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Abstract

Biological nitrogen fixation, conducted by soil diazotrophs, is the primary nitrogen source for natural grasslands. However, the diazotrophs in grassland soils are still far from fully investigated. Particularly, their regional-scale distribution patterns have never been systematically examined. Here, soils (0–5 cm) were sampled from 54 grasslands on the Tibetan Plateau to examine the diazotroph abundance, diversity, and community composition, as well as their distribution patterns and driving factors. The diazotroph abundance was expressed as nifH gene copies, measured using real-time PCR. The diversity and community composition of diazotrophs were analyzed through MiSeq sequencing of nifH genes. The results showed that Cyanobacteria (47.94%) and Proteobacteria (45.20%) dominated the soil diazotroph communities. Most Cyanobacteria were classified as Nostocales which are main components of biological crusts. Rhizobiales, most of which were identified as potential symbiotic diazotrophs, were also abundant in approximately half of the soil samples. The soil diazotroph abundance, diversity, and community composition followed the distribution patterns in line with mean annual precipitation. Moreover, they also showed significant correlations with prokaryotic abundance, plant biomass, vegetation cover, soil pH values, and soil nutrient contents. Among these environmental factors, the soil moisture, organic carbon, available phosphorus, and inorganic nitrogen contents could be the main drivers of diazotroph distribution due to their strong correlations with diazotroph indices. These findings suggest that autotrophic and symbiotic diazotrophs are the predominant nitrogen fixers in Tibetan grassland soils, and highlight the key roles of water and nutrient availability in determining the soil diazotroph distribution on the Tibetan Plateau.

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... Nitrogen (N) is a crucial nutrient for plants, and its availability for plants modulates ecosystem productivity and function. Globally, the primary productivity of terrestrial ecosystems is largely limited by the availability of soil N, particularly in highaltitude regions where environmental conditions exert important influences on biogeochemical cycling processes [1][2][3]. The Qinghai-Xizang Plateau, often referred to as the "Roof of the World", encompasses unique ecosystems that are sensitive to global climate change [3,4]. ...
... Globally, the primary productivity of terrestrial ecosystems is largely limited by the availability of soil N, particularly in highaltitude regions where environmental conditions exert important influences on biogeochemical cycling processes [1][2][3]. The Qinghai-Xizang Plateau, often referred to as the "Roof of the World", encompasses unique ecosystems that are sensitive to global climate change [3,4]. Despite its ecological significance, our current understanding regarding soil N status and the associated biogeochemical cycling across the extensive elevational gradients of the Qinghai-Xizang Plateau remains limited. ...
... The Hengduan Mountains, located in the southeastern Qinghai-Xizang Plateau, are characterized by distinctive geographical features with complex terrain, deep valleys, and extensive primeval forests under the influence of warm Indian monsoon [3]. This region undergoes a transition from arid and hot valleys to alpine climates with elevation increases, which makes the area rich in alpine flora, and it is recognized as one of the top 10 biodiversity hotspots and one of the areas most affected by climate change [18]. ...
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Alpine forest soil in the southeastern Qinghai–Xizang Plateau plays a crucial role in regional and global climate and biogeochemical cycles, yet the elevational distribution of soil nitrogen (N) availability and losing risk is unresolved. In this study, we characterized soil N composition and key N transformation processes across different elevations in 3 typical mounts of the Qinghai–Xizang Plateau. In contrast to previous suggestions, soil total N and ammonium are found to accumulate in high elevation zones. This accumulation of N at higher altitudes is due to a consistent soil net N mineralization rate coupled with an extremely low net nitrification rate, which is suppressed by low pH and high soil moisture in high elevation zones. Moreover, the elevated rates of biological N fixation along the elevation further contribute to N accumulation in high elevation regions in which the acid-tolerant Bradyrhizobium, plant-associated Herbaspirillum, and Klebsiella are identified as the key diazotrophic microbial taxa responsible for active N fixation. Collectively, our results suggest that total N and NH4⁺-N accumulation in higher altitude zone is a ubiquitous phenomenon in the southeast Qinghai–Xizang Plateau, with lower nitrification rates and higher biological nitrogen fixation being key processes enabling this occurrence.
... In addition, the Qinghai-Tibet grasslands receive a negligible amount of nitrogen from anthropogenic sources. Therefore, the main source of available nitrogen in the region is likely to be biological nitrogen fixation (Che et al., 2018). Over the years, soil microorganisms in the Qinghai-Tibet Plateau have received much attention due to their crucial role in nitrogen fixation (Deng et al., 2013;Che et al., 2015). ...
... Various soil microorganisms, including fungi, bacteria, and archaea, have been well studied in this region (Chu et al., 2016;Shi et al., 2016;Yang et al., 2017). Previous studies have explored the symbiotic diazotrophs associated with legumes on the Qinghai-Tibet Plateau (Che et al., 2018;Zhang et al., 2022;Sun et al., 2023). However, the free-living diazotrophs remain poorly understood. ...
... This contrasts with the findings of Keuter et al. (2014) and Li et al. (2019), which showed lower levels of nitrogen fixation rates in grassland soils, respectively. Diazotrophic abundance was significantly influenced by soil pH (Tai et al., 2013) and soil C: N ratio (Singh et al., 2011), and a strong correlation between soil moisture and the diazotrophic abundance was also observed (Che et al., 2018). Therefore, the high diazotrophic abundance could be attributed to the optimal environmental factors in bulk and rhizosphere soils on the plateau. ...
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Symbiotic diazotrophs form associations with legumes and substantially fix nitrogen into soils. However, grasslands on the Qinghai-Tibet Plateau are dominated by non-legume plants, such as Kobresia tibetica. Herein, we investigated the diazotrophic abundance, composition, and community structure in the soils and roots of three plants, non-legume K. tibetica and Kobresia humilis and the legume Oxytropis ochrocephala, using molecular methods targeting nifH gene. Diazotrophs were abundantly observed in both bulk and rhizosphere soils, as well as in roots of all three plants, but their abundance varied with plant type and soil. In both bulk and rhizosphere soils, K. tibetica showed the highest diazotroph abundance, whereas K. humilis had the lowest. In roots, O. ochrocephala and K. humilis showed the highest and the lowest diazotroph abundance, respectively. The bulk and rhizosphere soils exhibited similar diazotrophic community structure in both O. ochrocephala and K. tibetica, but were substantially distinct from the roots in both plants. Interestingly, the root diazotrophic community structures in legume O. ochrocephala and non-legume K. tibetica were similar. Diazotrophs in bulk and rhizosphere soils were more diverse than those in the roots of three plants. Rhizosphere soils of K. humilis were dominated by Actinobacteria, while rhizosphere soils and roots of K. tibetica were dominated by Verrumicrobia and Proteobacteria. The O. ochrocephala root diazotrophs were dominated by Alphaproteobacteria. These findings indicate that free-living diazotrophs abundantly and diversely occur in grassland soils dominated by non-legume plants, suggesting that these diazotrophs may play important roles in fixing nitrogen into soils on the plateau.
... The effects of grazing practice on the abundance of functional genes involved in soil N cycling have been documented in recent years (Ding et al., 2014;Song et al., 2019). Diazotrophs are highly diverse in phylogeny and in a wide distribution in the QTP, the abundance, Shannon diversity, and community composition of soil diazotrophs were significantly correlated with soil moisture (Che et al., 2018), while another study showed that N-fixing communities (nifH) were most affected by the soil C:N ratio (Singh et al., 2011). Moreover, the variation in diazotroph community composition has a greater impact on N-fixation rates than did soil characteristics (Hsu and Buckley, 2009), it is inconsistent with a research in a tallgrass prairie used primarily for cattle grazing and agriculture, where they found that abundance of nifH genes was not significantly correlated with N2-fixation rates (Caton et al., 2018). ...
... The copies of nifH gene were quantified using ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The quantification was conducted with universal primer sets for nifH, PolF: TGC GAY CCS AAR GCB GAC TC; PolR: ATS GCC ATC ATY TCR CCG GA (Poly et al., 2001;Che et al., 2018). The 10 μL reaction systems contained: 4.4 μL of SYBR Green Mix, 0.3 μL of forward primer (20 μmol L −1 ), 0.3 μL of reverse primer (20 μmol L −1 ) and 5 μL of template DNA. ...
... With an identify cutoff of 97%, we obtained 2,421 OTUs. More details of the taxonomic assignment for nifH OTUs was conducted similarly as described by (Che et al., 2018). The data were analyzed on the online platform of Majorbio Cloud Platform. 1 1 www.majorbio.com ...
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Grazing by local livestock is the traditional human practice in Qinghai-Tibetan Plateau grassland, and moderate intensity grazing can maintain high productivity and diversity of alpine grassland. Grazing ecosystems are often nitrogen-limited, but N2-fixing communities in response to yak grazing and Tibetan sheep grazing in Qinghai-Tibetan Plateau grassland have remained underexplored. In this study, we applied quantitative PCR quantitation and MiSeq sequencing of nifH under yak grazing and Tibetan grazing through a manipulated grazing experiment on an alpine grassland. The results showed that the grazing treatments significantly increased the soil ammonium nitrogen (AN) and total phosphorus (TP), but reduced the diazotrophs abundance. Compared with no grazing treatment, the composition of diazotrophs could be maximally maintained when the ratio of yak and Tibetan sheep were 1:2. The foraging strategies of grazing livestock reduced the legumes biomass, and thus reduced the diazotrophs abundance. Data analysis suggested that the direct key factors in regulating diazotrophs are AN and TP, and the changes of these two soil chemical properties were affected by the dung and urine of herbivore assemblages. Overall, these results indicated that the mixed grazing with a ratio of yak to Tibetan sheep as 1:2 can stabilize the soil diazotrophsic community, suggesting that MG12 are more reasonable grazing regimes in this region.
... The relative abundances of the order Nostocales and genus Nostoc were higher in the upper slopes than the lower slopes. This could be explained by the fact that Nostoc that belongs to Nostocales performs well in extreme situations [45,46], such as upper slopes with poor water-holding environments because of the shallow and discontinuous soil. Moreover, Nostocales also participate in the formation of biological crusts [47]. ...
... Moreover, Nostocales also participate in the formation of biological crusts [47]. Generally, biological crusts occur frequently in grasslands due to suitable shading and sunlight, leading to a high abundance of Nostocales on the upper slope [46]. For root AMF species, Paraglomerales play an important role in plant nutrient absorption and transfer under low AP levels, especially in the soil rhizosphere [48][49][50]. ...
... A random forest model was constructed to better understand the main contribution of soil properties and nutrients. Many previous studies reported that soil environment conditions (e.g., moisture) and nutrients (e.g., AP and AN) were strongly related to diazotroph abundance and diversity [2,25,46]. In the present study, the variation in diazotroph abundance was explained more by AN (Figure 4). ...
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Considering the crucial role of soil diazotrophs and root arbuscular mycorrhizal fungi (AMF) in soil nutrient cycling during ecosystem restoration, diazotroph and AMF communities may be determined by slope position. However, the effect of slope position on diazotroph and AMF abundance, diversity, and community composition of karst ecosystems remains unknown. In this study, soil diazotrophs and root AMF characteristics on varying slope positions were assessed in a karst shrub ecosystem. The results displayed that the abundance of soil diazotrophs and root AMF diversity were significantly affected by slope position. Diazotroph abundance accompanied by soil nutrient and plant richness was higher on the lower slopes than the upper slopes, whereas root AMF diversity displayed the opposite trend. The soil diazotroph and root AMF community composition differed among the upper, middle, and lower slopes. The dominant taxa of soil diazotrophs and root AMF at the order level were Rhizobiales and Glomerales, respectively. Moreover, the diazotroph order of Nostocales and the AMF order of Paraglomerales were richer on the upper slopes than on the lower slopes. The slope position directly affected the plant diversity and soil nutrient distribution, indirectly affecting the diazotroph and AMF communities. Increased available nitrogen on the lower slope caused great diazotroph abundance by stimulating plant growth with sufficient carbohydrates. However, low soil nutrients and plant diversity but high plant root biomass induced more root AMF diversity on the upper slope than on the lower slope. Therefore, this study expands the knowledge of soil diazotroph and root AMF ecological functions along different slope positions during vegetation recovery for the successive stages of grass and shrub in the karst region.
... Many factors may influence diazotrophs, including soil pH, moisture and nutrient availability (Che et al. 2018;Feng et al. 2019;Wang et al. 2017). For example, an increase in soil pH was found to increase soil diazotrophs' α-diversity (Wang et al. 2017). ...
... Field sampling was conducted in the growing season of 2019. We collected nodules, soil and kudzu leaves from each plot based on a zigzag sampling protocol (Carter and Gregorich 2007). Thirty nodules were sampled within a plot. ...
... Soil pH was assayed by a pH meter after extracting soil with water. Wet oxidation with K 2 CrO 7 + H 2 SO 4 was adopted to extract soil organic C (SOC) and leaf C and titrated using FeSO 4 solution (Carter and Gregorich 2007;Zhou et al. 2021). Soil inorganic N and available P were extracted with 2 M KCl and 0.5 M NaHCO 3 , respectively (Carter and Gregorich 2007;Zhou et al. 2021). ...
Article
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Biological N2 fixation (BNF) is a major pathway of external nitrogen input to terrestrial ecosystems. Nevertheless, the relative effects of diazotrophic community and soil properties on symbiotic (SNF) or free-living (FNF) N2 fixation remain poorly understood. Here, 20 sites of kudzu (Pueraria lobata) communities, which were at the early succession stage following agricultural abandonment, were selected across a karst area in southwest China. BNF rates were determined using the acetylene reduction assay calibrated with the ¹⁵N2 fixation method. The average rate was 17.81 ± 2.17 mg N g⁻¹ day⁻¹ nodule for SNF and 45.36 ± 5.15 ng N g⁻¹ day⁻¹ soil for FNF. Diazotrophic communities were dominated by Bradyrhizobium at the genus level in both nodule and soil. According to structural equation modeling, the relative abundances of two diazotrophic genera (Desulfovibrio and Geobacter) were the strongest explanatory variable for the variation of SNF rates with the second strongest variable being nifH gene abundance. Soil water content and available phosphorus indirectly affected SNF via their effects on diazotrophic community composition. In contrast, soil FNF rates were most pronouncedly affected by the availability of vanadium and iron, followed by soil water content, available phosphorus, and the relative abundances of two diazotrophic genera, i.e., Burkholderia and Cupriavidus. Our findings therefore help to improve the understanding of the relationship between BNF rates and their associated diazotrophic communities, and hence benefit a better prediction of BNF under global change.
... In recent years, the asymbiotic nitrogen fixation rate (ANFR) has been widely reported to be closely related to different soil physicochemical properties, such as organic carbon (Huang et al. 2022), nitrogen (Che et al. 2018;Wang et al. 2021), phosphorus (Tang et al. 2017) and trace elements (Vitousek et al. 2002). ANFR or nifH gene copy numbers in soil increased after the input of the above substances Tang et al. 2017;Wang et al. 2021). ...
... Since diazotrophic microbial community need carbon and energy supply for nitrogen fixing process, the high C:N ratio is benefit for the increase of diazotrophic microbial community abundance. The nitrogen levels in soil are a crucial factor influencing the diazotrophic community, although the relationship between nifH gene copy number and TN appears to be controversial (Che et al. 2018;Chen et al. 2021;Keshri et al. 2015). In this study, we found that total N and the C:N ratio had no effect on the nifH gene abundance but showed a significant correlation with the N fixing rate. ...
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Purpose To clarify the geographical distribution pattern of asymbiotic nitrogen activity and diazotrophic community in paddy soils, and to understand the primary environmental driving factors driving asymbiotic nitrogen fixation rate (ANFR) and community diversity. Materials and methods The ANFR and the environmental driving factors of diazotrophic community diversity were investigated in 76 paddy soils across 6 major rice-growing regions in China by ¹⁵N2 labeling and nifH gene high-throughput sequencing. Results and discussions Among all the sample sites, the northeast region had relatively higher ANFR (p < 0.05) compared to other regions. There was no significant correlation between soil ANFR and nifH abundance. The ANFR was significantly affected by Mo, total N and total P, while C:N ratio, exchangeable Ca and pH had a significant influence on nifH abundance. Proteobacteria, Thermodesulfobacteriota and Cyanobacteria dominated within the diazotrophic community across the paddy soils. Furthermore, 50.7% of the variance in community compositions could be attributed to different environmental variables. These multifactorial drivers, including Ca, C:N ratio, Mg, Na, Mn, N:P ratio, Mo and pH, significantly influence formation of specific diazotrophic microbial groups. Conclusions (1) No significant correlation between the rate of asymbiotic nitrogen fixation in paddy soil and the abundance of the nifH gene. (2) The ANFR was significantly positively correlated with the molybdenum, phosphorus, potassium, nitrogen and organic carbon contents. (3) The abundance of the nifH gene was primarily correlated with Ca content and the soil C:N ratio. (4) Distinct diazotrophic communities are influenced by different environmental drivers. (5) Alphaproteobacteria and Spirochaetota are more abundant in soils with high elevated nitrogen and molybdenum content, indicating their significant contributions to nitrogen fixation in paddy soil.
... This may be because the soil environment with good water or nutrients is beneficial to the survival and reproduction of Proteobacteria, while Actinobacteria is more suitable for poor soil. We also detected a high proportion of Verrucomicrobia in the soil of the alpine wetland, which may be attributed to its excellent survival ability in extreme environments (Che et al., 2018). However, their abundance did not change significantly during wetland degradation. ...
... nitrogen-fixing bacteria. First, it can be attributed to collinearity between soil nitrogen content and other soil properties (such as moisture, organic carbon, and total phosphorus), which cancels or even reverses the correlation between soil nitrogen content and nitrogen-fixing bacteria abundance (Che et al., 2018). Second, the positive correlation between soil nitrogen content and nitrogen-fixing bacteria may indicate that the community structure of nitrogen-fixing bacteria with different environmental preferences has changed due to the deterioration of the wetland soil environment. ...
Article
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Biological nitrogen fixation is a key process in the nitrogen cycle and the main source of soil available nitrogen. The number and diversity of nitrogen-fixing bacteria directly reflect the efficiency of soil nitrogen fixation. The alpine wetland on the Qinghai-Tibet Plateau (QTP) is degrading increasingly, with a succession toward alpine meadows. Significant changes in soil physicochemical properties accompany this process. However, it is unclear how does the soil nitrogen-fixing bacteria change during the degradation processes, and what is the relationship between these changes and soil physicochemical properties. In this study, the nifH gene was used as a molecular marker to further investigate the diversity of nitrogen-fixing bacteria at different stages of degradation (none, light, and severe degeneration) in the alpine wetland. The results showed that wetland degradation significantly reduced the diversity, altered the community composition of nitrogen-fixing bacteria, decreased the relative abundance of Proteobacteria, and increased the relative abundance of Actinobacteria. In addition to the dominant phylum, the class, order, family, and genus of nitrogen-fixing bacteria had significant changes in relative abundance. Analysis of Mantel test showed that most soil factors (such as pH, soil water content (SWC), the organic carbon (TOC), total nitrogen (TN), and soil C:P ratio) and abundance had a significant positive correlation. TOC, TN, total phosphorus (TP), soil C:P ratio and Shannon had a significant positive correlation with each other. The RDA ranking further revealed that TOC, SWC, and TN were the main environmental factors influencing the community composition of nitrogen-fixing bacteria. It is found that the degradation of the alpine wetland inhibited the growth of nitrogen-fixing bacteria to a certain extent, leading to the decline of their nitrogen-fixing function.
... For example, we used SIMPER analysis to identify Nostocales, a dominant diazotrophic taxon, as an indicator of the orders and as a dominant group (Fig. S3 and Table 2). This result was supported by several studies reporting the dominance of Nostocales in various environments and their significant contributions to N fixation (Dodds et al., 1995;Potts, 1994;Yeager et al., 2007;Che et al., 2018). The relative abundance of Nostocales was significantly lower in NK, NP and NPK than other treatments, similar to the 8-23 % of relative abundance lower after N fertilization reported by Kuppusamy et al. (2018). ...
... Gupta (2000) recognized Proteobacteria as a functionally diverse group of bacteria and divided it into chemoorganotrophs and chemolithotrophs, which are involved in nutrient transformation depending on the substrates they use. Dominant and active orders that are more abundant than the DNA-derived orders in our study could positively participate in biogeochemical cycling, such as N fixation (members of Rhizobiales and Rhodospirillales), nitrification (members of Nitrosomonadales) and C sequestration (members of Myxococcales), and the direct and indirect effects of N on these processes mediated by microbes have also been demonstrated previously Zhou et al., 2014;Che et al., 2018). ...
Article
Soil microbial communities play a vital role in mediating nutrient turnover, thus enhancing growth and development of plants. Understanding the dynamics of microorganisms in soils can provide insight into the influence of fertilization practices on improving soil fertility and plant growth in agricultural ecosystems. In this study, we compared the abundances and compositions of total (DNA-based, 16S rRNA gene) and active (RNA-based, 16S rRNA) bacterial communities at a 30-year experimental site in different inorganic fertilization treatments with different key elements (nitrogen, phosphorus, and potassium). The inorganic fertilizer amendments did not affect the abundance of total bacteria but significantly affected the abundance of active bacteria due to changes in microbial biomass carbon and NH4⁺-N contents. Cyanobacteria and Proteobacteria, especially for some dominant orders (e.g. Nostocales, Pseudanabaenales and Nitrosomonadales) were the dominant phyla in the active microbial community and differed proportionally in nitrogen and phosphorus fertilized soil. Soil N speciation (e.g. total N, NH4⁺-N and NO3⁻-N) were the main determinants controlling the Cyanobacteria and Proteobacteria communities. Our results indicated that the unbalanced fertilization could reduce the abundance of active bacteria and significantly changed the dominant phyla compared with balanced fertilization. These findings provided an insight of composition and ratio of nutrient elements including nitrogen, phosphorus and potassium for managing future fertilization regimes in agricultural ecosystem.
... The family Azospirillaceae was recently revised, based on genome sequence homologies, and the genus Skermanella is phylogenetically closely related to the genus Azospirillum, which comprises diazotrophic soil bacteria [2]. A metagenomic analysis of soil samples and a cDNA analysis of the nifH gene, which encodes the reductase component of Mo-nitrogenase, have shown that Skermanella strains are dominant and active diazotrophs in the rhizospheres of various crops, including cucumber [3], grapevine [4], tobacco [5], and coffee [6], as well as in Tibetan grassland soils [7]. Furthermore, recent research identified Skermanella as a core member of the diazotroph community in biological soil crusts (BSCs) from temperate semi-arid and arid deserts of China [8]. ...
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The genus Skermanella comprises important soil bacteria that are often associated with the crop rhizospheres, but its physiological traits remain poorly understood. This study characterizes Skermanella sp. TT6T, isolated from human skin, with a focus on its metabolic and environmental adaptations. Genome sequencing and phylogenomic analyses revealed that the strain TT6T is most closely related to S. rosea M1T, with average nucleotide identity and digital DNA–DNA hybridization values of 94.14% (±0.5%) and 64.7%, respectively. Comparative genomic analysis showed that the strains TT6T, S. rosea M1T and S. mucosa 8-14-6T share the Calvin cycle, and possess photosynthetic genes associated with the purple bacteria-type photosystem II. The strains TT6T and S. rosea M1T exhibited growth in a nitrogen-free medium under microaerobic conditions, which were generated in test tubes containing 0.1% soft agar. Under these conditions, with nitrate as a nitrogen source, S. rosea M1T formed gases, indicating denitrification. Strain TT6T also contains gene clusters involved in trehalose and carotenoid biosynthesis, along with salt-dependent colony morphology changes, highlighting its adaptive versatility. Genomic analyses further identified pathways related to hydrogenase and sulfur oxidation. Phenotypic and chemotaxonomic traits of strain TT6T were also compared with closely related type strains, confirming its genotypic and phenotypic distinctiveness. The new species, Skermanella cutis sp. nov., is proposed, with TT6T (=KCTC 82306T = JCM 34945T) as the type strain. This study underscores the agricultural and ecological significance of the genus Skermanella.
... A cultureindependent study of nitrogen-fixing bacteria associated with Switchgrass [50] using the nifH 3 and nifH 4 primers revealed diazotrophic species from Alpha-, Beta-, Delta-, Gamma-Proteobacteria, and Bacillota only. A study of Tibetan grassland soils using PolF/R primers reported Cyanobacteriota, Pseudomonadota, and Verrucomicrobiota [51]. The absence of diazotrophic Actinobacteriota nifH in culture-independent studies could be the result of nif primer bias. ...
Article
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Nitrogen inputs for sustainable crop production for a growing population require the enhancement of biological nitrogen fixation. Efforts to increase biological nitrogen fixation include bioprospecting for more effective nitrogen-fixing bacteria. As bacterial nitrogenases are extremely sensitive to oxygen, most primary isolation methods rely on the use of semisolid agar or broth to limit oxygen exposure. Without physical separation, only the most competitive strains are obtained. The distance between strains provided by plating on solid media in reduced oxygen environments has been found to increase the diversity of culturable potential diazotrophic bacteria. To obtain diverse nitrogen-fixing isolates from natural grasslands, we plated soil suspensions from 27 samples onto solid nitrogen-free agar and incubated them under atmospheric and oxygen-reducing conditions. Putative nitrogen fixers were confirmed by subculturing in liquid nitrogen-free media and PCR amplification of the nifH genes. Streaking of the 432 isolates on nitrogen-rich R2A revealed many cocultures. In most cases, only one community member then grew on NFA, indicating the coexistence of nonfixers in coculture with fixers when growing under nitrogen-limited conditions. To exclude isolates able to scavenge residual nitrogen, such as that from vitamins, we used a stringent nitrogen-free medium containing only 6.42 μmol/L total nitrogen and recultured them in a nitrogen-depleted atmosphere. Surprisingly, PCR amplification of nifH using various primer pairs yielded amplicons from only 17% of the 442 isolates. The majority of the nifH PCR-negative isolates were Bacillus and Streptomyces. It is unclear whether these isolates have highly effective uptake systems or nitrogen reduction systems that are not closely aligned with known nitrogenase families. We advise caution in determining the nitrogen fixation ability of plants from growth on nitrogen-free media, even where the total nitrogen is very limited.
... Dechloromonas [43], Paracoccus, and Thauera [44] exhibit metabolic activities associated with both nitrification and denitrification. Rhizobium fixes molecular nitrogen into organic ammonium [45], and Methylobacterium shares a similar metabolic pathway with Rhizobium [46]. The total percentage of these nitrification-related microorganisms was 45%, although each individual contribution was relatively small. ...
Article
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The removal of nitrogen compounds in wastewater has been successfully developed with various activated sludge-based processes. Microorganisms immobilized in media would enhance biological efficiency by the increase in biomass concentration; however, the microbial community composition in media has not been revealed. Attached microbial communities on immobilization media were analyzed after the operation of the wastewater treatment process, comparing aerobic and anoxic reactors. A modified Ludzack–Ettinger (MLE) process was operated with immobilized media with polyvinyl alcohol and polyethylene glycol. The mixed liquor suspended solid (MLSS) concentration in an aerobic reactor was maintained at 50,000 mg/L and 40,000 mg/L in an anoxic reactor by the media. A maximum of 99% of ammonium nitrogen from the influent was calculated to be oxidized; however, the organic nitrogen produced from microbial growth reduced the overall oxidation rate. The denitrification rate increased with the addition of glucose to adjust the carbon-to-nitrogen (C/N) ratio. Based on the total nitrogen concentration, the nitrogen removal efficiency was calculated to be 48.2% following the adjustment of the C/N ratio. A phylogenetic analysis of the microbial community in immobilized media using next-generation sequencing (NGS) revealed the dominance of nitrifying and denitrifying microorganisms in the aerobic and anoxic reactors, respectively. Sequences amplified using V3–V4 region primers of the 16S rRNA gene yielded 531,188 base pairs (bp) and 396,844 bp reads from the aerobic and anoxic reactors, respectively. Operational taxonomic units (OTUs) were identified at both the phylum and genus levels, with a total of 594 from the aerobic reactor and 375 from the anoxic reactor. Proteobacteria was the dominant phylum in both the aerobic and anoxic reactors, comprising 39.7% of the aerobic reactor and 65.9% of the anoxic reactor. The dominant genera in the aerobic reactor were Nitrospira and Povalibacter. Forty-five percent of the total number of OTUs consisted of known nitrification-related genera in the aerobic reactor. In contrast, the dominant genera in the anoxic reactor were Desulfomicrobium, Desulfobulbus, and Methyloversatilis. A total of 63% of the genera associated with denitrification, including Dechloromonas and Flavobacterium, were found in the anoxic reactor. The population of microorganisms in each reactor was compared in terms of diversity by the QIIME 2 algorithm. The Chao1 index values of α-diversity were 606.05 for the aerobic reactor and 415.53 for the anoxic reactor, indicating greater population diversity in the aerobic reactor compared to the anoxic one. The widespread distribution of nitrification activities among various groups has led to diverse population characteristics in the aerobic environment, particularly within the attached community. The microbiological community present in immobilized aerobic and anoxic media will contribute to future microbial studies on wastewater treatment processes.
... structure of diazotrophs in soil are strongly variable and are highly sensitive to multiple soil factors, such as pH [29], organic matter and available nutrient content [30]. Among these factors, soil pH and soil nutrients (N and P) are key factors affecting diazotroph diversity, community composition in different soils [31,32]. Therefore, it has been acknowledged that ecology has a significant role in inoculant survival, emphasis on target functional features in published literature, rather than establishment/survival traits indicates that this integrated perspective has not yet received enough attention [33]. ...
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Mesocosm based experiment was conducted to evaluate and compare the performance of selected three promising diazotrophic bacterial isolates (B1, B2, B3, identified as Brucella oryzae, Brucella ciceri and Pseudomonas nitroreducens respectively) isolated from acidic soils of Jharkhand; pH 5.5–6.3). The experiment aimed to assess their efficacy on improving plant growth and soil nutrient availability in wheat grown under contrasting soil pH conditions—acidic soil from Jharkhand (pH: 6.3) and neutral soils from New Delhi (pH: 7.8) conducted under controlled conditions at National Phytotron Facility, of Indian Agricultural Research Institute, New Delhi. Sampling done at 4 and 8 weeks after sowing revealed significant enhancement of 15–20% in N availability and organic C in soil, as well as improvement in plant biometrical and physiological attributes due to microbial inoculation. Additionally, there was a 15–30% increase in dehydrogenase activity and plant N content. Inoculation also led to a sharp increase in indole acetic acid (IAA) content in roots and leaves from plants grown in soils under both pH conditions, along with stimulation of leaf glutamine synthetase activity. Among the isolates, B1 was found to be superior in terms of plant growth enhancement and improving soil biological properties, while the combination of B1 + B2 was equally promising, as supported by PCA biplot analysis. Inoculation with these diazotrophic bacterial isolates positively influenced both soil properties and plant attributes, with more distinct effects observed under acidic soils of Jharkhand. The findings of this study provide a scientific foundation towards diazotrophic bacterial inoculation and development of region-specific growth invigorating inoculants for enhancing wheat production in acidic ecologies, with economic gains through the reduced use of N-fertilizers, and 25% N savings.
... From moderate to high N input significantly reduced the abundance of diazotrophic and their diversity, while P addition increased the abundance and association of diazotroph. At the same time, soil diazotrophic communities can cope with P limitation by changing their life history strategies (oligotrophic to symbiotic nutrients) [20]. Thus, understanding how the response of the diazotrophic community to P input during the green manure season in a green manure-rice rotation is essential. ...
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Chinese milk vetch (CMV) is widely recognized as the leading leguminous green manure utilized in the rice-green manure rotation system throughout southern China. While bacteria that form symbiotic relationships with CMV are responsible for fixing a significant portion of nitrogen (N) within agroecosystems. diazotrophic organisms play an essential role in the N cycle and enhance the pool of N readily accessible to plants. The goals of the current study were to investigate the effects of shifting partial phosphorus (P) fertilizer application from the rice season to the CMV season within a CMV-rice rotation system on soil nutrient levels, activity of soil enzymes and stoichiometric ratios, as well as diazotrophic community structure. The treatments consisted of a control group, a winter fallow-rice rotation without fertilizer application, and the treatments P0, P1, P2, and P3, representing 0, 1/3, 2/3, and the full dose, respectively, of phosphorus fertilizer (60 kg ha⁻¹ P2O5) added in a single rotation system during the CMV season, while combined with 60 % of regular N application rate during the rice season. In comparison to P0, the application of treatments P1, P2, and P3 resulted in higher CMV dry biomass and rice production across the seasons from 2018 to 2021 and the P2 treatment significantly increased the contents of total N (TN), soil organic matter (OM), and available P (AP) by 49 %, 48 %, and 110 %, respectively. The activities of alkaline phosphatase and L-leucine aminopeptidase showed a significant decrease when subjected to the P1 and P2 treatments. The P2 treatment enhanced the relative abundance of Frankia and Skermanella by 2.6 % and 1.6 %, respectively, comparing with P0 treatment. Furthermore, correlation analysis revealed a positive relationship between Skermanella and Mesorhizobium with the contents of TN, OM, AP, ammonium-N, and nitrate-N. In conclusion, the application of 1/3 to 2/3 of the full dose P fertilizer in CMV season reshaped soil diazotrophic community, improved soil N content, and thereby increased rice yield with 40 % N fertilizer reduction.
... Plants shape the surrounding microbiome by secreting exudates [84]. Soil nutrients are a key parameter affecting the nitrogen-fixing bacterial community, and the increase in soil nutrient content will lead to a substantial increase in nifH gene abundance [85]. Bradyrhizobium and Bacillus abundance increased after planting trees and shrubs ( Figures 5F and 6F). ...
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The remediation and exploitation of sandy saline soils, an underutilized resource, can be enhanced by a greater comprehension of the impact of plants and microorganisms on nutrient cycling. However, there is scant research information on the capacity of different trees and shrubs to improve carbon and nitrogen cycling in saline soils at different depth layers. This study investigated the effect of the trees Zelkova serrata (ZS) and Ligustrum lucidum (LL) and shrub Hibiscus syriacus (HS) on the carbon and nitrogen fractions, soil enzyme activities and microbial communities in sandy saline soils. Planting ZS, LL or HS improved soil quality, increased soil carbon and nitrogen content, changed rhizosphere soil metabolites and enhanced soil enzyme activities and microbial abundance and diversity. Compared to values in the bare soil, the highest reduction in soil salinity was noticed under Zelkova serrata (49%) followed by Ligustrum lucidum (48%). The highest increase in total soil organic carbon (SOC) was noted under Ligustrum lucidum and Hibiscus syriacus (62% each), followed by Zelkova serrata (43%), as compared to levels in the bare soil. In the 0–10 cm soil layer, the total N in bare soil was 298 ± 1.48 mg/kg, but after planting LL, ZS or HS, the soil total N increased by 101%, 56% and 40%, respectively. Compared with that of the bare soil, cbbL sequencing showed that the relative abundance of Bradyrhizobium increased and that of Bacillus decreased due to planting. Similarly, the nifH sequencing results indicated that the relative abundance of Bradyrhizobium and Motiliproteu increased and that of Desulfuromonas and Geoalkalibacter decreased. These findings suggested that soil microorganisms could play a pivotal role in the carbon and nitrogen cycle of saline soils by influencing the content of soil carbon and nitrogen.
... Although nitrogenase genes (nif) are conserved in a broad taxonomic range of prokaryotes [6], nif genes derived from Alphaproteobacteria, Betaproteobacteria, and Cyanobacteria have been frequently detected in various soil environments such as farmland, grassland, forests, rice paddy fields, riparian zones, and tundra by PCR amplicon surveys targeting nif genes [7][8][9][10][11]. Consequently, these bacteria are considered the primary nitrogen fixers in soil [12,13]. ...
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Background Biological nitrogen fixation is a fundamental process sustaining all life on earth. While distribution and diversity of N2-fixing soil microbes have been investigated by numerous PCR amplicon sequencing of nitrogenase genes, their comprehensive understanding has been hindered by lack of de facto standard protocols for amplicon surveys and possible PCR biases. Here, by fully leveraging the planetary collections of soil shotgun metagenomes along with recently expanded culture collections, we evaluated the global distribution and diversity of terrestrial diazotrophic microbiome. Results After the extensive analysis of 1,451 soil metagenomic samples, we revealed that the Anaeromyxobacteraceae and Geobacteraceae within Deltaproteobacteria are ubiquitous groups of diazotrophic microbiome in the soils with different geographic origins and land usage types, with particular predominance in anaerobic soils (paddy soils and sediments). Conclusion Our results indicate that Deltaproteobacteria is a core bacterial taxon in the potential soil nitrogen fixation population, especially in anaerobic environments, which encourages a careful consideration on deltaproteobacterial diazotrophs in understanding terrestrial nitrogen cycling. D657cgwaNSF-wLxbnJEePrVideo Abstract
... As mentioned previously, FF favors the establishment and development of biological crusts, leading to enhanced C exudation [26]. That explains the abundant presence of N-fixing Nostoc and Rhizobium in FF, supported by other studies that suggest biological crusts may result in high proportions of N-fixing Nostocales under grassland degradation in the Tibetan Plateau [42]. In addition, low soil nutrient (e.g., NO 3 − , AP, and AK) conditions may serve as the primary stimulus for the proliferation of cyanobacteria genera like Anabaena and Nostoc. ...
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The crucial functional arbuscular mycorrhizal fungi (AMF) and diazotrophs play pivotal roles in nutrient cycling during vegetation restoration. However, the impact of managed vegetation restoration strategies on AMF and diazotroph communities remains unclear. In this study, we investigated the community structure and diversity of AMF and diazotrophs in a karst region undergoing managed vegetation restoration from cropland. Soil samples were collected from soils under three vegetation restoration strategies, plantation forest (PF), forage grass (FG), and a mixture of plantation forest and forage grass (FF), along with a control for cropland rotation (CR). The diversity of both AMF and diazotrophs was impacted by managed vegetation restoration. Specifically, the AMF Shannon index was higher in CR and PF compared to FF. Conversely, diazotroph richness was lower in CR, PF, and FG than in FF. Furthermore, both AMF and diazotroph community compositions differed between CR and FF. The relative abundance of AMF taxa, such as Glomus, was lower in FF compared to the other three land-use types, while Racocetra showed the opposite trend. Among diazotroph taxa, the relative abundance of Anabaena, Nostoc, and Rhizobium was higher in FF than in CR. Soil properties such as total potassium, available potassium, pH, and total nitrogen were identified as the main factors influencing AMF and diazotroph diversity. These findings suggest that AMF and diazotroph communities were more sensitive to FF rather than PF and FG after managed vegetation restoration from cropland, despite similar levels of soil nutrients among PF, FG, and FF. Consequently, the integration of diverse economic tree species and forage grasses in mixed plantations notably altered the diversity and species composition of AMF and diazotrophs, primarily through the promotion of biocrust formation and root establishment.
... Increasing SOM in the topsoil may account for the higher abundance and diversity of diazotrophs in the topsoil than at soil-rock mixing layer. Moreover, topsoil provides an ideal light and oxygen environment for the growth of Nostoc (belonging to Cyanobacteria) because of their aerobic phototrophic N fixation (Che et al., 2018;Wang et al., 2021). In this study, the relative abundance of most diazotroph taxa (for example, Anabaena, Nostoc, and some unclassified taxa under the family) is higher in the topsoil than at soil-rock mixing layer. ...
... In an experiment, tomatoes were inoculated with Bacillus pumilus (PGPB) and planted with native soil without N fertilization and 150 mg N kg −1 urea soil in pots, which increased plant growth, N uptake, soil NH + 4 concentration, rhizosphere bacterial population, soil bacterial gene expression, and soil nitrogenase activity (Masood et al., 2020). According to previous reports, the contents of physiological and chemical factors such as available phosphorus, N, and pH in soil are related to the abundance of N-fixing bacteria (Orr et al., 2012;Che et al., 2018). The associative Nfixation process of N-fixing bacteria and plants requires more energy, and organic carbon is the main energy source for N-fixing bacteria in soil (Herbert, 1999). ...
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Sugarcane is an important sugar and energy crop worldwide, requiring a large amount of nitrogen (N). However, excessive application of synthetic N fertilizer causes environmental pollution in farmland. Endophytic nitrogen-fixing bacteria (ENFB) provide N nutrition for plants through biological N fixation, thus reducing the need for chemical fertilizers. The present study investigated the effect of the N-fixing endophytic strain Enterobacter roggenkampii ED5 on phytohormone indole-3-acetic acid (IAA), N-metabolism enzyme activities, microbial community compositions, and N cycle genes in sugarcane rhizosphere soil at different N levels. Three levels of ¹⁵N-urea, such as low N (0 kg/ha), medium N (150 kg/ha), and high N (300 kg/ha), were applied. The results showed that, after inoculating strain ED5, the IAA content in sugarcane leaves was significantly increased by 68.82% under low N condition at the seedling stage (60 days). The nitrate reductase (NR) activity showed a downward trend. However, the glutamine synthase (GS) and NADH-glutamate dehydrogenase (NADH-GDH) activities were significantly enhanced compared to the control under the high N condition, and the GS and NR genes had the highest expression at 180 and 120 days, respectively, at the low N level. The total N content in the roots, stems, and leaves of sugarcane was higher than the control. The ¹⁵N atom % excess of sugarcane decreased significantly under medium N condition, indicating that the medium N level was conducive to N fixation in strain ED5. Metagenome analysis of sugarcane rhizosphere soil exhibited that the abundance of N-metabolizing microbial richness was increased under low and high N conditions after inoculation of strain ED5 at the genus level, while it was increased at the phylum level only under the low N condition. The LefSe (LDA > 2, p < 0.05) found that the N-metabolism-related differential microorganisms under the high N condition were higher than those under medium and low N conditions. It was also shown that the abundance of nifDHK genes was significantly increased after inoculation of ED5 at the medium N level, and other N cycle genes had high abundance at the high N level after inoculation of strain ED5. The results of this study provided a scientific reference for N fertilization in actual sugarcane production.
... Non-metric multidimensional scaling (NMDS), principal coordinate analysis (PCoA), and permutational multivariate analysis of variance (PERMA-NOVA) were performed to test the effects of raw material and composting time on microbial community profiles. The temporal and raw material ratio effects were further reflected by multivariate regression tree (MRT) (Che et al., 2018a) and principle response curves (PRCs) method, respectively (Nopp-Mayr et al., 2020;Van den Brink and Braak, 1999), using vegan, mvpart, and psych packages in R. ...
Article
Composting with five levels of green waste and sewage sludge was compared to examine how feeding ratios affected composting performance with special focus on humification, and the underlying mechanisms. The results showed that the raw material ratio persistently affected compost nutrients and stability. Humification and mineralization were promoted by higher proportion of sewage sludge. Bacterial community composition and within-community relationships were also significantly affected by the raw material feeding ratio. Network analysis indicated that clusters 1 and 4 which dominated by Bacteroidetes, Proteobacteria, and Acidobacteria shown significantly positive correlation with humic acid concentration. Notably, the structural equational model and variance partitioning analysis demonstrated that bacterial community structure (explained 47.82% of the variation) mediated the effect of raw material feeding ratio on humification, and exceeded the effect of environmental factors (explained 19.30% of the variation) on humic acid formation. Accordingly, optimizing the composting raw material improves the composting performance.
... Soil nitrate-N (NO 3 − ) and ammonium-N (NH 4 + ) were extracted 8.0 g fresh soil with 40 mL potassium chloride (KCl) (2 mol L −1 ) (soil mass: solution = 1:5) and determined by a continuous flow analyzer (Autoanalyzer AA3, Seal, Berlin, Germany) (Che et al. 2018). Fresh soil sample were air-dried at room temperature for 15 d and passed through a 2-mm sieve for further determine of the soil physicochemical properties. ...
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Aims The aims of the current study were to understand the variation in the abundance, diversity and structure of the diazotrophic communities in the rhizosphere soil of these three dominant plant species around Siding Pb–Zn mine. Methods Three dominant plant species (Pteris vittata, Miscanthus floridulus and Phragmites australis) were randomly selected, and rhizosphere soils were sampled from the rhizosphere of the plants. Results The nifH gene abundance in the rhizosphere soil of Pteris vittata was the highest among the three plant species. Variations in rhizosphere soil diazotrophic communities were mainly due to the changes in soil nutrient contents through plant‒soil system interactions. Diversity and structure of soil diazotrophic communities, including Alphaproteobacteria, Deltaproteobacteria and Cyanobacteria, were strongly influenced by soil heavy metals, ammonium nitrogen, soil moisture and available phosphorus contents. In addition, soil enzymes, especially urease, protease and alkaline phosphatase activities, also contributed to the structure of the diazotrophic communities. Alphaproteobacteria and Cyanobacteria play vital roles in the soil biological nitrogen fixation process. Heavy metal enrichment in mines provides electron donors for diazotrophs to support their activities in harsh environments. Diazotrophs can provide N to support plant growth in mines to help restore heavy metal-containing soil by dominant plants. Conclusions Our results showed the variations in diazotrophic community compositions in rhizosphere soil of three dominant plants and their impact on heavy metal accumulation. This study will help to determine the role and importance of soil properties and plant species in the soil biological nitrogen fixation process in highly contaminated mine areas.
... Three commonest legumes of the central QTP grasslands could account for 14-60% total aboveground biomass (Xu-Ri et al. 2021) and 70-88% N in these legume's underground biomass was xed from the atmosphere (Xu-Ri et al. 2021). The potential of asymbiotic nitrogen xation on QTP grasslands has also been reported (Che et al. 2018;Rui et al. 2022), however, the N contribution and the response to grazing/grazing exclosure remain unknown for this type of BNF. The relative abundance of nitrogen-xing bacterial increasing with the time extension of grazing exclusion is reported for an alpine grassland in eastern QTP (Cao et al. 2022). ...
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Grazing exclusion is the commonest degradation-recovering practice for alpine grasslands on Qing-Tibetan Plateau (QTP). A general response of legumes is the reduction in abundance, cover or biomass (esp. in relative scale), which is supposed to decrease in biological nitrogen fixation (BNF). Here, we want to estimate whether the capacity of BNF in legumes (measured by unit biomass) would be changed by grazing exclusion. Two ¹⁵ N isotope techniques were adopted to compare the BNF capacity of Astragalus arnoldii (a legume) between inside and outside animal-exclusion fence. For natural abundance (NA) method, plants were sampled at three timepoints, while for isotope dilution (ID) method, plants were collected after one day (24h), one month and one year of labeling. The two consistent in estimating percentage of N derived from atmosphere (%Ndfa) (77.52 ± 1.96% in NA vs. 72.39 ± 2.59% in ID), except an underestimation in ID for the first-time sampling (24h after labeling), seemingly owing to insufficient recovery time for reference plants. No significant difference in %Nfda inside (74.36 ± 2.36%) and outside (75.57 ± 2.26%) of the fence was found. Given that the biomass of legumes inside the fence (33.96 ± 2.71 g m-2) was significantly lower than that outside (19.62 ± 1.25 g m-2), we conclude that if total BNF were reduced by grazing exclusion, it would be due to the population size of legumes rather than the capacity.
... Thus, the decrease in their relative abundance contributed to the decrease in subsurface soil nitrogen concentrations, thereby limiting vegetation growth (Fig. 2b). This finding was supported by several existing studies (Che et al., 2019b;Luo et al., 2020), and it could be mainly caused by the decreased soil TOC concentration in degraded grasslands (Che et al., 2018b;Cao et al., 2021b). Similarly, the degradation also significantly decreased the relative abundances of Claroidoglomus, Glomus, and Paraglomus ( Fig. 4c and d), which are identified as AMF (Tisserant et al., 2013). ...
Article
Grassland degradation seriously affects the health of our planet, thereby weakening the ability of grasslands to provide multiple ecosystem services. Soil microbes have been recognized as game changers in the succession of degraded lands; therefore, the determination of their responses to grassland degradation is crucial for restoring degraded grasslands. However, soil microbial responses to grassland degradation are still far from being well understood, especially for low-latitude meadows. Hence, this study was performed in one natural and two artificial degraded subalpine meadows in Southern China to identify the soil microbial responses to the degradation of low-latitude subalpine meadows. At each site, soils were collected from nondegraded, moderately degraded, and heavily degraded subalpine meadows. Soil microbial abundance and diversity were analyzed via real-time PCR and amplicon NovaSeq sequencing, respectively. We found that the degradation of subalpine meadows significantly decreased the concentrations of almost all of the soil nutrients and considerably changed soil pH and texture. We also found that soil microbes demonstrated almost consistent responses to the degradation of the natural and artificial subalpine meadows. Specifically, degradation did not affect soil microbial abundance but significantly changed microbial community profiles. The relative abundance of most plant growth-promoting microbial taxa significantly decreased with subalpine meadow degradation, whereas the microbial taxa that were adverse to plant health exhibited a reverse trend. Soil microbial richness was also significantly decreased by the heavy degradation of the subalpine meadows. Additionally, the heavy degradation of the subalpine meadows significantly decreased the complexity and stability of the microbial co-occurrence network. The aforementioned soil microbiome changes also showed the significant correlations with plant biomass. Collectively, the changes in the soil microbiome can be critical factors affecting the degradation of subalpine meadows, and the regulation of soil microbiome is a promising restoration strategy.
... The characterization of determining factors is crucial for improving crop production and resource utilization rates (Che et al., 2018). Although recent well-designed experimental studies have demonstrated that lint yield is usually positively correlated with soil physicochemical parameters (Ghimire et al., 2019;Zhai et al., 2019), it is still challenging to quantify the complex relationships between belowground soil properties and aboveground crop productivity. ...
Article
The advantages of cover crops in enhancing soil organic C (SOC) sequestration and curbing reactive N losses have been verified. However, there is still a lack of related research evaluating the effects of cover crops and N fertilization on soil properties and crop performance as well as their potential relationships, especially in the continuous cotton cropland of the Yellow River Basin. Herein, we examined the impacts of two cropping systems (cotton (Gossypium hirsutum L.)-cotton (C-C) continuous cropping and Orychophragmus violaceus (O. violaceus)/ cotton (O/C) relay intercropping) and four N application rates (0 (N0), 112.5 (N1), 168.75 (N2), and 225 (N3) kg N ha − 1) on soil chemical properties, cotton yield, biomass, N uptake and N use efficiency (NUE) during 2017-2019 in the Yellow River Basin. In addition, we determined important soil chemical properties and revealed the relationship between soil parameters and lint yield. Compared with the CC system, the O/C relay intercropping system significantly improved the soil total N (STN), soil organic N (SON), microbial biomass N (MBN), lint yield (1312.84 kg ha − 1), biomass (10130.04 kg ha − 1) and N uptake (195.68 kg ha − 1) by 2%, 2%, 8%, 6%, 7% and 7%, respectively. N application notably increased the soil chemical properties associated with C and N, lint yield, biomass and N uptake, while the NUE of N3 was significantly lower than that of N2. Moreover, the key driving factors that significantly influenced the lint cotton yield were particulate organic N (PON), dissolved organic N (DON), soil ammonia N (SAN), SON, SOC and soil nitrate N (SNN). DON, PON, and SOC were key factors that directly impacted lint yield. According to the Z score of soil parameters, cotton productivity and resource use efficiency, the combination of O/C relay intercropping and N2 yielded the highest total Z score (10.97). Our results demonstrated that O. violaceus/cotton relay intercropping with 75% N application (168.75 kg N ha − 1) can maintain higher lint cotton yield, biomass accumulation, N uptake and NUE by increasing soil C and N fractions. Because non-legume crop straw requires a long period of time to decompose, it is necessary to evaluate the legacy impacts of O. violaceus cover cops in long-term experiments in the future.
... These results revealed that fungal and bacterial rare and abundant taxa showed distinct biogeographic distribution patterns in the proso millet fields. Similar results have also been reported in previous literature revealing that environmental factors (such as soil pH, water, and nutrient availability) are essential variables affecting the distribution patterns of soil microbial communities in various ecosystems [46][47][48][49]. We speculated that environmental selection may be more stringent and become even more pronounced in governing the biogeographic patterns of microbial communities [50]. ...
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Unraveling how microbial interactions and assembly process regulate the rhizosphere abundant and rare taxa is crucial for determining how species diversity affects rhizosphere microbiological functions. We assessed the rare and abundant taxa of rhizosphere fungal and bacterial communities in proso millet agroecosystems to explore their biogeographic patterns and co-occurrence patterns based on a regional scale. The taxonomic composition was significantly distinct between the fungal and bacterial abundant and rare taxa. Additionally, the rare taxa of bacteria and fungi exhibited higher diversity and stronger phylogenetic clustering than those of the abundant ones. The phylogenetic turnover rate of abundant taxa of bacteria was smaller than that of rare ones, whereas that of fungi had the opposite trend. Environmental variables, particularly mean annual temperature (MAT) and soil pH, were the crucial factors of community structure in the rare and abundant taxa. Furthermore, a deterministic process was relatively more important in governing the assembly of abundant and rare taxa. Our network analysis suggested that rare taxa of fungi and bacteria were located at the core of maintaining ecosystem functions. Interestingly, MAT and pH were also the important drivers controlling the main modules of abundant and rare taxa. Altogether, these observations revealed that rare and abundant taxa of fungal and bacterial communities showed obvious differences in biogeographic distribution, which were based on the dynamic interactions between assembly processes and co-occurrence networks.
... N 2 fixation stems from soil diazotrophs (N 2 -fixing bacteria and archaea) that convert dinitrogen (N2) gas to ammonia (NH3) using the nitrogenase protein complex (Nif) (Reed et al., 2011;Vitousek et al., 2013;Van Groenigen et al., 2015). Diazotrophs are common in soils but their abundance, diversity and community structure are still poorly explored in grasslands in spite of their key role in global N input (Patra et al., 2006;Regan et al., 2017;Che et al., 2018). Variations in the diversity and composition of soil diazotrophs are strongly associated with vegetation type, climatic and soil parameters, whereas soil pH and moisture were the main influencing parameters (Collavino et al., 2014;Tu et al., 2016). ...
Article
Anthropogenic nitrogen (N) input is known to alter the soil microbiome, but how N enrichment influences the abundance, alpha-diversity and community structure of N-cycling functional microbial communities in grasslands remains poorly understood. Here, we collected soils from plant communities subjected to up to 9 years of annual N-addition (10 g N m⁻² per year using urea as a N-source) and from unfertilized plots (control) in 30 grasslands worldwide spanning a large range of climatic and soil conditions. We focused on three key microbial groups responsible for two essential processes of the global N cycle: N2 fixation (soil diazotrophs) and nitrification (AOA: ammonia-oxidizing archaea and AOB: ammonia-oxidizing bacteria). We targeted soil diazotrophs, AOA and AOB using Illumina MiSeq sequencing and measured the abundance (gene copy numbers) using quantitative PCR. N-addition shifted the structure of the diazotrophic communities, although their alpha-diversity and abundance were not affected. AOA and AOB responded differently to N-addition. The abundance and alpha-diversity of AOB increased, and their community structure shifted with N-addition. In contrast, AOA were not affected by N-addition. AOA abundance outnumbered AOB in control plots under conditions of low N availability, whereas N-addition favoured copiotrophic AOB. Overall, N-addition showed a low impact on soil diazotrophs and AOA while effects for AOB communities were considerable. These results reveal that long-term N-addition has important ecological implications for key microbial groups involved in two critical soil N-cycling processes. Increased AOB abundance and community shifts following N-addition may change soil N-cycling, as larger population sizes may promote higher rates of ammonia oxidation and subsequently increase N loss via gaseous and soil N-leaching. These findings bring us a step closer to predicting the responses and feedbacks of microbial-mediated N-cycling processes to long-term anthropogenic N-addition in grasslands.
... They even play a more dominant role in C fixation in plant-constrained ecosystems with stressful conditions, such as cold desert (Thomas, 2005;Novis et al., 2007;Namsaraev et al., 2010;Ferrenberg et al., 2015), hot desert (Zaady et al., 2000;Gunnigle et al., 2017), temperate desert (Rajeev et al., 2013), and deglaciated soils (Strauss et al., 2012;Liu et al., 2016). Many soil autotrophs are diazotrophs, such as Nostocales and Rhizobiales, and are associated with nitrogen cycling in terrestrial ecosystems (Steven et al., 2012;Che et al., 2018). They thus conduct biological nitrogen fixation and are a primary nitrogen source for semi-arid and arid ecosystems (Belnap, 2003). ...
Article
CO 2-fixing by soil autotrophic microbes is as important as by plants in semi-arid and arid ecosystems, such as the Tibetan Plateau grassland. CO 2-fixing microbial community characteristics, capacity and their driving environmental factors remain unclear. Here we investigated the autotrophic microbial community in grassland surface soils on the Tibetan Plateau using molecular methods targeting the large subunit gene (cbbL) of ribulose-1, 5-bisphosphate carboxylase/oxygenase. The CO 2 fixation capacity was assessed by the 13 CO 2 probing method. The results showed that soil autotrophic microbial abundance substantially increased from desert, steppe to meadow. The autotrophic abundance significantly increased with enhancing mean annual precipitation (MAP), soil ammonium concentration and aboveground plant biomass (APB). Forms IAB and IC autotrophic microbial communities strongly varied with grassland types. Variation partitioning analysis revealed that the structure variations were mainly explained by MAP and aridity, which explained 4.2% and 2.6% for the IAB community, and 7.6% and 8.5% for the IC community. Desert and steppe soils exhibited significantly higher atmospheric 13 CO 2 fixation rate than meadow soils (29 versus 18 mg kg −1 soil d −1). The 13 CO 2 fixation rate negatively correlated with APB and soil ammonium concentration, demonstrating the substantially important role of au-totrophic microbes in oligotrophic soils. Form IAB autotrophs were phylogenetically affiliated with Cyanobacteria. Form IC autotrophs were affiliated with Rhizobiales and Actinobacteria, the former gradually increased and the latter decreased from desert, steppe to meadow. Our findings offer new insight into the importance of MAP in driving soil autotrophic microbial community and highlight microbial roles in carbon cycling in dryland ecosystems.
... This revealed that 23 MAGs, most prevalent in the moderate-salinity G3 sediments (t test, all P , 0.05), possessed the nifDKH genes encoding nitrogenase, suggesting that they were autotrophic diazotrophs. In previous studies, the fixation of inorganic carbon and nitrogen by such microorganisms was recognized as a crucial process for community assembly in extreme environments (49). In the moderate-salinity G3 sediments, autotrophic communities were dominated by diazotrophs, which was also found in some Tibetan soils (50), and the reason for this phenomenon is still mysterious. ...
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The compositional and physiological responses of autotrophic microbiotas to salinity in lakes remain unclear. In this study, the community composition and carbon fixation pathways of autotrophic microorganisms in lacustrine sediments with a salinity gradient (82.6 g/L to 0.54 g/L) were investigated by using metagenomic analysis. A total of 117 metagenome-assembled genomes (MAGs) with carbon fixation potentially belonging to 20 phyla were obtained. The abundance of these potential autotrophs increased significantly with decreasing salinity, and the variation of sediment autotrophic microbial communities was mainly affected by salinity, pH, and total organic carbon. Notably, along the decreasing salinity gradient, the dominant lineage shifted from Desulfobacterota to Proteobacteria. Meanwhile, the dominant carbon fixation pathway shifted from the Wood-Lungdahl pathway to the less-energy-efficient Calvin-Benson-Bassham cycle, with glycolysis shifting from the Embden-Meyerhof-Parnas pathway to the less-exergonic Entner-Doudoroff pathway. These results suggest that the physiological efficiency of autotrophic microorganisms decreased when the environmental salinity became lower. Metabolic inference of these MAGs revealed that carbon fixation may be coupled to the oxidation of reduced sulfur compounds and ferrous iron, dissimilatory nitrate reduction at low salinity, and dissimilatory sulfate reduction in hypersaline sediments. These results extend our understanding of metabolic versatility and niche diversity of autotrophic microorganisms in saline environments and shed light on the response of autotrophic microbiomes to salinity. These findings are of great significance for understanding the impact of desalination caused by climate warming on the carbon cycle of saline lake ecosystems. IMPORTANCE The Qinghai-Tibetan lakes are experiencing water increase and salinity decrease due to climate warming. However, little is known about how the salinity decrease will affect the composition of autotrophic microbial populations and their carbon fixation pathways. In this study, we used genome-resolved metagenomics to interpret the dynamic changes in the autotrophic microbial community and metabolic pathways along a salinity gradient. The results showed that desalination drove the shift of the dominant microbial lineage from Desulfobacterota to Proteobacteria, enriched autotrophs with lower physiological efficiency pathways, and enhanced coupling between the carbon cycle and other element cycles. These results can predict the future response of microbial communities to lake desalination and improve our understanding of the effect of climate warming on the carbon cycle in saline aquatic ecosystems.
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Introduction Nitrogen-fixing bacteria (NFB) have a pivotal impact on the nitrogen cycle within agroforestry systems. The organic management of the Panax notoginseng (sanqi)-Pinus armandii agroforestry (SPA) system resulted in nitrogen deficiency because of the lack of application of chemical fertilizers. Therefore, assessing the variability in NFB due to the cultivation of sanqi in the SPA system becomes crucial. Methods The seasonal dynamics in the abundance, diversity, and community structure of NFB in the soil of monocropping pine (MP) and SPA systems were assessed using real-time quantitative polymerase chain reaction and high-throughput sequencing technology. Results and discussion Sanqi cultivation triggered a decrease in the abundance of NFB but increased α diversity. Additionally, significant differences in the community structure of NFB were noted between the MP and SPA systems. Moreover, the abundance of Bradyrhizobium and Azospirillum increased in the soil after sanqi was cultivated. Furthermore, the cultivation of sanqi broadened the ecological niche breadth of NFB and increased the stochasticity in its community structure assembly (i.e., dispersal limitation). Additionally, the SPA system increased the network complexity but not the stability of NFB. The structural equation model (SEM) revealed that pH directly impacted the network complexity and stability of NFB in the SPA system. Sanqi cultivation positively influences the community characteristics of NFB in the soil in the SPA system. Our study provides new insights into nitrogen cycling and utilization in the SPA system.
Article
Legume species are essential components of plant diversity and affect soil biodiversity across various ecosystems. Their effect on the diversity and traits of soil bacteria, particularly in degraded grasslands, remains unknown. This study analysed the relationships among plant diversity, soil traits and legume‐associated rhizobacterial communities in Xiahe (XH) and Maqu (MQ) in Gansu Province, Haibei (HB) in Qinghai Province and Hongyuan (HY) in Sichuan Province in the eastern Qinghai‐Tibetan Plateau (QTP). The diversity index values (coverage, richness, Shannon index and evenness) of legume species were positively correlated with plant diversity. Several soil nutrients (ammonia‐nitrogen, nitrate‐nitrogen, total nitrogen, available potassium, available phosphorus and soil organic matter) and enzymes (urease, sucrase, peroxidase and dehydrogenase) were lower in HB and HY than in XH and MQ. The Shannon index for rhizobacterial diversity was higher in HB and HY than in XH and MQ. In contrast, the diversity index values were higher for geographical locations than for sympatric plant species. Additionally, HB and HY showed 50% fewer positive and negative associations with rhizobacteria than XH and MQ. Functional Annotation of Prokaryotic Taxa analysis indicated a higher relative abundance of nitrate reduction occurred in HB and HY than in XH and MQ, whereas nitrogen fixation occurred at a lower level in HB and HY than in XH and MQ. The Simpson index value for bacterial diversity was positively correlated with plant diversity, legume species diversity and soil multifunctionality. However, the Shannon index value was negatively correlated with these parameters. Changes in the composition of legume‐associated rhizobacteria across different geographical locations are strongly influenced by plant diversity and soil nutrients, reflecting the distribution characteristics of legumes in alpine grasslands.
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Removal of nitrogen compound in wastewater has been successfully developed with various activated sludge based processes. The microorganism immobilized on media enhanced biological efficiency by increase in biomass concentration, however the microbial community composition in the media was little revealed. A modified Ludzack-Ettinger (MLE) process was operated with immobilized media with polyvinyl alcohol and polyethylene glycol. MLSS in aerobic reactor was maintained at 50,000 mg/L and 40,000 mg/L in anoxic reactor by the media. 99% of ammonium in influent was oxidized but organic nitrogen resulted from microbial growth reduced total oxidation rate during the operation. Nitrate reduction rate increased by the addition of glucose for C/N ratio adjustment to 4.5. Based on total nitrogen concentration, the removal efficiency of nitrogen was 48.2% after C/N ratio adjustment, which showed the mid-range of nitrogen removal efficiency in MLE. Microbial community composition was compared between aerobic and anoxic media by NGS technique with V3-V4 region of 16S rRNA gene. Proteobacteria was the dominant phylum both in aerobic and anoxic media, and the ratio was 39.7% in aerobic media and 65.9% in anoxic media. Bacteroidetes was secondly largest phylum. The dominant genera in aerobic media were Nitrospira and Povalibacter. Ratio of nitrification-related genera was 45%. On the contrary, the dominant genera in anoxic media were Desulfomicrobium, Desulfobulbus, and Methyloversatilis in sequence of dominance. Total genera related with denitrification, including Dechloromonas and Flavobacterium amounted 63%. Population of microorganisms in each reactor was compared in terms of diversity by QIIME 2 algorithm. The Chao1 index values of α-diversity were 606.05 and 415.53 for aerobic and anoxic media, respectively, which showed higher population diversity in aerobic media than in anoxic. The microbiological community on the immobilized aerobic and anoxic media would help future microbial studies in wastewater treatment process.
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Soil biological nitrogen fixation (BNF) plays a significant role in N input in terrestrial ecosystems, and can be largely altered by management. In semi-arid region of Northern China, natural grasslands rely on soil BNF to maintain nitrogen input under long-mowing, but the effects of mowing on BNF remain unclear. Here we assessed the soil BNF rate and the nifH gene abundance in soil (0–10 cm) of the grasslands subject to long-term (19 years) annual mowing (MO) versus no mowing (NM) in a semiarid natural steppe grassland. Our results indicated that mowing significantly increased the BNF rate (P < 0.01) from 11.48 g N ha−1 d−1 (NM) to 25.16 g N ha−1 d−1 (MO); mowing also significantly increased average N fixation activity per nifH gene (P < 0.05), while reduced the nifH gene abundance (P < 0.05). The nifH gene abundance was not significantly correlated with the BNF rate (P > 0.05), suggesting that the nifH gene abundance based on DNA analysis was not indicative for BNF rate; while soil ammonium nitrogen (-N) content was identified by stepwise multiple regression the only variable that can significantly explain the variation in BNF rate. Our results suggest that soil -N content is the most efficient predictor of BNF rate instead of nifH gene abundance, and it is more crucial to quantify the impacts of soil -N than the effects of diazotrophic abundance in predicting the changes in BNF rate in response to mowing management in semiarid grassland.
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Functional characteristics of microbiomes are good predictors of nitrogen (N) biogeochemistry, but soil microbial N-functional profiles under different land use practices and their dominant drivers at continental scale remain unclear. For this study, we selected soils having a broad range of edaphic factors across different land use practices and soil types in China and used metagenomic sequencing to investigate N-functional profiles of microbiomes. The N-pathway frequencies were highest in paddy soils, and the composition of N-cycling genes in paddy soils significantly diverged from upland and forest soils. Differential abundance analysis showed that genes associated with nitrogen dissimilatory pathways exhibited enrichment in paddy soils, whereas those linked to assimilatory pathways were enriched in forest soils. Aggregated boosted tree analysis revealed that the frequencies of N-pathways and relative abundances of N-cycling genes were mainly explained by soil moisture and pH. Partial Mantel and redundancy analysis consistently showed that N-functional composition correlated strongest with moisture and pH. Overall, our results demonstrated that soil moisture and pH were vital drivers of microbial N-functional profiles, providing novel evidence for soil moisture and pH’s role in driving whole nitrogen cycling. Considering the large fluctuations in moisture over time, soil pH, with minor seasonality, could be used as indicators to reflect microbial N-functional profiles at the continental scale.
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Revealing the mechanisms underlying soil microbial community assembly is a fundamental objective in molecular ecology. However, despite increasing body of research on overall microbial community assembly mechanisms, our understanding of subcommunity assembly mechanisms for different prokaryotic and fungal taxa remains limited. Here, soils were collected from more than 100 sites across southwestern China. Based on amplicon high-throughput sequencing and iCAMP analysis, we determined the subcommunity assembly mechanisms for various microbial taxa. The results showed that dispersal limitation and homogenous selection were the primary drivers of soil microbial community assembly in this region. However, the subcommunity assembly mechanisms of different soil microbial taxa were highly variable. For instance, the contribution of homogenous selection to Crenarchaeota subcommunity assembly was 70%, but it was only around 10% for the subcommunity assembly of Actinomycetes, Gemmatimonadetes and Planctomycetes. The assembly of subcommunities including microbial taxa with higher occurrence frequencies, average relative abundance and network degrees, as well as wider niches tended to be more influenced by homogenizing dispersal and drift, but less affected by heterogeneous selection and dispersal limitation. The subcommunity assembly mechanisms also varied substantially among different functional guilds. Notably, the subcommunity assembly of diazotrophs, nitrifiers, saprotrophs and some pathogens were predominantly controlled by homogenous selection, while that of denitrifiers and fungal pathogens were mainly affected by stochastic processes such as drift. These findings provide novel insights into understanding soil microbial diversity maintenance mechanisms, and the analysis pipeline holds significant value for future research.
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Biochar amendment can be adopted to improve soil substrate, in turn facilitated phytoremediation. However, improvements to the properties of tailings following different feedstocks of biochar amendment in phytoremediation, particularly the impacts on nitrogen cycle and the related nitrogen-fixing microorganisms remain unclear. In this study, a 100-day pot experiment was designed to determine the co-effects of different combinations of woody and non-woody biochar, namely hibiscus cannabinus core biochar (HB), sewage sludge biochar (SB), chicken manure biochar (MB) and two crops (Cassia alata L., Boehmeria nivea L.). It was found that, on the one hand, biochar amendment directly immobilized heavy metal (loid) contamination in the tailings; on the other hand, biochar amendment, particularly non-woody SB, improved soil properties (i.e., the combination of SB with crops increased the total nitrogen content by 4.7-7.5 times). This indirectly improved phytostabilization (i.e., SB increased crop height 1.5-1.8 fold, root length 3.3-3.7 fold, decreased NH4NO3-extractable Pb, Cu, Cd and also increased the relative abundance of nitrogen-fixing bacteria such as Mesorhizobium, Bradyrhizobium, and Rhizobium). Besides this, redundant analysis shown that the carbon, nitrogen sources, and pH provided by the biochar were identified as the key factors associated with the nitrogen-fixing bacteria. Through the comprehensive evaluation of different biochar amendment in phytoremediation, it was found that the non-woody SB got higher comprehensive score (3.1-3.6) in biochar amendment in phytoremediation, especially in Boehmeria nivea L. Thus, the combination of non-woody SB and Boehmeria nivea L. improved microbial function, while the microorganisms in turn promoted crop growth. Our results revealed the prospect of using non-woody SB assisted Boehmeria nivea L. for phytoremediation in multi-metal mine tailings.
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Coal is the main source of energy for China's economic development, but coal gangue dumps are a major source of heavy metal pollution. Bacterial communities have a major effect on the bioremediation of heavy metals in coal gangue dumps. The effects of different concentrations of heavy metals on the composition of bacterial communities in coal gangue sites remain unclear. Soil bacterial communities from four gangue sites that vary in natural heavy metal concentrations were investigated using high-throughput sequencing in this study. Correlations among bacterial communities, heavy metal concentrations, physicochemical properties of the soil, and the composition of dissolved organic matter of soil in coal gangue dumps were also analyzed. Our results indicated that Actinobacteriota, Proteobacteria, Chloroflexi, Acidobacteriota, and Gemmatimonadota were the bacterial taxa most resistant to heavy metal stress at gangue sites. Heavy metal contamination may be the main cause of changes in bacterial communities. Heavy metal pollution can foster mutually beneficial symbioses between microbial species. Microbial-derived organic matter was the main source of soil organic matter in unvegetated mining areas, and this could affect the toxicity and transport of heavy metals in soil. Polar functional groups such as hydroxyl and ester groups (A226-400) play an important role in the reaction of cadmium (Cd) and lead (Pb), and organic matter with low molecular weight (SR) tends to bind more to mercury (Hg). In addition to heavy metals, the content of nitrogen (N), phosphorus (P), and total organic carbon (TOC) also affected the composition of the bacterial communities; TOC had the strongest effect, followed by N, SOM, and P. Our findings have implications for the microbial remediation of heavy metal-contaminated soils in coal gangue sites and sustainable development.
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The diversity and community structure of rhizospheric microbes are largely affected by soil physicochemical properties and plant species. In this work, high throughput sequencing and quantitative real-time PCR targeting nifH gene were used to assess the abundance and diversity of diazotrophic community in the coastal saline soils of Yellow River Delta (YRD). We demonstrated that the copy number of nifH gene encoding the Fe protein subunit of the nitrogenase in the nitrogen fixation process was significantly affected by soil physiochemical factors, and the abundance of diazotrophs in the rhizospheric soil samples collected from different locations was positively related with soil physicochemical properties. Soil salinity (P=0.003) and moisture (P=0.003) were significantly co-varied with the OTU-based community composition of diazotrophs. Taxonomic analysis showed that most diazotrophs belonged to the Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. Linear discriminant analysis (LDA) effect size (LEfSe) and canonical correspondence analysis (CCA) showed that diazotrophic community structure significantly varied with soil salinity, moisture, pH and total nitrogen, carbon, sulphur and nitrite (NO2 –N) content. Our findings provide direct evidence toward the understanding of different effects of soil physicochemical properties and host plant traits such as halophytes types, life span and cotyledon type, on the community composition of diazotrophic populations in the rhizosphere of plants grown in coastal saline soils.
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Global warming can trigger dramatic glacier area shrinkage and change the flux of glacial runoff, leading to the expansion and subsequent retreat of riparian wetlands. This elicits the interconversion of riparian wetlands and their adjacent ecosystems (e.g., alpine meadows), probably significantly impacting ecosystem nitrogen input by changing soil diazotrophic communities. However, the soil diazotrophic community differences between glacial riparian wetlands and their adjacent ecosystems remain largely unexplored. Here, soils were collected from riparian wetlands and their adjacent alpine meadows at six locations from glacier foreland to lake mouth along a typical Tibetan glacial river in the Namtso watershed. The abundance and diversity of soil diazotrophs were determined by real-time PCR and amplicon sequencing based on nifH gene. The soil diazotrophic community assembly mechanisms were analyzed via iCAMP, a recently developed null model-based method. The results showed that compared with the riparian wetlands, the abundance and diversity of the diazotrophs in the alpine meadow soils significantly decreased. The soil diazotrophic community profiles also significantly differed between the riparian wetlands and alpine meadows. For example, compared with the alpine meadows, the relative abundance of chemoheterotrophic and sulfate-respiration diazotrophs was significantly higher in the riparian wetland soils. In contrast, the diazotrophs related to ureolysis, photoautotrophy, and denitrification were significantly enriched in the alpine meadow soils. The iCAMP analysis showed that the assembly of soil diazotrophic community was mainly controlled by drift and dispersal limitation. Compared with the riparian wetlands, the assembly of the alpine meadow soil diazotrophic community was more affected by dispersal limitation and homogeneous selection. These findings suggest that the conversion of riparian wetlands and alpine meadows can significantly alter soil diazotrophic community and probably the ecosystem nitrogen input mechanisms, highlighting the enormous effects of climate change on alpine ecosystems.
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Clarifying the response of soil microbial communities to the change of different vegetation types on a small regional scale is of great significance for understanding the sustainability of grassland development. However, the distribution patterns and driving factors of the microbial community are not well understood in the Qilian Mountains. Therefore, we characterized and compared the soil microbial communities underlying the four vegetation types in a national natural reserve (reseeded grassland, swamp meadow, steppe meadow, and cultivated grassland) using high-throughput sequencing of the 16S rRNA and ITS. Meanwhile, the plant community and soil physicochemical characteristics were also determined. The results showed that bacterial and fungal communities in all vegetation types had the same dominant species, but the relative abundance differed substantially, which caused significant spatial heterogeneities on the small regional scale. Specifically, bacteria showed higher variability among different vegetation types than fungi, among which the bacterial and fungal communities were more sensitive to the changes in soil than to plant characteristics. Furthermore, soil organic carbon affected the widest portion of the microbial community, nitrate-nitrogen was the main factor affecting bacteria, and aboveground plant biomass was the main factor affecting fungi. Collectively, these results demonstrate the value of considering multiple small regional spatial scales when studying the relationship between the soil microbial community and environmental characteristics. Our study may have important implications for grassland management following natural disturbances or human alterations.
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Biological nitrogen fixation (BNF) is a vital approach to replenishing soil nitrogen (N) pools by converting atmospheric N2. Agronomic practice of growing legumes frequently includes stover return, which generally induces the growth of N2-fixers (i.e., diazotrophs) and stimulates BNF; however, there is uncertainty regarding the stover return effects along soil depth. Here, soil samples were collected from 0 to 10, 10–20 and 20–30 cm in the peanut (Arachis hypogaea) field experiment (established in 2013) comprising treatments with no fertilizer (CK), maize (Zea mays) stover (MS), and MS + chemical fertilizers (SNPK). The abundance of 16S rRNA and nifH genes was determined by real-time PCR, the rate of N2 fixation in soil (RNfix) was measured by acetylene reduction assay, and diazotroph community structure was explored by high-throughput sequencing. Results showed that maize stover application alone increased nifH gene abundance in the 0–10 cm layer, and combined with chemical fertilizer application increased RNfix in the 0–20 cm layers, affecting diazotroph community structure succession in the 0–20 cm layers. This is consistent with the effects of stover on soil properties such as dissolved organic carbon and nitrogen, soil organic matter (SOM) and carbon-to‑nitrogen ratio, with SOM playing a dominant role in governing the abundance, structure and activity of diazotroph communities in the soil. In addition, total nitrogen (TN) was the key factor in shaping the vertical stratification of diazotroph community structure, the abundance of diazotrophs and bacteria as well as the structure and RNfix of the diazotroph community were highly correlated. This study demonstrated that maize stover incorporation caused significant positive changes in the vertical stratification of soil diazotroph communities; although these effects decreased with soil depth and were minor when stover was combined with chemical fertilizers, they still highlighted the crucial role of stover return in enhancing BNF in soil where legumes were present.
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Climate change and human activities have led to the degradation of desert wetlands. Free-living diazotrophs are vital to soil nitrogen input. However, a comprehensive understanding of how soil free-living diazotrophic community and co-occurrence patterns respond to desert wetland degradation is lacking. Here, quantitative polymerase chain reaction (qPCR) and amplicon sequencing targeted on nitrogenase (nifH) genes and network analysis were used to investigate soil free-living diazotrophic abundance, diversity and composition and co-occurrence patterns along the wetland degradation gradient [non-degraded (ND), lightly degraded (LD), moderately degraded (MD), and severely degraded (SD) wetlands] in the southeastern of Mu Us Desert, northern China. Results showed the abundance, Shannon, Simpson, Chao1 and Ace indexes decreased (P < 0.05) by 14.6%, 20.7%, 2.1%, 46.5% and 45.0% at SD, respectively, whereas no significant difference (P > 0.05) was observed between ND and LD. The relative abundance of Proteobacteria generally decreased across the degradation (53.5%–19.7%), while the variation was opposite for Cyanobacteria from ND to MD (6.2%–40.1%). Soil organic carbon had the strongest linkage with abundance, diversity and composition, followed by total nitrogen, moisture and pH. The lowest network nodes, edges and density were observed at MD and SD, indicating the complexity of free-living diazotrophic networks was reduced by continued degeneration. Overall, severe desert wetland degradation more negatively affects soil free-living diazotrophic abundance, diversity and network complexity than light degradation. The degradation promotes the growth of autotrophic diazotrophs, inhibiting heterotrophic diazotrophs. These changes are mostly related to the loss of soil organic carbon.
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We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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The vast expanses of rangeland on the Tibetan Plateau, which support the livelihood of c . 9.8 million local inhabitants, have experienced rapid climate warming over the past 50 years. At the same time, precipitation has increased in large parts of the Plateau but decreased in other parts, particularly in the northwest. These trends are predicted to continue into the future. However, their potential effects on rangeland quality remain unclear. We conducted a two‐factor field experiment in which we manipulated temperature (control or warming by 1.5–1.8°C) and precipitation (control or 50% reduction or increase in rainfall) in an alpine grassland on the northeastern Tibetan Plateau, starting in 2011. From 2014 to 2016, we measured forage production and community composition, and in 2015 forage quality (crude protein, cell‐soluble contents, hemicellulose, cellulose, lignin and digestibility) was represented by seven abundant species. Overall, warming did not change total forage production at plant community level, but increased legume production and decreased non‐legume forb production. Increased and reduced precipitation enhanced and decreased forage production by 18.2% and 12.9% respectively. Increased precipitation in particular increased grass and sedge production, but not legume production. Forage quality showed species‐specific responses to the simulated climate changes. At community level, warming and reduced precipitation improved forage quality, which were mainly caused by a shift in community composition towards more legumes, rather than the direct effects of simulated climate changes. Meanwhile, increased precipitation did not reduce forage quality, despite the precipitation‐induced increase in forage production. Integrating forage production and quality into nutrient production as a measure of rangeland quality, we found that warming and increased precipitation additively improved rangeland quality, while reduced precipitation decreased it. Synthesis and applications . Rangeland quality, an important ecosystem provisioning service, will benefit from a warmer climate on the Tibetan Plateau in the regions with a predicted increase in precipitation, but not in those regions where precipitation might be reduced in the future. We suggest management strategies, including reseeding native legumes, establishing sustainable pastures and assisting the exchange of harvested forage, to cope with the challenges posed by these different climate change scenarios.
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Alpine meadows on the Tibetan Plateau have experienced severe degradation in recent decades. Although the effects of alpine meadow degradation on soil properties have been well documented, there is still a paucity of knowledge regarding the responses of nitrogen-cycling microbes (NCMs) to degradation and their links to the changes in soil properties. Here, we systematically determined the effects of degraded patch formation on soil properties (i.e., total carbon, total nitrogen, ammonium nitrogen, nitrate nitrogen, available phosphorus, dissolved organic carbon, moisture, δ15N, δ13C, and pH) and NCMs (based on nifH, amoA, narG, nirK, and nirS genes and their transcripts) across three Tibetan alpine meadows at different degradation stages. Results showed that compared to the original grassed patches, the contents of most soil nutrients (e.g., carbon, nitrogen, and phosphorus) were significantly decreased in the degraded patches across the study sites. Degraded patches also tended to have higher soil δ15N values and nitrate contents. Among the aforementioned NCMs, soil diazotrophs and denitrifiers only showed weak responses to the patch formation, while ammonia-oxidizing microbes showed the highest consistency and sensitivity in response to the patch formation across the study sites. The abundance of amoA gene and archaeal amoA mRNA significantly increased in the degraded patches, and they were positively correlated with soil δ15N values and nitrate nitrogen contents, but negatively correlated with soil total and inorganic nitrogen contents. These results suggest that the increased abundance of ammonia-oxidizing microbes may be an important driver of soil nitrogen loss during degraded patch formation in alpine meadows.
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A novel Gram-stain-negative, small rod-shaped bacterium (strain 8-14-6T) was isolated from hydrocarbon contaminated desert soils collected from Kuwait. Strain 8-14-6T grew at 5-37 ºC, pH 6.0-8.8 and 0-2 % (w/v) of NaCl concentration. Casein, starch, Tween 20 and Tween 80 were hydrolyzed while urea, chitin, DNA and carboxymethyl-cellulose were not hydrolyzed by strain 8-14-6T. The major cellular fatty acids were identified as C18:1ω6c/C18:1ω7c, C16:0 and iso-C16:1I/ C14:03-OH. Strain 8-14-6T produced diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unidentified phospholipids, two unidentified lipids and five unidentified amino lipids as polar lipids. Genomic G+C content was 73.5 mol %. 16S rRNA gene sequence comparisons indicated that strain 8-14-6T represents a member of the genus Skermanella with in family Rhodospirillaceae of the class Alphaproteobacteria. Strain 8-14-6T has a sequence similarity of 98.9 % with Skermanella rosea M1T, 97.4 % with Skermanella aerolata 5416T-32T, 96.9 % with Skermanella stibiiresistens SB22T and <95.4 % with other two species of the genus Skermanella. The DNA-DNA relatedness values between strain 8-14-6T and the type strains of the nearest species were clearly below the 70 % threshold. From the combination of phenotypic and genotypic characteristics and distinct phylogenetic position, the strain is considered to represent a novel species of the genus Skermanella, for which the name Skermanella mucosa sp. nov. is proposed. The type strain is 8-14-6T (=KEMB 2255-438T= JCM 31590T).
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Previous studies have revealed inconsistent correlations between fungal diversity and plant diversity from local to global scales, and there is a lack of information about the diversity–diversity and productivity–diversity relationships for fungi in alpine regions. � Here we investigated the internal relationships between soil fungal diversity, plant diversity and productivity across 60 grassland sites on the Tibetan Plateau, using Illumina sequencing of the internal transcribed spacer 2 (ITS2) region for fungal identification. � Fungal alpha and beta diversities were best explained by plant alpha and beta diversities, respectively, when accounting for environmental drivers and geographic distance. The best ordinary least squares (OLS) multiple regression models, partial least squares regression (PLSR) and variation partitioning analysis (VPA) indicated that plant richness was positively correlated with fungal richness. However, no correlation between plant richness and fungal richness was evident for fungal functional guilds when analyzed individually. � Plant productivity showed a weaker relationship to fungal diversity which was intercorrelated with other factors such as plant diversity, and was thus excluded as a main driver. Our study points to a predominant effect of plant diversity, along with other factors such as carbon : nitrogen (C : N) ratio, soil phosphorus and dissolved organic carbon, on soil fungal richness.
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A widespread pattern of the Tibetan plateau is mosaics of grasslands of Cyperaceae and grasses with forbs, interspersed with patches covered by lichen crusts induced by overgrazing. However, the fate of inorganic and organic N in non-crusted and crusted patches in Kobresia grasslands remains unknown. We reported on a field 15N-labeling experiment in two contrasting patches to compare retention of organic and inorganic N over a period of 29 days. 15N as KNO3, (NH4)2SO4 or glycine was sprayed onto soil surface. Crusted patches decreased plant and soil N stocks. More 15N from three N forms was recovered in soil than plants in both patches 29 days after the labeling. In non-crusted patches, 15N recovery by the living roots was about two times higher than in crusted ones, mainly because of higher root biomass. Microorganisms in non-crusted patches were N-limited because of more living roots and competed strongly for N with roots. Inorganic N input to non-crusted patches could alleviate N limitation to plants and microorganisms, and leads to higher total 15N recovery (plant + soil) for inorganic N forms. Compared to non-crusted patches, microorganisms in crusted patches were more C-limited because of depletion of available C caused by less root exudation. Added glycine could activate microorganisms, together with the hydrophobicity of glycine and crusts, leading to higher 15N-glycine than inorganic N. We conclude that overgrazing-induced crusts in Kobresia grasslands changed the fate of inorganic and organic N, and lead to lower total recovery from inorganic N but higher from organic N.
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We examined the impact of arbuscular mycorrhizal fungi and rhizobia on the living microbial community and microbial necromass under different long-term fertilization treatments at the long-term Static Fertilization Experiment Bad Lauchstädt (Germany). Phospholipid fatty acids (PLFA) and amino sugars plus muramic acid, were used as biomarkers for soil microbial bio- and necromass, respectively, and analyzed from six treatments imposed on two crop rotations, varying only in the inclusion/non-inclusion of a legume. Treatments included: two levels of only farmyard manure (FYM), only mineral fertilizer (NPK), the combined application of both fertilizer types and a non-fertilized control. PLFA profiles differed clearly between the investigated crop rotations and were significantly related to labile C, mineral N, and soil pH. This emphasizes the role of carbon, and of mycorrhizal and rhizobial symbioses, as driver for changes in the microbial community composition due to effects on the living conditions in soil. We found some evidence that legume associated symbiosis with arbuscular mycorrhizal fungi and rhizobia act as a buffer, reducing the impact of varying inputs of mineral nutrients on the decomposer community. While our results support former findings that living microbial populations vary within short-term periods and are reflective of a given crop grown in a given year, soil necromass composition indicates longer term changes across the two crop rotation types, mainly shaped by fertilizer related effects on the community composition and C turnover. However, there was some evidence that specifically the presence of a legume, affects the soil necromass composition not only over the whole crop rotation but even in the short-term.
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Abstract The biogeographical distribution of soil bacterial communities has been widely investigated. However, there has been little study of the biogeography of soil archaeal communities on a regional scale. Here, using high-throughput sequencing, we characterized the archaeal communities of 94 soil samples across the eastern Tibetan Plateau. Thaumarchaeota was the predominant archael phylum in all the soils, and Halobacteria was dominant only in dry soils. Archaeal community composition was significantly correlated with soil moisture content and C:N ratio, and archaeal phylotype richness was negatively correlated with soil moisture content (r= -0.47, P < 0.01). Spatial distance, a potential measure of the legacy effect of evolutionary and dispersal factors, was less important than measured environmental factors in determining the broad scale archaeal community pattern. These results indicate that soil moisture and C:N ratio are the key factors structuring soil archaeal communities on the eastern Tibetan Plateau. Our findings suggest that archaeal communities have adjusted their distributions rapidly enough to reach range equilibrium in relation to past environmental changes e.g. in water availability and soil nutrient status. This responsiveness may allow better prediction of future responses of soil archaea to environmental change in these sensitive ecosystems. Keywords: Biogeographical distribution, Soil archaeal communities, High through-put sequencing, Tibetan Plateau
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N2 fixation and ammonia oxidation (AO) are the two most important processes in the nitrogen (N) cycle of biological soil crusts (BSCs). We studied the short-term response of acetylene reduction assay (ARA) rates, an indicator of potential N2 fixation, and AO rates to temperature (T, -5°C to 35°C) in BSC of different successional stages along the BSC ecological succession and geographic origin (hot Chihuahuan and cooler Great Basin deserts). ARA in all BSCs increased with T until saturation occurred between 15 and 20°C, and declined at 30–35°C. Culture studies using cyanobacteria isolated from these crusts indicated that the saturating effect was traceable to their inability to grow well diazotrophically within the high temperature range. Below saturation, temperature response was exponential, with Q10 significantly different in the two areas (~ 5 for Great Basin BSCs; 2–3 for Chihuahuan BSCs), but similar between the two successional stages. However, in contrast to ARA, AO showed a steady increase to 30–35°C in Great Basin, and Chihuhuan BSCs showed no inhibition at any tested temperature. The T response of AO also differed significantly between Great Basin (Q10 of 4.5–4.8) and Chihuahuan (Q10 of 2.4–2.6) BSCs, but not between successional stages. Response of ARA rates to T did not differ from that of AO in either desert. Thus, while both processes scaled to T in unison until 20°C, they separated to an increasing degree at higher temperature. As future warming is likely to occur in the regions where BSCs are often the dominant living cover, this predicted decoupling is expected to result in higher proportion of nitrates in soil relative to ammonium. As nitrate is more easily lost as leachate or to be reduced to gaseous forms, this could mean a depletion of soil N over large landscapes globally.
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Global warming has resulted in substantial glacier retreats in high-elevation areas, exposing deglaciated soils to harsh environmental conditions. Autotrophic microbes are pioneering colonizers in the deglaciated soils and provide nutrients to the extreme ecosystem devoid of vegetation. However, autotrophic communities remain less studied in deglaciated soils. We explored the diversity and succession of the cbbL gene encoding the large subunit of form I RubisCO, a key CO2-fixing enzyme, using molecular methods in deglaciated soils along a 10-year deglaciation chronosequence on the Tibetan Plateau. Our results demonstrated that the abundance of all types of form I cbbL (IA/B, IC and ID) rapidly increased in young soils (0 to 2.5 years old) and kept stable in old soils. Soil total organic carbon (TOC) and total nitrogen (TN) gradually increased along the chronosequence and both demonstrated positive correlations with the abundance of bacteria and autotrophs, indicating that soil TOC and TN originated from autotrophs. Form IA/B autotrophs, affiliated with cyanobacteria, exhibited a substantially higher abundance than IC and ID. Cyanobacterial diversity and evenness increased in young soils (<6 years old) and then remained stable. Our findings suggest that cyabobacteria play an important role in accumulating TOC and TN in the deglaciated soils.
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Microorganisms drive much of the Earth's nitrogen (N) cycle, but we still lack a global overview of the abundance and composition of the microorganisms carrying out soil N processes. To address this gap, we characterized the biogeography of microbial N traits, defined as eight N-cycling pathways, using publically available soil metagenomes. The relative frequency of N pathways varied consistently across soils, such that the frequencies of the individual N pathways were positively correlated across the soil samples. Habitat type, soil carbon, and soil N largely explained the total N pathway frequency in a sample. In contrast, we could not identify major drivers of the taxonomic composition of the N functional groups. Further, the dominant genera encoding a pathway were generally similar among habitat types. The soil samples also revealed an unexpectedly high frequency of bacteria carrying the pathways required for dissimilatory nitrate reduction to ammonium, a little-studied N process in soil. Finally, phylogenetic analysis showed that some microbial groups seem to be N-cycling specialists or generalists. For instance, taxa within the Deltaproteobacteria encoded all eight N pathways, whereas those within the Cyanobacteria primarily encoded three pathways. Overall, this trait-based approach provides a baseline for investigating the relationship between microbial diversity and N cycling across global soils.
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The biological crusts on lateritic soils, red soils and mine-waste burdened soils in the eastern region of India covering a transect of about 800 km were principally composed of sheathed cyanobacteria of the genera Scytonema, Tolypothrix and Lyngbya along with few other species of Cylindrospermum, Nostoc, Calothrix and Fischerella. Molecular phylogeny based on 16S rRNA gene sequence of these cyanobacteria along with those occurring in different habitats of four different continents formed a distinct clade, however, were clustered close to other filamentous cyanobacteria.
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