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Coniferous forest nitrogen (N) budgets indicate unknown sources of N. A consistent association between limber pine (Pinus flexilis) and potential N₂‐fixing acetic acid bacteria (AAB) indicates that native foliar endophytes may supply subalpine forests with N. To assess whether the P. flexilis–AAB association is consistent across years, we re‐sampled P. flexilis twigs at Niwot Ridge, CO and characterized needle endophyte communities via 16S rRNA Illumina sequencing. To investigate whether endophytes have access to foliar N₂, we incubated twigs with ¹³N₂‐enriched air and imaged radioisotope distribution in needles, the first experiment of its kind using ¹³N. We used the acetylene reduction assay to test for nitrogenase activity within P. flexilis twigs four times from June to September. We found evidence for N₂ fixation in P. flexilis foliage. N₂ diffused readily into needles and nitrogenase activity was positive across sampling dates. We estimate that this association could provide 6.8–13.6 μg N m⁻² d⁻¹ to P. flexilis stands. AAB dominated the P. flexilis needle endophyte community. We propose that foliar endophytes represent a low‐cost, evolutionarily stable N₂‐fixing strategy for long‐lived conifers. This novel source of biological N₂ fixation has fundamental implications for understanding forest N budgets.

The ability of non-nodulating pioneer plants on newly exposed glacial till to obtain atmospheric N2 was investigated using both acetylene reduction assay (ARA) and 15N2 gas incorporation methods. Plant tissues were also examined for the presence of endophytic diazotrophic bacteria using high-throughput nifH amplification sequencing. Both ARA and 15N2 gas incorporation demonstrated that non-nodulating pioneer plants can obtain atmospheric N2, but the amount was highly variable depending on the plant organ, with clearly higher values in leaves relative to twigs and roots. High-throughput amplification sequencing of nifH genes indicated that a great variety of diazotrophic communities developed in pioneer plant tissues under primary succession. The N2 fixation rate (based on both 15N2 incorporation and ARA) was not directly linked to the abundance of any specific diazotrophs, but was correlated with the Chao1 index and the number of observed species, suggesting that different diazotrophs jointly accounted for N2 fixation activity. Our results highlight that non-nodulating pioneer plants may acquire N through endophytic N2-fixing bacteria as a supplement for other potential N sources in oligotrophic environments in early stages of primary succession following glacier retreat.

Accumulating evidence indicates that fertilizing soybean with nickel (Ni) can enhance biological nitrogen fixation (BNF) and plant productivity. Seed application is ideal for promoting nodule formation and early plant development, but this practice raises the risk of toxicity by reducing the difference between beneficial and harmful doses. Unfortunately, studies of the effects of Ni on the survival of spp. applied as inoculants or on BNF have not yielded a consensus on an optimal dose. The objective of this study was to establish Ni thresholds that maximize physiological and productivity benefits for soybean, balancing Ni's positive effects on BNF and plant growth against risks of phytotoxicity and bacterial inhibition. Soybean seeds were treated with nickel sulfate (NiSO ·6H O) at six doses: 0, 60, 120, 180, 240, and 300 mg Ni kg . We then assessed the effects of seed treatment with Ni on the recovery of cells from treated seeds, BNF as assessed by continuous-flow analysis of acetylene reduction activity (ARA), and soil CO evolution in greenhouse experiments. In addition, we evaluated the impact of Ni dose on the physiological, nutritional, agronomic traits, and grain yield of soybean in multi-site field trials over two cropping seasons. Ni doses of up to 60 mg kg enhanced nitrogenase activity, nodulation, nodule biomass, and grain yield without compromising viability. Doses exceeding this threshold reduced bacterial survival, nodulation, and yield, indicating Ni toxicity. The field trials exhibited a natural gradient in soil Ni levels and texture (0.4-0.6 mg dm sandy to clayey), which helps explain differences in response magnitude and reinforces the need for contextualized recommendations. Applying a second-order polynomial regression to mean standardized Z-scores of integrated agronomic traits revealed a significant quadratic response ( < 0.05). Consequently, an agronomically optimal range of 50-100 mg Ni kg is recommended to sustainably optimize soybean growth and N fixation by balancing the benefits and risks of Ni application.

Background and aim Biological soil crusts (biocrusts) play numerous crucial roles in drylands, which comprise over 40% of Earth's terrestrial surface. Among these key contributions is the fixation of atmospheric nitrogen. Yet, relatively little is known about the N2 fixation capabilities of different lichen species that are found in late successional biocrust communities across diylands globally. Methods In order to improve our species-specific understanding of biocrust lichen N2 fixation, we collected biocrusts dominated by four common species of lichens – Collema spp., Gyalolechia desertorum, Psora decipiens, and Squamarina lentigera – that represent a range of lichen families and morphological types. Nitrogenase activity of the biocrust community dominated by these lichens was evaluated using the acetylene reduction assay. Additionally, biocrust community composition was assessed using the point-intercept method along transects at varied distances from exposed bedrock. Results As expected, Collema spp.-dominated biocrusts had the highest rates of nitrogenase activity, with rates up to seven times larger than those of the other three target species. Nitrogen concentrations and carbon:nitrogen ratios of lichen tissue differed among lichen species. However, when the composite biocrust profile was assessed (i.e., biocrust tissue, microbial cells, and mineral soil to a 2 cm depth) these among-species differences in total nitrogen disappeared. Community composition changed according to distance from exposed bedrock, with a higher diversity of lichens closer to the bedrock. Conclusion Multiple drivers, including climate and land use change, affect biocrust community composition and species-specific functional information, even within a group such as late successional biocrusts, could help in forecasting the potential effects of global change on N2 fixation, and consequently, soil fertility in drylands.

Poor soil fertility is one of the major constraints for crop production. Nitrogen is the most limiting nutrient for increasing crop productivity. Therefore, there is a need to identify diazotrophic inoculants as an alternative or supplement to N-fertilizers for sustainable agriculture. In the current study, a number of free-living diazotrophic bacteria were isolated from soils collected from maize rhizosphere and from leaves and roots of maize within the KwaZulu-Natal Province, Republic of South Africa. Ninety-two isolates were selected for further screening because they were able to grow on N-free media containing different carbon sources. Isolates that were very slow to grow on N-free media were discarded. The isolates were screened in vitro for diazotrophic potential tests for ammonia production and acetylene reduction. Ethylene (C2H4) production was quantified and ranged from 4 to 73 nmoles of C2H4h(-1) culture(-1). The top 20 isolates were re-screened on maize seedlings, and eight isolates significantly (P = 0.001) enhanced some growth parameters of maize above the un-inoculated control. Isolates that showed significant effect on at least two growth parameters were identified at species or genera level. In conclusion, selected diazotrophic isolates may be potentially beneficial but they should be tested more in greenhouse and field conditions with maize to confirm their potential for application as biofertilizers.

Drought is the most important factor limiting the activity of rhizobia during N-fixation and plant growth. In the present study, we isolated Bradyrhizobium spp. from root nodules of higher trehalose-accumulating soybean genotypes and examined for moisture stress tolerance on a gradient of polyethylene glycol (PEG 6000) amended in yeast extract mannitol (YEM) broth. In addition, the bradyrhizobial strains were also evaluated for symbiotic effectiveness on soybean. Based on 16S rDNA gene sequences, four bradyrhizobial species were recovered from high trehalose-accumulating genotypes, i.e., two Bradyrhizobium liaoningense strains (accession number KX230053, KX230054) from EC 538828 and PK-472, respectively, one Bradyrhizobium daqingense (accession number KX230052) from PK-472, and one Bradyrhizobium kavangense (accession number MN197775) from Valder genotype having low trehalose. These strains, along with two native strains, viz., Bradyrhizobium japonicum (JF792425), Bradyrhizobium liaoningense (JF792426), and one commercial rhizobium, were studied for nodulation, leghaemoglobin, and N-fixation abilities on soybean under sterilized sand microcosm conditions in a completely randomized design. Among all the strains, D-4A ( B. daqingense ) followed by D-4B ( B. liaoningense ) was found to have significantly higher nodulation traits and acetylene reduction assay (ARA) activity when compared to other strains and commercial rhizobia. The bradyrhizobia isolates showed plant growth promotion traits such as indole acetic acid (IAA), exopolysaccharide (EPS), and siderophore production, phosphate-solubilizing potential, and proline accumulation. The novel species B. daqingense was reported for the first time from Indian soil and observed to be a potential candidate strain and should be evaluated for conferring drought tolerance in soybean under simulated stress conditions .

Biological desert crusts are relatively common in the arid deserts of the Sultanate of Oman; however, little is known about their microbial community composition and role in soil fertilization. We compared three crusts from geographically different locations for their soil texture, bacterial community structure, pigment composition and nitrogenase activity. The crusts were growing on alkaline (pH 7.6-8.7) loamy sand and silty loam soils. Microscopically, Microcoleus vaginatus was the most abundant cyanobacterium, but Nostoc and Scytonema types dominated in cultures. The 16S rRNA gene sequences showed close similarities in the crusts' bacterial composition, with 77-81% of the total clones belonging to cyanobacteria and the rest distributed among Alpha- and Deltaproteobacteria, Bacteriodetes, Gemmatimonas and Planctomycetes. Thirty-seven percent of the cyanobacterial clones were affiliated with heterocystous types such as Nostoc, Scytonema, Brasilonema and Petalonema. Chlorophyll a concentrations suggest a similar abundance of phototrophs in all crusts. High levels of the UVA sunscreen scytonemin were detected in the exposed crusts. The three crusts exhibited comparable acetylene reduction rates in the light and in the dark, with a maximum rate of 58.5±2.6 μmol C₂H₂ reduced m⁻² h⁻¹. We conclude that the crusts, regardless of their geographical location, were rich in heterocystous cyanobacteria that can fix nitrogen and could possibly improve soil stability and productivity.

While nitrogen (N 2 ) fixation is an important process in nitrogen (N) biogeochemical cycling, supplying a significant portion of the N in natural ecosystems, few quantitative constraints exist concerning its contribution to the N enrichment and export from river ecosystems. This study estimates the N 2 fixation rates of urban rivers in the Yangtze Estuary area using acetylene reduction. The results demonstrate that the prominent spatiotemporal variability of river N 2 fixation rates is driven by various environmental factors. River N 2 fixation rates are significantly higher in the summer (90.57 ± 14.60 ngN·L −1 ·h −1 ) than in the winter (57.98 ± 15.73 ngN·L −1 ·h −1 ). Spatially, rivers draining urban and suburban areas have higher N 2 fixation rates than those draining rural areas. The N 2 fixation rates are positively correlated with the N 2 fixing cyanobacteria density, water temperature, light, and the water phosphorus (P) concentration, but they are negatively correlated with the dissolved N concentration (NH 4 + -N and NO 3 − -N). The N 2 fixation rates annually range from 53.20 to 89.24 ngN·L −1 ·h −1 for all of the sampling rivers, which is equivalent to a depth integrated (0–0.6 m) N input of 0.163–0.274 gN·m −2 ·a −1 . The determined annual N input via N 2 fixation is generally higher than that of marine systems, but it is lower than that of eutrophic lakes. This study provides robust evidence that N 2 fixation can supply a substantial portion of the N input to human-impacted river ecosystems, which has not been sufficiently accounted for when determining the N mass balance of riverine ecosystems. A high N 2 fixation rate may increase the ratio of N to P input to river systems, and therefore render P the limiting factor in aquatic eutrophication.

Background and aims Infection of tall fescue with the common toxic fungal endophyte Epichloë coenophiala harms livestock via toxic alkaloid production; therefore, non-toxic 'novel' strains of the endophyte have been developed and released. How different endophyte strains impact biological nitrogen fixation (BNF) in mixed species pastures is unknown. We asked whether novel endophyte or common toxic endophyte-infected (NE+; CTE+) tall fescue affects symbiotic and nonsymbiotic BNF, and utilization of biologically-fixed nitrogen in tall fescue. Methods Tall fescue was planted either endophyte-free (E-), infected with CTE, two non-toxic strains AR542 NE, AR584 NE, or a blend of endophyte treatments. We measured natural abundance of ¹⁵N in plant and soil samples, and conducted soil acetylene reduction assays. Results Endophyte presence and strain significantly affected the δ¹⁵N of tall fescue. Near red clover, CTE+ and AR584 NE+ tall fescue were most ¹⁵N-depleted; but away, E-tall fescue was most ¹⁵N-depleted. Endophyte strain significantly influenced N concentration in red clover, but not symbiotic or non-symbiotic BNF. Conclusions Endophyte strains produce different effects on tall fescue's competitive ability and nitrogen utilization. In mixed pastures, deployment of NE strains for decreased alkaloid toxicity will differentially impact use of biologically fixed nitrogen in tall fescue and nitrogen concentration in red clover.

Adoption of innovative crop establishment practices relies on the type of impact on soil-plant-water complex, rather than just crop productivity. Hence, a field experiment was conducted to study the impact of wheat establishment methods (WEMs) on soil biological properties as well as crop and water productivity. Three wheat establishment methods (WEMs), viz., drill-sown wheat (DW), wheat intensification system (WIS) and zero till-drill wheat (ZTDW) were selected along with two sources of nutrients, viz. chemical fertilizers and microbial inoculation Significant changes in the soil and plant-related aspects, particularly an improvement in soil microbiological properties such as acetylene reduction activity (ARA) (2.7–11.8%), soil chlorophyll (0.6–21.1%), dehydrogenase activity (1.1–10.8%), microbial biomass carbon (0.5–2.1%) and alkaline phosphatase activity (1.3–9.0%) at 90 days after sowing (DAS) in ZTDW over DW and WIS were recorded in the two-year field study. The highest values were observed with the application of 75% recommended dose of nutrients (RDN) (90 kg nitrogen ha−1 and 19.35 kg phosphorus ha−1) + Anabaena–Pseudomonas biofilm formulation + Zn. The soil microbiological parameters such as dehydrogenase activity, microbial biomass carbon and alkaline phosphatase activity also showed a significant increase due to the application of 100% RDN indicating that the application of fertilizers induced beneficial changes in soil-plant-water system. Interestingly, microbial inoculation had an economic impact on wheat which recorded an increase in grain yield by 200–230 kg ha−1 and water productivity by 0.45–0.53 kg ha-mm−1. The contribution of zero till-drill wheat to improvement in grain yield and water productivity was 240–260 kg ha−1 and 0.54–0.58 kg ha-mm−1, respectively. ZTDW and application of Anabaena–Pseudomonas biofilm formulation with 75% RDN led to significant and positive impacts on the biological activities in the rhizosphere of wheat.