Effect of different long-term fertilizer managements on soil nitrogen fixing bacteria community in a double-cropping rice paddy field of southern China.

Soil microorganism plays an important role in nitrogen (N) fixation process of paddy field, but the related information about how soil microorganism that drive N fixation process response to change of soil phy-chemical characteristics under the double-cropping rice (Oryza sativa L.) paddy field in southern of China is need to further study. Therefore, the impacts of 34-years different long-term fertilization system on soil N-fixing bacteria community under the double-cropping rice paddy field in southern of China were investigated by taken chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) method in this paper. The field experiment were set up four different fertilizer treatments: chemical fertilizer alone (MF), rice straw and chemical fertilizer (RF), 30% organic manure and 70% chemical fertilizer (OM), and unfertilized as a control (CK). This results showed that compared with CK treatment, the diversity index of cbbLR and nifH genes with OM and RF treatments were significantly increased (p<0.05), respectively. Meanwhile, the abundance of cbbLR gene with OM, RF and MF treatments were increased by 23.94, 12.19 and 6.70×107 copies g-1 compared to CK treatment, respectively. Compared with CK treatment, the abundance of nifH gene with OM, RF and MF treatments were increased by 23.90, 8.82 and 5.40×109 copies g-1, respectively. This results indicated that compared with CK treatment, the soil autotrophic azotobacter and nitrogenase activities with OM and RF treatments were also significantly increased (p<0.05), respectively. There were an obvious difference in features of soil N-fixing bacteria community between application of inorganic fertilizer and organic manure treatments. Therefore, this results demonstrated that abundance of soil N-fixing bacteria community in the double-cropping rice paddy field were increased by long-term applied with organic manure and crop residue managements.

Genomic characterization and computational phenotyping of nitrogen-fixing bacteria isolated from Colombian sugarcane fields.

Previous studies have shown the sugarcane microbiome harbors diverse plant growth promoting microorganisms, including nitrogen-fixing bacteria (diazotrophs), which can serve as biofertilizers. The genomes of 22 diazotrophs from Colombian sugarcane fields were sequenced to investigate potential biofertilizers. A genome-enabled computational phenotyping approach was developed to prioritize sugarcane associated diazotrophs according to their potential as biofertilizers. This method selects isolates that have potential for nitrogen fixation and other plant growth promoting (PGP) phenotypes while showing low risk for virulence and antibiotic resistance. Intact nitrogenase (nif) genes and operons were found in 18 of the isolates. Isolates also encode phosphate solubilization and siderophore production operons, and other PGP genes. The majority of sugarcane isolates showed uniformly low predicted virulence and antibiotic resistance compared to clinical isolates. Six strains with the highest overall genotype scores were experimentally evaluated for nitrogen fixation, phosphate solubilization, and the production of siderophores, gibberellic acid, and indole acetic acid. Results from the biochemical assays were consistent and validated computational phenotype predictions. A genotypic and phenotypic threshold was observed that separated strains by their potential for PGP versus predicted pathogenicity. Our results indicate that computational phenotyping is a promising tool for the assessment of bacteria detected in agricultural ecosystems.

Elevated level of arsenic negatively influences nifH gene expression of isolated soil bacteria in culture condition as well as soil system.

Comprehensive studies on the effect of arsenic (As) on free-living diazotrophs that play a crucial role in soil fertility by nitrogen fixation are still scanty. Here, we isolated three free-living bacteria from rice field with potential nitrogen-fixing ability and investigated the impact of As on their nifH gene expression and extracellular polysaccharide (EPS) production in culture condition and soil system. 16S rRNA sequence analysis showed that the isolated bacteria were affiliated to ß-Proteobacteria, γ-Proteobacteria and Firmicutes. As(III) exposure to bacterial isolates followed by RT-qPCR analysis revealed that elevated levels of As reduced the expression of nifH gene in selective bacteria, both in culture medium and soil condition. We also noticed reduced production of EPS under higher concentration of As. All the three bacteria showed high tolerance to As(III), able to oxidize As and exhibited significant plant growth-promoting traits. This investigation indicated that an environment exposed with higher concentration of As might perturbed the activity of free-living diazotrophs in agricultural soil system.

Long-Term Exposure of Agricultural Soil to Veterinary Antibiotics Changes the Population Structure of Symbiotic Nitrogen-Fixing Rhizobacteria Occupying Nodules of Soybeans (Glycine max).

Antibiotics are entrained in agricultural soil through the application of manures from medicated animals. In the present study, a series of small field plots was established in 1999 that receive annual spring applications of a mixture of tylosin, sulfamethazine, and chlortetracycline at concentrations ranging from 0.1 to 10 mg · kg-1 soil. These antibiotics are commonly used in commercial swine production. The field plots were cropped continuously for soybeans, and in 2012, after 14 annual antibiotic applications, the nodules from soybean roots were sampled and the occupying bradyrhizobia were characterized. Nodules and isolates were serotyped, and isolates were distinguished using 16S rRNA gene and 16S to 23S rRNA gene intergenic spacer region sequencing, multilocus sequence typing, and RSα fingerprinting. Treatment with the antibiotic mixture skewed the population of bradyrhizobia dominating the nodule occupancy, with a significantly larger proportion of Bradyrhizobium liaoningense organisms even at the lowest dose of 0.1 mg · kg-1 soil. Likewise, all doses of antibiotics altered the distribution of RSα fingerprint types. Bradyrhizobia were phenotypically evaluated for their sensitivity to the antibiotics, and there was no association between in situ treatment and a decreased sensitivity to the drugs. Overall, long-term exposure to the antibiotic mixture altered the composition of bradyrhizobial populations occupying nitrogen-fixing nodules, apparently through an indirect effect not associated with the sensitivity to the drugs. Further work evaluating agronomic impacts is warranted.IMPORTANCE Antibiotics are entrained in agricultural soil through the application of animal or human waste or by irrigation with reused wastewater. Soybeans obtain nitrogen through symbiotic nitrogen fixation. Here, we evaluated the impact of 14 annual exposures to antibiotics commonly used in swine production on the distribution of bradyrhizobia occupying nitrogen-fixing nodules on soybean roots in a long-term field experiment. By means of various sequencing and genomic fingerprinting techniques, the repeated exposure to a mixture of tylosin, sulfamethazine, and chlortetracycline each at a nominal soil concentration of 0.1 mg · kg-1 soil was found to modify the diversity and identity of bradyrhizobia occupying the nodules. Nodule occupancy was not associated with the level of sensitivity to the antibiotics, indicating that the observed effects were not due to the direct toxicity of the antibiotics on bradyrhizobia. Altogether, these results indicate the potential for long-term impacts of antibiotics on this agronomically important symbiosis.

Effects of decabromodiphenyl ether and planting on the abundance and community composition of nitrogen-fixing bacteria and ammonia oxidizers in mangrove sediments: A laboratory microcosm study.

While nitrogen (N) fixation and ammonia oxidation by microorganisms are two important N cycling processes, little is known about how the microbes that drive these two processes respond when sediments are contaminated with persistent organic pollutants. In this study, we carried out a laboratory microcosm experiment to examine the effects of decabromodiphenyl ether (BDE-209), either on its own or combined with a common mangrove species, Avicennia marina, on the abundance, diversity, and community composition of N-fixing bacteria (NFB) and ammonia-oxidizing archaea (AOA) and bacteria (AOB) in mangrove sediments. The sediments were very N-limited after one year. The rates of N fixation and NFB abundance were significantly higher in the sediments that contaminated by BDE-209, especially in the planted sediment, indicating that both BDE-209 and planting stimulated N fixation in N-limited mangrove sediments. In contrast, the potential nitrification rate and abundance of AOA and AOB decreased significantly under BDE-209 and planting, and the inhibitory effects were stronger in the sediment with both planting and BDE-209 than in the sediments with either BDE-209 or planting. The results from pyrosequencing showed that the richness and diversity of NFB increased, while those of AOA and AOB decreased, in the sediments treated with BDE-209 only and with BDE-209 combined with planting. The community compositions of NFB, AOA, and AOB in the sediments shifted significantly because of BDE-209, either alone or particularly when combined with planting, as shown by the increases in some NFB from the Proteobacteria phylum and decreases in AOA in the Nitrosopumilus genus and AOB in the Nitrosospira genus, respectively.

Regulation of Differentiation of Nitrogen-Fixing Bacteria by Microsymbiont Targeting of Plant Thioredoxin s1.

Legumes associate with rhizobia to form nitrogen (N2)-fixing nodules, which is important for plant fitness [1, 2]. Medicago truncatula controls the terminal differentiation of Sinorhizobium meliloti into N2-fixing bacteroids by producing defensin-like nodule-specific cysteine-rich peptides (NCRs) [3, 4]. The redox state of NCRs influences some biological activities in free-living bacteria, but the relevance of redox regulation of NCRs in planta is unknown [5, 6], although redox regulation plays a crucial role in symbiotic nitrogen fixation [7, 8]. Two thioredoxins (Trx), Trx s1 and s2, define a new type of Trx and are expressed principally in nodules [9]. Here, we show that there are four Trx s genes, two of which, Trx s1 and s3, are induced in the nodule infection zone where bacterial differentiation occurs. Trx s1 is targeted to the symbiosomes, the N2-fixing organelles. Trx s1 interacted with NCR247 and NCR335 and increased the cytotoxic effect of NCR335 in S. meliloti. We show that Trx s silencing impairs bacteroid growth and endoreduplication, two features of terminal bacteroid differentiation, and that the ectopic expression of Trx s1 in S. meliloti partially complements the silencing phenotype. Thus, our findings show that Trx s1 is targeted to the bacterial endosymbiont, where it controls NCR activity and bacteroid terminal differentiation. Similarly, Trxs are critical for the activation of defensins produced against infectious microbes in mammalian hosts. Therefore, our results suggest the Trx-mediated regulation of host peptides as a conserved mechanism among symbiotic and pathogenic interactions.

Effects of oxytetracycline on the abundance and community structure of nitrogen-fixing bacteria during cattle manure composting.

The effects of oxytetracycline (OTC) on nitrogen-fixing bacterial communities were investigated during cattle manure composting. The abundance and community structure of nitrogen-fixing bacteria were determined by qPCR and denaturing gradient gel electrophoresis (DGGE), respectively. The matrix was spiked with OTC at four levels: no OTC, 10mg/kg dry weight (DW) OTC (L), 60mg/kg DW OTC (M), and 200mg/kg DW OTC (H). The high temperature period of composting was shorter with M and H, and the decline in temperature during the cooling stage was accelerated by OTC. OTC had a concentration-dependent inhibitory effect on the nitrogenase activity during early composting, and the nifH gene abundance declined significantly during the later composting stage. The DGGE profile and statistical analysis showed that OTC changed the nitrogen-fixing bacterial community succession and reduced the community richness and dominance. The nitrogen-fixing bacterial community structure was affected greatly by the high level of OTC.

Symbiosis between nitrogen-fixing bacteria and Medicago truncatula is not significantly affected by silver and silver sulfide nanomaterials.

Silver (Ag) engineered nanomaterials (ENMs) are being released into waste streams and are being discharged, largely as Ag2S aged-ENMs (a-ENMs), into agroecosystems receiving biosolids amendments. Recent research has demonstrated that biosolids containing an environmentally relevant mixture of ZnO, TiO2, and Ag ENMs and their transformation products, including Ag2S a-ENMs, disrupted the symbiosis between nitrogen-fixing bacteria and legumes. However, this study was unable to unequivocally determine which ENM or combination of ENMs and a-ENMs was responsible for the observed inhibition. Here, we examined further the effects of polyvinylpyrollidone (PVP) coated pristine Ag ENMs (PVP-Ag), Ag2S a-ENMs, and soluble Ag (as AgSO4) at 1, 10, and 100 mg Ag kg(-1) on the symbiosis between the legume Medicago truncatula and the nitrogen-fixing bacterium, Sinorhizobium melliloti in biosolids-amended soil. Nodulation frequency, nodule function, glutathione reductase production, and biomass were not significantly affected by any of the Ag treatments, even at 100 mg kg(-1), a concentration analogous to a worst-case scenario resulting from long-term, repeated biosolids amendments. Our results provide additional evidence that the disruption of the symbiosis between nitrogen-fixing bacteria and legumes in response to a mixture of ENMs in biosolids-amended soil reported previously may not be attributable to Ag ENMs or their transformation end-products. We anticipate these findings will provide clarity to regulators and industry regarding potential unintended consequences to terrestrial ecosystems resulting from of the use of Ag ENMs in consumer products.