Search Results (31)

Lentil is a nutritionally valuable legume crop, rich in protein, carbohydrates, amino acids, and vitamins, and is also used as green manure. Symbiotic nitrogen fixation (SNF) plays a crucial role in lentil growth and development, yet there is limited research on isolating and identifying lentil rhizobia related to nodulation and nitrogen fixation. This study employed tissue block isolation, line purification, and molecular biology to isolate, purify, and identify rhizobial strains from lentils, analyzing their physiological characteristics, including bromothymol blue (BTB) acid and alkali production capacity, antibiotic resistance, salt tolerance, acid and alkali tolerance, growth temperature range, and drought tolerance simulated by PEG6000. Additionally, the nodulation capacity of these rhizobia was assessed through inoculation experiments using the identified candidate strains. The results showed that all isolated rhizobial strains were resistant to Congo red, and nifH gene amplification confirmed their potential as nitrogen fixers. Most strains were positive for H₂O₂ and BTB acid and base production, with a preference for alkaline environments. In terms of salt tolerance, the strains grew normally at 0.5–2% NaCl, and six strains were identified as salt stress resistant at 4% NaCl. The temperature range for growth was between 4 °C and 49 °C. Antibiotic assays revealed resistance to ampicillin and low concentrations of streptomycin, while kanamycin significantly inhibited growth. Two drought-tolerant strains, TG25 and TG55, were identified using PEG6000-simulated drought conditions. Inoculation with candidate rhizobial strains significantly increased lentil biomass, highlighting their potential for enhancing crop productivity. Full article

Moss–cyanobacteria associations serve as significant nitrogen fixers and represent the primary nitrogen sink in boreal forests. Fungi, which are essential for soil biogeochemical cycling, have community structures intrinsically linked to forest ecosystem health and productivity. Using high-throughput sequencing, we investigated differences between moss-covered and non-moss soils in two alpine forests (both plantation and natural forests) by examining soil nitrogen contents, fungal community structure, composition, and functional guilds. Results demonstrated that moss cover enhanced soil nutrient contents, including total carbon, total nitrogen, and inorganic nitrogen. It also altered fungal community characteristics, resulting in higher Chao1 and Shannon diversity indices, as well as a more complex fungal network. Notable changes in functional guilds included an increase in saprotrophic fungi abundance and a decrease in ectomycorrhizal fungi. Our findings support the concept that moss cover creates distinct soil environments: moss-covered soils attract decomposers and nutrient-mobilizing fungi (particularly saprotrophs and ectomycorrhiza), while non-moss soils favor ectomycorrhizal fungi that relieve nutrient limitation through extensional mycelial networks. These findings highlight the critical role of moss cover in sustaining forest soil health and resilience, positioning it as a cornerstone of carbon and nutrient cycling within forest ecosystems. Full article

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. Full article

Background: Lake Al-Asfar in KSA was used as a sink for wastewater for decades and suffered from pollution. The lake is a habitat to different microbial species that play important ecological roles, some of which are good, and some are bad and even pathogenic. In a previous investigation, algal-bacteria consortia have proven to be beneficial in bioremediating heavy metals and hydrocarbons in Lake Al-Asfar. The identity of algae was revealed to be Chlorella sp. and Geitlernema sp. in the consortia. The identity of the heterotrophic bacterial partners, on the other hand, awaits investigation and is addressed in the present research. On the other hand, investigating the diversity of Protozoa and parasites is also tackled as they represent indicators of pollution. Some pose serious health risks, but some of them also contribute to reducing some of the pollution levels. Methods: Bacteria associated with algae were isolated in pure form. The polyphasic approach was used to identify bacterial samples, including staining procedures, the use of Vitek technology, and scanning electron microscopy. This information was integrated with structure information such as capsule presence, endospore formation, and wall characteristics indicated by Gram stain. With regard to protists including Protozoa and parasites, Light microscopy and taxonomic books of identification were used to reveal their identity. Results: three main bacterial strains belonging to the following genera were identified: Sphingomonas, Rhizobium, and Enterbacter. The last is potentially pathogenic and poses health risks to Lake goers. Rhizobium, on the other hand, is most likely found in the lake from agricultural wastewater and is a nitrogen fixer that increases the fertility of crops. The first bacterium is associated with special lipid metabolism and is hardly pathogenic. Several diverse microscopic forms of protists, mainly Protozoa and parasites, were identified, which included Entamoeba histolytica, Balantidium coli, Ascaris lumbricoides, Amoeba, Paramecium, Euglena, and Gymnodinium sp. Discussion: The three types of bacteria identified have metabolic activities that are associated with bioremediation. On the other hand, protists, including Protozoa and parasites, are regular members of wastewater communities and help in scavenging solid wastes, but they cause hazards such as secreting toxins, causing disease, and impacting the bioremediation potential by feeding on beneficial bioremediating algae and bacteria. This is part of the wastewater ecosystem dynamics, but efforts must be exerted to minimize, if not completely eliminate, pathogenic parasites in order to maximize the growth of algal consortia. Conclusions: Vitek technology is an emerging less time- and effort-consuming fast technology for identifying bacteria. Bacteria identified have significant ecological bioremediating roles, together with their algal partners, but some pose pathogenic risks. Identifying co-inhabitants like protists and parasites helps to shed light on their impact on one another and pave the way for restoration efforts that minimize the biological hazards and maximize the use of beneficial local microorganisms. Full article

Nitrogen fertilizers have a significant impact on the growth of rice. The overuse and inappropriate application of nitrogen fertilizers have resulted in environmental pollution, in addition to subjecting both humans and livestock to negative health hazards. Finding a viable substitute for traditional nitrogen fertilizers is crucial and essential to help improve crop yield and minimize environmental damage. Nano-nitrogen fertilizers offer a possible alternative to traditional fertilizers due to a slow/controlled release of nitrogen. The present work aimed to study the effect of a slow-release urea nanofertilizer on soil ammonical (NH₄-N) and nitrate-N (NO₃-N) content, culturable soil microflora, and soil enzyme activities in three different soil samples procured from Ludhiana and Patiala districts through a soil column study. Seven treatments, including 0, 50 (75 kg/ha N), 75 (112.5 kg/ha N), and 100% (150 kg/ha N) of the recommended dose (RD) of conventional urea and nano-urea fertilizer were applied. The leachate samples collected from nano-urea treatment exhibited NH₄-N for the first two weeks, followed by NO₃-N appearance. The higher NH₄-N and NO₃-N contents in the leachate were recorded for light-textured soil as compared to medium- and heavy-textured soil samples. The soil microbial counts and enzyme activities were recorded to be maximum in light-textured soils. Therefore, this slow-release formulation could be more useful for light-textured soils to decrease applied N-fertilizer losses, as well as for improving the soil microbial viable cell counts and soil enzyme activities. The effect of urea nanofertilizer on the growth and yield of direct-seeded rice (Oryza sativa L.) was also evaluated under field conditions. Both studies were performed independently. Numerically, the highest shoot height, fresh and dry shoot weight, and significantly maximum total chlorophyll, carotenoid, and anthocyanins were recorded in the T2 (100% RDF through nano-urea) treatment. The yield-attributing traits, including the number of filled grains and thousand-grain weight, were also recorded to have increased in T2 treatment. A numerical increase in NPK for plant and grain of rice at 100% RDN through nano-urea was recorded. The soil application of the product exhibited no negative effect on the soil microbial viable cell count on different doses of nano-urea fertilizer. The soil nitrogen fixer viable counts were rather improved in nano-urea treatments. The results reflect that nano-urea fertilizer could be considered as a possible alternative to conventional fertilizer. Full article

In this study, the changes in yield, nutrient content, and amino acid levels in legume–cereal grass mixtures were qualitatively evaluated depending on the legume–cereal combination and inoculation with preparations based on Rhizobium. This study, taking into account the biological characteristics of legume forage crops, used inoculations with strains of nodule bacteria and associative nitrogen fixers to enhance the process of the nitrogen fixation of mixed crops of legumes and cereal. The aim of this study was to compare the yields and nutritional values of monocultures and mixed crops, as well as to determine the effects of preparations based on strains of nodule bacteria and the associated nitrogen fixer on the photosynthetic activity and yield of combined annual legume–grain crops. A comparative study of forage crop biomass was conducted to analyze crude protein, fiber, carotene, and amino acid content in monocultures and legume–cereal mixtures, with and without the use of nodule-bacteria-based preparations (Rhizotorphin, Mizorine, Flavobactrin, and Azolene). The combined effect of crop mixtures and biological products led to increased green mass yield, protein content, and feed productivity. Notably, two-component mixtures with Rhizotorphin inoculation increased green mass yield by 8.79%, while three-component mixtures saw a 16.49% increase. The oat–pea mixture showed the most significant amino acid improvements, with lysine increasing by 6.26% and tyrosine by 3.24%. The general conclusion reached by the two-year experiment of 2022–2023 in the hill–plain zone of northern Kazakhstan is that double grass mixtures treated with nodule bacteria are more productive than monoculture crops in this area. These results suggest that inoculation with bacterial strains can effectively enhance the productivity of forage crops in northern Kazakhstan, providing a basis for future recommendations on optimizing herbaceous crop combinations. It is recommended to grow annual forage crops in mixtures with legumes to produce highly nutritious feeds with high metabolic energy in terms of biochemical composition. Full article

The expanding global population has increased the demand for sustainable protein sources, and microbial protein (MP) has emerged as a promising alternative. However, conventional carbon (glucose) and nitrogen (ammonia, urea) sources needed for MP production pose environmental and economic issues. This study aims to produce protein using lignocellulosic biomass (LCB) as a carbon source and the nitrogen fixation ability of Klebsiella oxytoca M5A1 as a nitrogen source. The study investigates the pretreatment of LCB (switchgrass), enzymatic hydrolysis, protein quantification, nitrogen fixation, glucose utilization and organic acids production. K. oxytoca M5A1 harnessed free nitrogen from the atmosphere and used abundant, cheap glucose from LCB to produce MP and organic acids as by-products. Protein production occurred in two phases: first within the initial 8 h and secondly, within the last 16 h. The highest protein concentration was at 40 h, with approximately 683.46 µg/mL protein content. High-performance liquid chromatography system (HPLC) analysis revealed a dynamic profile of glucose utilization and organic acids (Lactic acid, Propionic acid, Acetic acid, and Succinic acid) production. K. oxytoca M5A1 exhibited an early high rate of glucose consumption, and conversion to organic acids, that were later used for second-phase protein production. The acids profile revealed intra-conversion from one acid to another via metabolic pathways (glycolysis and tricarboxylic acid cycle). Overall, leveraging LCB and the nitrogen-fixing ability of K. oxytoca M5A1 for MP production offers an eco-friendly and cost-effective alternative to traditional protein sources, contributing to a sustainable circular economy. Full article

Given the importance of biological nitrogen (N) fixation in agroecosystems, using inoculants with phyllosphere N fixers effective across various crops would revolutionize agriculture. In this study, the application of an inoculant prepared from Methylobacterium symbioticum was tested on young olive trees. The pot experiment was arranged in a factorial design, with inoculant (Yes and No) and mineral N applied to the soil [0 (N0), 25 (N25), 50 (N50), and 100 (N100) kg ha⁻¹], and four replicates. The inoculant application did not increase plant dry matter yield (DMY), whereas the application of mineral N had a significant and pronounced effect. The inoculant also did not significantly increase N concentration in tissues, unlike the strong increase observed with N applied to the soil. The inoculant significantly increased plant N recovery, a cumulative effect resulting from small increases in DMY and N concentration in tissues. This increase represented 5.2% more N in plants receiving the inoculant compared to untreated ones. However, only treatments receiving mineral N recorded positive values of fixed N, with the highest value observed in the N50 treatment (12.4%), whereas a negative value (−7.7%) was observed in the N0 treatment. Overall, these low values of fixed N question the economic rationale of using this inoculant by farmers and especially render it unsuitable for organic farming systems, where plants tend to have lower N levels in tissues. Full article

Under salinity conditions, growth and productivity of grain crops decrease, leading to inhibition and limited absorption of water and elements necessary for plant growth, osmotic imbalance, ionic stress, and oxidative stress. Microorganisms in bio-fertilizers have several mechanisms to provide benefits to crop plants and reduce the harmful effect of salinity. They can be effective in dissolving phosphate, fixing nitrogen, promoting plant growth, and can have a combination of all these qualities. During two successful agricultural seasons, two field experiments were conducted to evaluate the effect of bio-fertilizer applications, including phosphate solubilizing bacteria (PSB), nitrogen fixation bacteria and a mix of phosphate-solubilizing bacteria and nitrogen fixation bacteria with three rates, 50, 75 and 100% NPK, of the recommended dose of minimal fertilizer on agronomic traits, yield and nutrient uptake of barley (Hordeum vulgare) under saline condition in Village 13, Farafra Oasis, New Valley Governorate, Egypt. The results showed that the application of Microbein + 75% NPK recorded the highest values of plant height, spike length, number of spikes/m², grain yield (Mg ha⁻¹), straw yield (Mg ha⁻¹), biological yield (Mg ha⁻¹), protein content %, nitrogen (N), phosphorus (P), potassium (K) uptakes in grain and straw (kg ha⁻¹), available nitrogen (mg/kg soil), available phosphorus (mg/kg soil), total microbial count of soil, antioxidant activity of soil (AOA), dehydrogenase, nitrogen fixers, and PSB counts. The application of bio-fertilizers led to an increase in plant tolerance to salt stress, plant growth, grain yield, and straw yield, in addition to the application of the bio-fertilizers, which resulted in a 25% saving in the cost of mineral fertilizers used in barley production. Full article

Lentil (Lens culinaris Medik.) is an essential legume crop providing healthy and nutritious food for people in low- to middle-income countries, worldwide. Lentil roots support symbiotic interactions with soil rhizobia species fostering nitrogen fixation; however, assemblage and diversity of the complete microbial rhizosphere community and the effect of seed genotype and origin remain largely unexplored. In this study we examined, via metagenomic analysis, the effects of seed origin on the rhizosphere’s communities in samples of the famous Greek lentil landrace, Eglouvis, derived from different local farmers and farming systems (including a Gene Bank sample), in comparison to a commercial variety. The landrace exhibited higher rhizosphere microbiome diversity compared to the commercial variety for all indexes. A core microbiome comprised of 158 taxa was present in all samples, while a greater number of unique bacterial taxa was recorded in the landrace samples compared to the commercial cultivar. Notably, landrace samples originated from organic farming had more than double the number of unique taxa compared to conventional counterparts. The study revealed a higher diversity of N₂ fixers and archaea, Crenarchaeota and Thaumarchaeota, in landrace samples and particularly in those derived from organic farming, underpinning the distinct recruiting efficiency of beneficial soil microbes by the landrace. Full article

Presently, as human activity and climate warming gradually increase, straw burning leads to more accidental burning in neighbouring wetlands, which threatens wetland carbon stores. Plants are important carbon fixers in wetlands, converting carbon dioxide to biomass through photosynthesis and releasing carbon into the soil as plants die off. Nitrogen and phosphorus limitation in wetlands is a key factor affecting plant growth, and different burning seasons have different effects on mitigating this limitation. To further elucidate the effects of nitrogen and phosphorus distribution on wetland inter-month nutrient dynamics after different burning seasons, we selected a Calamagrostis angustifolia wetland in the Sanjiang Plain that was burned in spring and autumn, respectively, and conducted a monthly survey from May to September. We found that the leaf nitrogen content in September at spring burning sites was 3.59 ± 2.69 g/kg, which was significantly lower than that in July, while the difference at the unburned sites was only 0.60 ± 3.72 g/kg, and after the autumn burning, soil nitrogen and phosphorus contents remained higher than at the unburned sites in August, being 0.55 ± 1.74 g/kg and 0.06 ± 0.12 g/kg, respectively. Our results indicate that spring burning immediately increased the nitrogen and phosphorus contents in soil and plants but that these effects only lasted for a short time, until June. In comparison, autumn burning had a long-term effect on soil nitrogen and phosphorus levels and significantly increased the aboveground biomass. Thus, we recommend that conducting autumn burning before the commencement of agricultural burning not only reduces combustible accumulation to prevent fires but also promotes nitrogen and phosphorus cycling in wetlands, and the increase in plant biomass after autumn burning also enhances the carbon fixation capacity of the wetland. Full article

Alterations to the functions and connectivity of mangrove–seagrass–patch reef (MSP) seascapes have the potential to impact the survival, foraging activities, and movement of reef-dependent invertebrates (e.g., crabs and shrimp) and fishes. In the current study, we examined carbon flow in the Guánica Biosphere Reserve in southwestern Puerto Rico using pigment analysis of particulate organic matter and stable isotope analysis of carbon (δ¹³C) and nitrogen (δ¹⁵N) in flora and fauna. Several lines of evidence pointed to N₂ fixers (cyanobacteria) being important for fueling primary productivity in this oligotrophic ecosystem including low (<0.7 µg L⁻¹) chlorophyll, prevalence of cyanobacteria based on pigment signatures, and the isotope signatures of seagrass and red mangrove leaf tissue (enriched δ¹⁵N values) and consumers (depleted δ¹⁵N values). Food web mixing models based on stable isotopes (δ¹³C and δ¹⁵N) revealed that multiple producers (phytoplankton, benthic microalgae, seagrasses, etc.) contributed organic matter to the consumers (zooplankton, invertebrates, and fishes) in the MSP seascape at the center of the reserve. Contribution estimates for common benthic invertebrates (crabs and shrimp) were taxon-specific, and the highest input was generally linked to particulate organic matter (POM) and benthic microalgae (BMA)/seagrass producer categories, although meaningful mangrove contribution was observed for some taxa. Similarly, contribution estimates for fishes were highest for POM and BMA/seagrass, with the latter producer category being more important for species known to migrate from mangroves or patch reefs to seagrass beds at night (bluestriped grunt, French grunt, and white grunt). Although all fish investigated were observed in mangrove prop-root habitats, input of organic matter from mangroves to these consumers was typically limited for most of the species examined. Understanding these complex seascapes contributes to our understanding of the ecology of these vital ecosystems. Full article

Global warming and eutrophication are the main factors driving the development of cyanobacterial dominance in aquatic ecosystems. We used a model linking water temperature, oxygen saturation, concentrations of PO₄³⁻, NO₃⁻, NH₄⁺, total dissolved iron (TDFe), and SO₄²⁻ to cyanobacteria to test the turnover patterns of cyanobacterial dominance of non-nitrogen-fixing (chroococcal species) and nitrogen-fixing (filamentous diazotrophic) species. Statistical analysis was performed using decision trees. The dominance patterns of the two morphologically and ecologically distinct cyanobacterial species were associated with different environmental factors. However, SO₄²⁻ was the most important factor that explained whether non-nitrogen-fixing or nitrogen-fixing species would dominate. Other important factors were water temperature, phosphate concentration, and oxygen saturation. The model for dominance of non-nitrogen-fixing species used SO₄²⁻, PO₄³⁻, and water temperature (upper layers), and SO₄²⁻, the ratio of PO₄³⁻/NH₄⁺, and oxygen saturation (bottom layers). In contrast, water temperature, SO₄²⁻, and NH₄⁺ in the upper layers and SO₄²⁻, NH₄⁺, and water temperature in the bottom layers were used for the dominance of nitrogen-fixing species. The dominance of Aphanizomenon flos-aquae was explained by different sets of variables, indicating the presence of different strains of this species. The other cyanobacteria species showed dominance patterns that could be explained by one set of variables. As cyanobacterial blooms proliferate due to climate change, it is important to know which factors, in addition to phosphorus and nitrogen, are crucial for the mass development of the various cyanobacterial species. Full article

Evaluating how different tree species and substrates affect the microbial and physicochemical properties of technosols from combustion wastes and reclaimed mine soil (RMS) is vital in species selection to enhance restored ecosystem services. This research aimed to evaluate the impact of pioneer and N-fixing tree species and substrates on the post-mining soil microbial and physicochemical properties. Common birch (Betula pendula Roth) and Scots pine (Pinus sylvestris L.), as the commonly introduced species on reclaimed mine soils (RMS) in eastern and central Europe, were selected as pioneer species, whereas black alder (Alnus glutinosa (L) Gaernt.) and black locust (Robinia pseudoacacia L.) were selected as N-fixer species. Soil samples were collected from different RMS developed from three substrates (fly ashes, clay, and sand) and measured for the content of total nitrogen (N_t), organic carbon (C_org), exchangeable calcium (Ca²⁺), exchangeable potassium (K⁺), exchangeable magnesium (Mg²⁺), C to N ratio (C:N), basal respiration rate (RESP), and microbial biomass carbon (C_mic). The research indicated that tested tree species influenced water holding capacity (WHC), N_t, C:N, and RESP value. The highest N_t accumulation in soil was observed under N-fixing, but it did not transfer into higher organic carbon content under N-fixers. The soil under pine had a greater C:N ratio than the soil under birch, alder, and locust. The RESP rate was highest under birch. In terms of substrate type, RMS developed on Miocene clays exhibited higher carbon and macronutrient contents followed by ashes, whereas sands exhibited the lowest values of both physicochemical and microbial properties. The study suggested that both tree species and substrates affect microbial activities and physicochemical properties of RMS; however, the substrate effect is stronger. Full article

Soil extracellular enzyme stoichiometry (EES) is the essential predictor in nutrient status and resource limitation of soil microorganisms, whose metabolism has a vital role in biogeochemical cycling and ecosystem function. However, little is known about how N₂-fixer tree species with different planting patterns affect soil nutrient resources in terms of extracellular enzyme activity (EEA) or EES within aggregates in degraded karst ecosystems. In this study, we evaluated soil EEA and EES related to carbon (C), nitrogen (N), and phosphorus (P) cycles across two eight-year-old pure plantations of legume species [Dalbergia odorifera T. Chen (PD) and Acrocarpus fraxinifolius Wight ex Arn. (PA)] and a mixed plantation of the two tree species listed above (MP). Meanwhile, a nearby undisturbed shrubland was used as a control (CK). We concluded that the activities of C-, N-, and P-acquiring enzyme increased to different degrees in the N₂-fixer tree species stands (particularly in MP) compared to CK in all aggregates. Compared to CK, MP significantly increased by 39.0%, 54.0%, 39.3%, and 24.8% in total C-acquiring EEA, 41.1%, 60.5%, 47.8%, and 12.5% in total N-acquiring EEA, and 100.4%, 79.7%, 69.2%, and 56.4% in total P-acquiring EEA within >2 mm, 1–2 mm, 0.25–1 mm, and <0.25 mm aggregates, respectively. Furthermore, the logarithmic transformed ratio of C-, N-, and P-acquiring enzyme activities was 1.20:1.08:1, which deviated from the global ratio (1:1:1). Vector analysis of EEA showed that the vector length (VL) within aggregates was significantly lower than that of CK in all stands of N₂-fixer species except PD; while in all treatments, vector angle (VA) was <45° for all aggregate sizes, except in MP, where VA reached 45° for <0.25 mm aggregate. These indicated soil microbes were limited by C and N together. However, MP significantly alleviated microbial C and N limitation than CK (p < 0.05). There were obvious positive relationships between enzyme C:N, C:P, and N:P ratios. VL was markedly negatively linked to VA. EES was markedly related to most soil nutrients and microbial biomass stoichiometry ratios. Changes in soil EEA and EES were primarily driven by available phosphorus (AP), microbial biomass carbon (MBC), soil C:N and MBN:MBP ratios. Together, our results demonstrate the influences after introducing N₂-fixer tree species (particularly MP) for vegetation recovery on soil microbial nutrient limitation and ecological processes in aggregate level and will contribute to the development of ecological restoration practices and fertility management in degraded karst ecosystems of southwest China. Full article