions, respectively, in methanotrophs remain unclear. No study has demonstrated the cooccurrence of both bioactivities in a single methanotroph bacterium in its natural environment. Here, we demonstrate that both bioactivities in type II methanotrophs occur at the single-cell level in the root tissues of paddy rice (Oryza sativa L. cv. Nipponbare). We first verified that difluoromethane, an inhibitor of methane monooxygenase, affected methane oxidation in rice roots. The results indicated that methane assimilation in the roots mostly occurred due to oxygen-dependent processes. Moreover, the results indicated that methane oxidation-dependent and methane oxidation-independent nitrogen fixation concurrently occurred in bulk root tissues. Subsequently, we performed fluorescence in situ hybridization and NanoSIMS analyses, which revealed that single cells of type II methanotrophs (involving six amplicon sequence variants) in paddy rice roots simultaneously and logarithmically fixed stable isotope gases 15N2 and 13CH4 during incubation periods of 0, 23, and 42 h, providing in vivo functional evidence of nitrogen fixation in methanotrophic cells. Furthermore, 15N enrichment in type II methanotrophs at 42 h varied among cells with an increase in 13C accumulation, suggesting that either the release of fixed nitrogen into root systems or methanotroph metabolic specialization is dependent on different microenvironmental niches in the root. IMPORTANCE Atmospheric methane concentrations have been continually increasing, causing methane to become a considerable environmental concern. Methanotrophy may be the key to regulating methane fluxes. Although research suggests that type II methanotrophs are involved in methane oxidation aerobically and nitrogen fixation anaerobically, direct evidence of simultaneous aerobic and anaerobic bioreactions of methanotrophs in situ is still lacking. In this study, a single-cell isotope analysis was performed to demonstrate these in vivo parallel functions of type II methanotrophs in the root tissues of paddy rice (Oryza sativa L. cv. Nipponbare). The results of this study indicated that methanotrophs might provide fixed nitrogen to root systems or depend on cells present in the spatially localized niche of the root tissue. Furthermore, our results suggested that single type II methanotrophic cells performed simultaneous methane oxidation and nitrogen fixation in vivo. Under natural conditions, however, nitrogen accumulation varied at the single-cell level.

Methanotrophs Contribute to Nitrogen Fixation in Emergent Macrophytes.

Root-associated aerobic methanotroph plays an important role in reducing methane emissions from wetlands. In this study, we examined the activity of methane-dependent nitrogen fixation and active nitrogen-fixing bacterial communities on the roots of Typha angustifolia and Scirpus triqueter using a 15N-N2 feeding experiment and a cDNA-based clone library sequence of the nifH gene, respectively. A 15N-N2 feeding experiment showed that the N2 fixation rate of S. triqueter (1.74 µmol h-1 g-1 dry weight) was significantly higther than that of T. angustifolia (0.48 µmol h-1 g-1 dry weight). The presence of CH4 significantly increased the incorporation of 15N-labeled N2 into the roots of both plants, and the rate of CH4-dependent N2 fixation of S. triqueter (5.6 µmol h-1 g-1 dry weight) was fivefold higher than that of T. angustifolia (0.94 µmol h-1 g-1 dry weight). The active root-associated diazotrophic communities differed between the plant species. Diazotrophic Methylosinus of the Methylocystaceae was dominant in S. triqueter, while Rhizobium of the Rhizobiaceae was dominant in T. angustifolia. However, there were no significant differences in the copy numbers of nifH between plant species. These results suggest that N2 fixation was enhanced by the oxidation of CH4 in the roots of macrophytes grown in natural wetlands and that root-associated Methylocystacea, including Methylosinus, contribute to CH4 oxidation-dependent N2 fixation.

Salinity significantly affects methane oxidation and methanotrophic community in Inner Mongolia lake sediments.

Methanotrophs oxidize methane (CH4) and greatly help in mitigating greenhouse effect. Increased temperatures due to global climate change can facilitate lake salinization, particularly in the regions with cold semiarid climate. However, the effects of salinity on the CH4 oxidation activity and diversity and composition of methanotrophic community in the sediment of natural lakes at a regional scale are still unclear. Therefore, we collected lake sediment samples from 13 sites in Mongolian Plateau; CH4 oxidation activities of methanotrophs were investigated, and the diversity and abundance of methanotrophs were analyzed using real-time quantitative polymerase chain reaction and high throughput sequencing approach. The results revealed that the diversity of methanotrophic community decreased with increasing salinity, and community structure of methanotrophs was clearly different between the hypersaline sediment samples (HRS; salinity > 0.69%) and hyposaline sediment samples (HOS; salinity < 0.69%). Types II and I methanotrophs were predominant in HRS and HOS, respectively. Salinity was significantly positively correlated with the relative abundance of Methylosinus and negatively correlated with that of Methylococcus. In addition, CH4 oxidation rate and pmoA gene abundance decreased with increasing salinity, and salinity directly and indirectly affected CH4 oxidation rate via regulating the community diversity. Moreover, high salinity decreased cooperative association among methanotrophs and number of key methanotrophic species (Methylosinus and Methylococcus, e.g). These results suggested that salinity is a major driver of CH4 oxidation in lake sediments and acts by regulating the diversity of methanotrophic community and accociation among the methanotrophic species.

Multiple factors influence bacterial community diversity and composition in soils with rare earth element and heavy metal co-contamination.

The effects of long-term rare earth element (REE) and heavy metal (HM) contamination on soil bacterial communities remains poorly understood. In this study, soil samples co-contaminated with REEs and HMs were collected from a rare-earth tailing dam. The bacterial community composition and diversity were analyzed through Illumina high-throughput sequencing with 16S rRNA gene amplicons. Bacterial community richness and diversity were lower in the co-contaminated soils than in the uncontaminated soils, with clearly different bacterial community compositions. The results showed that total organic carbon and available potassium were the most important factors affecting bacterial community richness and diversity, followed by the REE and HM contents. Although the canonical correspondence analysis results showed that an REE alone had no obvious effects on bacterial community structures, we found that the combined effects of soil physicochemical properties and REE and HM contents regulated bacterial community structure and composition. The effects of REEs and HMs on bacterial communities were similar, whereas their combined contributions were greater than the individual effects of REEs or HMs. Some bacterial taxa were worth noting. These specifically included the plant growth-promoting bacteria Exiguobacterium (sensitive to REEs and HMs) and oligotrophic microorganisms with metal tolerance (prevalent in contaminated soil); moreover, relative abundance of JTB255-Marine Benthic Group, Rhodobacteraceae, Erythrobacter, and Truepera may be correlated with REEs. This study was the first to investigate the responses of bacterial communities to REE and HM co-contamination. The current results have major implications for the ecological risk assessment of environments co-contaminated with REEs and HMs.

Exploring Microbial Resource of Different Rhizocompartments of Dominant Plants Along the Salinity Gradient Around the Hypersaline Lake Ejinur.

Lake littoral zones can also be regarded as another extremely hypersaline environment due to hypersaline properties of salt lakes. In this study, high-throughput sequencing technique was used to analyze bacteria and fungi from different rhizocompartments (rhizosphere and endosphere) of four dominant plants along the salinity gradient in the littoral zones of Ejinur Salt Lake. The study found that microbial α-diversity did not increase with the decrease of salinity, indicating that salinity was not the main factor on the effect of microbial diversity. Distance-based redundancy analysis and regression analysis were used to further reveal the relationship between microorganisms from different rhizocompartments and plant species and soil physicochemical properties. Bacteria and fungi in the rhizosphere and endosphere were the most significantly affected by SO4 2-, SOC, HCO3 -, and SOC, respectively. Correlation network analysis revealed the potential role of microorganisms in different root compartments on the regulation of salt stress through synergistic and antagonistic interactions. LEfSe analysis further indicated that dominant microbial taxa in different rhizocompartments had a positive response to plants, such as Marinobacter, Palleronia, Arthrobacter, and Penicillium. This study was of great significance and practical value for understanding salt environments around salt lakes to excavate the potential microbial resources.

Elevated Atmospheric CO2 and Nitrogen Fertilization Affect the Abundance and Community Structure of Rice Root-Associated Nitrogen-Fixing Bacteria.

Elevated atmospheric CO2 (eCO2) results in plant growth and N limitation, yet how root-associated nitrogen-fixing bacterial communities respond to increasing atmospheric CO2 and nitrogen fertilization (eN) during the growth stages of rice is unclear. Using the nifH gene as a molecular marker, we studied the combined effect of eCO2 and eN on the diazotrophic community and abundance at two growth stages in rice (tillering, TI and heading, HI). Quantitative polymerase chain reaction (qPCR) showed that eN had no obvious effect on nifH abundance in rice roots under either ambient CO2 (aCO2) or eCO2 treatment at the TI stage; in contrast, at the HI, nifH copy numbers were increased under eCO2 and decreased under aCO2. For rhizosphere soils, eN significantly reduced the abundance of nifH under both aCO2 and eCO2 treatment at the HI stage. Elevated CO2 significantly increased the nifH abundance in rice roots and rhizosphere soils with nitrogen fertilization, but had no obvious effect without N addition at the HI stage. There was a significant interaction [CO2 × N fertilization] effect on nifH abundance in root zone at the HI stage. In addition, the nifH copy numbers in rice roots were significantly higher at the HI stage than at the TI stage. Sequencing analysis indicated that the root-associated diazotrophic community structure tended to cluster according to the nitrogen fertilization treatment and that Rhizobiales were the dominant diazotrophs in all root samples at the HI stage. Additionally, nitrogen fertilization significantly increased the relative abundance of Methylosinus (Methylocystaceae) under eCO2 treatment, but significantly decreased the relative abundance of Rhizobium (Rhizobiaceae) under aCO2 treatment. Overall, the combined effect of eN and eCO2 stimulates root-associated diazotrophic methane-oxidizing bacteria while inhibits heterotrophic diazotrophs.

Evaluation of Microbial Assemblages in Various Saline-Alkaline Soils Driven by Soluble Salt Ion Components.

Land degraded by salinization and alkalization is widely distributed globally and involves a wide range of ecosystem types. However, the knowledge of the indigenous microbial assemblages and their roles in various saline-alkaline soils is limited. This study demonstrated microbial assemblages in various saline-alkaline soils from different regions of Inner Mongolia and revealed the key driving factors to influence microbiome. The correlation network analysis indicates the difference in adaptability of bacterial and fungal communities under stimulation by saline-alkaline stress: fungal community shows higher tolerance, stability, and resilience to various saline-alkaline soils than a bacterial community. The keystone bacteria and fungi that have potential adaptability to various saline-alkaline environments are further identified, and they may confer benefits in restoring saline-alkaline soils by their own effects or assisting plants. For salt-rich soils in different regions, the soluble salt ion components are the major determinant to drive microbial assemblages of different saline-alkaline soils, rather than salinity. Thus, these saline-alkaline soils are clustered into sulfated, chlorinated, and soda-type saline-alkaline soils. Multivariate analysis reveals unique, dominant, and common microbial taxa in three saline-alkaline soils. This result of the conceptual mode indicates that potential roles of unique and dominant microbial taxa on regulating saline-alkaline functions are more vital.

Arbuscular mycorrhizal fungi alter microbiome structure of rhizosphere soil to enhance maize tolerance to La.

Rhizosphere microbes are essential partners for plant stress tolerance. Recent studies indicate that arbuscular mycorrhizal fungi (AMF) can facilitate the revegetation of soils contaminated by heavy metals though interacting with rhizosphere microbiome. However, it is unclear how AMF affect rhizosphere microbiome to improve the growth of plant under rare earth elements (REEs) stress. AMF (Claroideoglomus etunicatum) was inoculated to maize grown in soils spiked with Lanthanum (0 mg kg-1, La0; 10 mg kg-1, La10; 100 mg kg-1, La100; 500 mg kg-1, La500). Plant biomass, nutrient uptake, REE uptake and rhizosphere bacterial and fungal community were evaluated. The results indicated that La100 and La500 decreased significantly root colonization rates and nutrition uptake (K, P, Ca and Mg content). La500 decreased significantly α-diversity indexes of bacterial and fungal community. AMF enhanced significantly the shoot and root fresh and dry weight of maize in all La treatments (except for the root fresh and dry weight of La0 and La10 treatment). For La100 and La500 treatments, AMF increased significantly nutrition uptake (K, P, Ca and Mg content) in shoot of maize by 27.40-441.77%. For La500 treatment, AMF decreased significantly shoot La concentration by 51.53% in maize, but increased significantly root La concentration by 30.45%. In addition, AMF decreased bacterial and fungal Shannon index in La0 treatment, but increased bacterial Shannon index in La500 treatment. Both AMF and La500 affected significantly the bacterial and fungal community composition, and AMF led to more influence than La. AMF promoted the enrichment of bacteria, including Planomicrobium, Lysobacter, Saccharothrix, Agrococcus, Microbacterium, Streptomyces, Penicillium and other unclassified genus, and fungi (Penicillium) in La500, which showed the function for promoting plant growth and tolerance of heavy metal. The study revealed that AMF can regulate the rhizosphere bacterial and fungal composition and foster certain beneficial microbes to enhance the tolerance of maize under La stress. Phytoremediation assisted by AMF is an attractive approach to ameliorate REEs-contaminated soils.

Pseudomonas pratensis sp. nov., Isolated from Grassland Soil from Inner Mongolia, China.

A novel bacterial strain, designated MHJ-10JT, was isolated from a soil sample obtained from a grassland in Inner Mongolia, China. MHJ-10JT strain could grow at 4-37 °C (optimum: 30 °C) and pH 4-9 (optimum: pH 6), as well as in the presence of 0-6% NaCl (optimum: 1%). Cells of strain MHJ-10JT are Gram-negative, rod-shaped, and motile. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MHJ-10JT was most closely related to Pseudomonas lutea OK2T (98.5% 16S rRNA gene sequence similarity). The values of the average nucleotide identities (ANI) and digital DNA-DNA hybridization (dDDH) between strain MHJ-10JT and its related species were all below 80.5% and 24.4%, respectively, which are significantly lower than the thresholds of 95% for ANI and 70% for DDH for species delineation. The genomic G + C content of the MHJ-10JT strain is 64.8 mol%. Based on the phenotypic, genotypic, chemotaxonomic, and phylogenetic analyses, strain MHJ-10JT can be assigned to the genus Pseudomonas. In this study, we propose that strain MHJ-10JT be classified as a novel species belonging to the genus Pseudomonas with the species name Pseudomonas pratensis sp. nov. The type strain of the proposed novel species is MHJ-10JT (= KCTC 82206T = CGMCC 17322T).

Soil fungal networks are more sensitive to grazing exclusion than bacterial networks.

Soil microbial communities play a crucial role in ecological restoration, but it is unknown how co-occurrence networks within these communities respond to grazing exclusion. This lack of information was addressed by investigating the effects of eight years of grazing exclusion on microbial networks in an area of Stipa glareosa P. Smirn desert steppe in northern China. Here, we show that fungal networks were more sensitive to grazing exclusion than bacterial networks. Eight years of grazing exclusion decreased the soil fungal community stability via changes in plant composition and reductions in soil total organic carbon, in this case triggering negative effects on the S. glareosa desert steppe. The results provide new insights into the response mechanisms of soil microbes to grazing exclusion and offer possible solutions for management issues in the restoration of degraded desert steppe.

Diversity of active root-associated methanotrophs of three emergent plants in a eutrophic wetland in northern China.

Root-associated aerobic methanotrophs play an important role in regulating methane emissions from the wetlands. However, the influences of the plant genotype on root-associated methanotrophic structures, especially on active flora, remain poorly understood. Transcription of the pmoA gene, encoding particulate methane monooxygenase in methanotrophs, was analyzed by reverse transcription PCR (RT-PCR) of mRNA isolated from root samples of three emergent macrophytes, including Phragmites australis, Typha angustifolia, and Schoenoplectus triqueter (syn. Scirpus triqueter L.) from a eutrophic wetland. High-throughput sequencing of pmoA based on DNA and cDNA was used to analyze the methanotrophic community. Sequencing of cDNA pmoA amplicons confirmed that the structure of active methanotrophic was not always consistent with DNA. A type I methanotroph, Methylomonas, was the most active group in P. australis, whereas Methylocystis, a type II methanotroph, was the dominant group in S. triqueter. In T. angustifolia, these two types of methanotroph existed in similar proportions. However, at the DNA level, Methylomonas was predominant in the roots of all three plants. In addition, vegetation type could have a profound impact on root-associated methanotrophic community at both DNA and cDNA levels. These results indicate that members of the genera Methylomonas (type I) and Methylocystis (type II) can significantly contribute to aerobic methane oxidation in a eutrophic wetland.

Enrichment of Type I Methanotrophs with nirS Genes of Three Emergent Macrophytes in a Eutrophic Wetland in China.

The pmoA gene, encoding particulate methane monooxygenase in methanotrophs, and nirS and nirK genes, encoding bacterial nitrite reductases, were examined in the root and rhizosphere sediment of three common emergent macrophytes (Phragmites australis, Typha angustifolia, and Scirpus triqueter) and unvegetated sediment from eutrophic Wuliangsuhai Lake in China. Sequencing analyses indicated that 334 out of 351 cloned pmoA sequences were phylogenetically the most closely related to type I methanotrophs (Gammaproteobacteria), and Methylomonas denitrificans-like organisms accounted for 44.4% of the total community. In addition, 244 out of 250 cloned nirS gene sequences belonged to type I methanotrophs, and 31.2% of nirS genes were the most closely related to paddy rice soil clone SP-2-12 in Methylomonas of the total community. Three genera of type I methanotrophs, Methylomonas, Methylobacter, and Methylovulum, were common in both pmoA and nirS clone libraries in each sample. A quantitative PCR (qPCR) analysis demonstrated that the copy numbers of the nirS and nirK genes were significantly higher in rhizosphere sediments than in unvegetated sediments in P. australis and T. angustifolia plants. In the same sample, the nirS gene copy number was significantly higher than that of nirK. Furthermore, type I methanotrophs were localized in the root tissues according to catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Thus, nirS-carrying type I methanotrophs were enriched in macrophyte root and rhizosphere sediment and are expected to play important roles in carbon/nitrogen cycles in a eutrophic wetland.

Molecular Analyses of the Distribution and Function of Diazotrophic Rhizobia and Methanotrophs in the Tissues and Rhizosphere of Non-Leguminous Plants.

Biological nitrogen fixation (BNF) by plants and its bacterial associations represent an important natural system for capturing atmospheric dinitrogen (N2) and processing it into a reactive form of nitrogen through enzymatic reduction. The study of BNF in non-leguminous plants has been difficult compared to nodule-localized BNF in leguminous plants because of the diverse sites of N2 fixation in non-leguminous plants. Identification of the involved N2-fixing bacteria has also been difficult because the major nitrogen fixers were often lost during isolation attempts. The past 20 years of molecular analyses has led to the identification of N2 fixation sites and active nitrogen fixers in tissues and the rhizosphere of non-leguminous plants. Here, we examined BNF hotspots in six reported non-leguminous plants. Novel rhizobia and methanotrophs were found to be abundantly present in the free-living state at sites where carbon and energy sources were predominantly available. In the carbon-rich apoplasts of plant tissues, rhizobia such as Bradyrhizobium spp. microaerobically fix N2. In paddy rice fields, methane molecules generated under anoxia are oxidized by xylem aerenchyma-transported oxygen with the simultaneous fixation of N2 by methane-oxidizing methanotrophs. We discuss the effective functions of the rhizobia and methanotrophs in non-legumes for the acquisition of fixed nitrogen in addition to research perspectives.

Intrathecal circHIPK3 shRNA alleviates neuropathic pain in diabetic rats.

Neuropathic pain is one of the most common diabetic complications and significantly decrease the quality of life. The aetiology of the painful diabetic neuropathic pain is not fully clear. Circular RNAs (circRNAs) have been identified as miRNA sponges and involved in various biological processes, including pain. CircHIPK3 is a circRNA that have been shown to be an oncogene or tumor suppressor to regulate cancer cells growth by sponging multiple miRNAs. However, the role of circHIPK3 in diabetic neuropathic pain remains unknown. The aim of the present study was to elucidate the possible role of circHIPK3 in the control of diabetic neuropathic pain. We found that circHIPK3 are highly abundant in serum from diabetes patients who suffered from neuropathic pain and in dorsal root ganglion from STZ-induced diabetes rats. Upregulation of circHIPK3 was positively associated with grade neuropathic pain in patients with type 2 diabetes. Silencing circHIPK3 alleviated neuropathic pain in diabetic rats, which was involved in neuroinflammation. Further mechanistic investigation demonstrated that circHIPK3 interacted with miR-124 and negatively regulated its expression. MiR-124 inhibitor can reverse circHIPK3 knockdown-mediated alleviation of neuropathic pain and inhibition of neuroinflammation in diabetic rats. We present the first evidence that intrathecal circHIPK3 shRNA treatment can be used to treat neuropathic pain of diabetic rats.