Inoculation with Bacillus thuringiensis reduces uptake and translocation of Pb/Cd in soil-wheat system: A life cycle study.

Microbial inoculation is an important strategy to reduce the supply of heavy metals (HMs) in soil-crop systems. However, the mechanisms of microbial inoculation for the availability of HMs in soil and their accumulation/transfer in crops remain unclear. Here, the inhibitory effect of inoculation with Bacillus thuringiensis on the migration and accumulation of Pb/Cd in the soil-wheat system during the whole growth period was investigated by pot experiments. The results showed that inoculation with Bacillus thuringiensis increased soil pH and available nutrients (including carbon, nitrogen, and phosphorus), and enhanced the activities of nutrient-acquiring enzymes. Dominance analysis showed that dissolved organic matter (DOM) is the key factor affecting the availability of HMs. The content of colored spectral clusters and humification characteristics of DOM were significantly improved by inoculation, which is conducive to reducing the availability of Pb/Cd, especially during the flowering stage, the decrease was 12.8 %. Inoculation decreased Pb/Cd accumulation in the shoot and the transfer from root to shoot, with the greatest decreases at the jointing and seedling stages (27.0-34.1 % and 6.9-11.8 %), respectively. At the maturity stage, inoculation reduced the Pb/Cd accumulation in grain (12.9-14.7 %) and human health risk (4.1-13.2 %). The results of Pearson correlation analysis showed that the availability of Pb/Cd was positively correlated with the humification of DOM. Least square path model analysis showed that Bacillus thuringiensis could significantly reduce Pb/Cd accumulation in the grain and human health risks by regulating DOM spectral characteristics, the availability of HMs in soil and metals accumulation/transport in wheat at different growth stages. This study revealed the inhibition mechanism of Bacillus thuringiensis on migration of Pb/Cd in a soil-wheat system from a viewpoint of a full life cycle, which offers a valuable reference for the in-situ remediation of HM-contaminated soil and the safe production of food crops in field.

New perspectives on microbiome and nutrient sequestration in soil aggregates during long-term grazing exclusion.

Grazing exclusion alters grassland soil aggregation, microbiome composition, and biogeochemical processes. However, the long-term effects of grazing exclusion on the microbial communities and nutrient dynamics within soil aggregates remain unclear. We conducted a 36-year exclusion experiment to investigate how grazing exclusion affects the soil microbial community and the associated soil functions within soil aggregates in a semiarid grassland. Long-term (36 years) grazing exclusion induced a shift in microbial communities, especially in the <2 mm aggregates, from high to low diversity compared to the grazing control. The reduced microbial diversity was accompanied by instability of fungal communities, extended distribution of fungal pathogens to >2 mm aggregates, and reduced carbon (C) sequestration potential thus revealing a negative impact of long-term GE. In contrast, 11-26 years of grazing exclusion greatly increased C sequestration and promoted nutrient cycling in soil aggregates and associated microbial functional genes. Moreover, the environmental characteristics of microhabitats (e.g., soil pH) altered the soil microbiome and strongly contributed to C sequestration. Our findings reveal new evidence from soil microbiology for optimizing grazing exclusion duration to maintain multiple belowground ecosystem functions, providing promising suggestions for climate-smart and resource-efficient grasslands.

Microbial metabolic limitation of rhizosphere under heavy metal stress: Evidence from soil ecoenzymatic stoichiometry.

Slow nutrient turnover and destructed soil function were the main factors causing low efficiency in phytoremediation of heavy metal (HM)-contaminated soil. Soil ecoenzymatic stoichiometry can reflect the ability of soil microorganisms to acquire energy and nutrients, and drive nutrient cycling and carbon (C) decomposition in HM-contaminated soil. Therefore, for the first time, we used the enzymatic stoichiometry modeling to examine the microbial nutrient limitation in rhizospheric and bulk soil of different plants (Medicago sativa, Halogeton arachnoideus and Agropyron cristatum) near the Baiyin Copper Mine. Results showed that the main pollutants in this area were Cu, Zn, Cd, and Pb, while Cd and Zn have the greatest contribution according to the analysis of pollution load index (PLI). The activities of soil C-, nitrogen (N)-, and phosphorus (P)-acquiring enzymes in the rhizosphere of plants were significantly greater than that in bulk soil. Moreover, microbial C and P limitations were observed in all plant treatments, while the lower limitation was generally in the rhizosphere compared to bulk soil. The HM stress significantly increased microbial C limitation and decreased microbial P limitation, especially in the rhizospheric soil. The partial least squares path modeling (PLS-PM) further indicated that HM concentration has the greatest effects on microbial P limitation (-0.64). In addition, the highest enzyme activities and the lowest P limitation were observed in the rhizospheric and bulk soil of M. sativa, thereby implying that soil microbial communities under the remediation of M. sativa were steadier and more efficient in terms of their metabolism. These findings are important for the elucidation of the nutrient cycling and microbial metabolism of rhizosphere under phytoremediation, and provide guidance for the restoration of HM-contaminated soil.

Reduction of Cu and nitrate leaching risk associated with EDDS-enhanced phytoextraction process by exogenous inoculation of plant growth promoting rhizobacteria.

Biodegradable chelant (S,S)-N,N'-ethylenediaminedisuccinic acid (EDDS) has the more advantages of enhanced metal mobility, rapid degradation, environmental friendliness, and ammonium release. However, the risk of metal and/or nitrate residues and leaching within EDDS biodegradation remains as the bottleneck for the widespread application of EDDS-induced phytoremediation. This study aims to explore if the inoculation of plant growth-promoting rhizobacteria (PGPRs) can eliminate the risk associated with the short-term application of EDDS by investigating Cu phytoextraction and soil nitrate content. Results showed that EDDS application significantly increased the copper (Cu) concentration in shoots, soil total Cu, NH4+-N and NO3--N content, but decreased plant biomass. The inoculation of PGPRs in the soil showed a strong ability to increase plant biomass, Cu phytoextraction and soil NH4+-N content, and decrease soil Cu and NO3--N content. Moreover, bacterial dominant taxa were found to be the largest contributors to soil NH4+-N and NO3--N variation, and the abundance of denitrifying bacteria (Bacteroidetes and Stenotrophomonas) decreased in the treatment with PGPRs. The risk of residual Cu and nitrate leaching was reduced by the inoculation of PGPRs without significantly changing the stability of the bacterial community. These new findings indicate that the exogenous application of beneficial rhizobacteria can provide an effective strategy to reduce the risk in metal-contaminated soils of chelant-assisted phytoextraction.

Associated soil aggregate nutrients and controlling factors on aggregate stability in semiarid grassland under different grazing prohibition timeframes.

Grazing prohibition is an effective measure in improving soil stability and ecological quality. However, only a limited number of studies have been published on the dominant factors that impact soil aggregate stability and their associated effects on nutrient distribution for different size soil aggregates under long-term grazing prohibition management. In this study, we investigated variation in soil aggregate stability and nutrient distribution characteristics in semiarid grassland sites under different grazing prohibition timeframes (0 years [GP0], 11 years [GP11], 26 years [GP26], and 36 years [GP36]). Results showed that organic carbon (C) and total nitrogen (TN) concentrations in soil aggregates decreased at GP11 before progressively increasing and reaching its highest value at GP36, and the total phosphorus (TP) concentration did not change significantly. Most nutrients accumulated in macroaggregates (> 0.25 mm) under grazing prohibition, and the nutrient stoichiometry in soil aggregates increased after 26 years. Compared to the control (GP0), the mean weight diameter (MWD) value of the soil stability index increased at GP11 (21.7%) and decreased at GP26 (18.9%). Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) also showed that the proportion of stable organic C-related functional groups (i.e., alkene-C and aromatic-C) in macroaggregates were higher at GP11 and GP36 than at GP26. Furthermore, principal component analysis (PCA), partial least squares path modeling (PLS-PM), and the relative importance of regressors all showed that glomalin-related soil proteins (GRSP) and nutrients indirectly improved aggregate stability in semiarid grassland through their influence on the GRSP accumulation potential and nutrient stoichiometry. Generally, after 26 years grazing prohibition had a positive effect on soil aggregate stability and nutrient accumulation in the semiarid grassland sites investigated for this study. Results from this study provide a theoretical basis to select appropriate grazing prohibition timeframes under grassland management initiatives to optimize ecological quality measures in semiarid regions.

Exogenous application of signaling molecules to enhance the resistance of legume-rhizobium symbiosis in Pb/Cd-contaminated soils.

Being signaling molecules, nitric oxide (NO) and hydrogen sulfide (H2S) can mediate a wide range of physiological processes caused by plant metal toxicity. Moreover, legume-rhizobium symbiosis has gained increasing attention in mitigating heavy metal stress. However, systematic regulatory mechanisms used for the exogenous application of signaling molecules to alter the resistance of legume-rhizobium symbiosis under metal stress are currently unknown. In this study, we examined the exogenous effects of sodium nitroprusside (SNP) as an NO donor additive and sodium hydrosulfide (NaHS) as a H2S donor additive on the phytotoxicity and soil quality of alfalfa (Medicago sativa)-rhizobium symbiosis in lead/cadmium (Pb/Cd)-contaminated soils. Results showed that rhizobia inoculation markedly promoted alfalfa growth by increasing chlorophyll content, fresh weight, and plant height and biomass. Compared to the inoculated rhizobia treatment alone, the addition of NO and H2S significantly reduced the bioaccumulation of Pb and Cd in alfalfa-rhizobium symbiosis, respectively, thus avoiding the phytotoxicity caused by the excessive presence of metals. The addition of signaling molecules also alleviated metal-induced phytotoxicity by increasing antioxidant enzyme activity and inhibiting the level of lipid peroxidation and reactive oxygen species (ROS) in legume-rhizobium symbiosis. Also, signaling molecules improved soil nutrient cycling, increased soil enzyme activities, and promoted rhizosphere bacterial community diversity. Both partial least squares path modeling (PLS-PM) and variation partitioning analysis (VPA) identified that using signaling molecules can improve plant growth by regulating major controlling variables (i.e., soil enzymes, soil nutrients, and microbial diversity/plant oxidative damage) in legume-rhizobium symbiosis. This study offers integrated insight that confirms that the exogenous application of signaling molecules can enhance the resistance of legume-rhizobium symbiosis under metal toxicity by regulating the biochemical response of the plant-soil system, thereby minimizing potential health risks.

A novel extracellular enzyme stoichiometry method to evaluate soil heavy metal contamination: Evidence derived from microbial metabolic limitation.

Heavy metal contaminates have become a significant threat to soil ecosystems due to their chronicity and universality in soil. Soil microbial metabolism plays a vital role in biogeochemical cycles and soil functions. However, the response of microbial metabolism to heavy metal contamination in soil remains elusive despite potentially offering important insight into the health and ecological consequences of soil ecosystems under such contamination. This study used extracellular enzyme stoichiometry models to identify the response of microbial metabolism to various heavy metal contaminants, while also revealing potential implications of heavy metal contaminates in soil ecosystems. Results showed that microbial metabolism was restricted by soil carbon (C) and phosphorus (P) within a heavy metal polluted area in Northwest China. Heavy metal stress significantly increased microbial C limitation while decreasing microbial P limitation. However, microbial C and P limitations both responded consistently to different heavy metals (i.e., Cd, Pb, Zn, and Cu). Heavy metals had the greatest effect on microbial C limitation (i.e., 0.720 of the total effects) compared to other soil properties, and soil with the lowest heavy metal concentration exhibited the lowest microbial C limitation, and vice versa. These results indicated that microbial metabolic limitation can robustly and sensitively reflect the degree of heavy metals pollution in soil. Additionally, increased microbial C limitation caused by heavy metal contaminants could potentially escalate C release by promoting soil C decomposition as well as increasing investments in enzyme production and the maintenance of metabolic processes. Consequently, potential C loss induced by heavy metal pollution on soil ecosystems may be extensive and significant. Generally, our results suggest the usefulness of extracellular enzyme stoichiometry as a new method from which to evaluate heavy metal soil pollution, while microbial metabolic limitation could potentially be a promising indicator.

Co-inoculation effect of plant-growth-promoting rhizobacteria and rhizobium on EDDS assisted phytoremediation of Cu contaminated soils.

Chelants application can increase the bioavailability of metals, subsequently limiting plant growth and reducing the efficiency of phytoremediation. Plant growth-promoting rhizobacteria (PGPRs) and rhizobium have substantial potential to improve plant growth and plant tolerance to metal stress. We evaluated the effects of co-inoculation with a PGPR strain (Paenibacillus mucilaginosus) and a Cu-resistant rhizobium strain (Sinorhizobium meliloti) on the efficiency of biodegradable chelant (S,S-ethylenediaminedisuccinic acid; EDDS) assisted phytoremediation of a Cu contaminated soil using alfalfa. The highest total Cu extraction by alfalfa was observed in the EDDS-treated soil upon co-inoculation with the PGPR and rhizobium strains, which was 1.2 times higher than that without co-inoculation. Partial least squares path modeling identified plant oxidative damage and soil microbial biomass as the key variables influencing Cu uptake by alfalfa roots. Co-inoculation significantly reduced the oxidative damage to alfalfa by mitigating the accumulation of malondialdehyde and reactive oxygen species, and improving the antioxidation capacity of the plant in the presence of EDDS. EDDS application decreased microbial diversity in the rhizosphere, whereas co-inoculation increased microbial biomass carbon and nitrogen, and microbial community diversity. Increased relative abundances of Actinobacteria and Bacillus and the presence of Firmicutes taxa as potential biomarkers demonstrated that co-inoculation increased soil nutrient content, and improved plant growth. Co-inoculation with PGPR and rhizobium can be useful for altering plant-soil biochemical responses during EDDS-enhanced phytoremediation to alleviate phytotoxicity of heavy metals and improve soil biochemical activities. This study provides an effective strategy for improving phytoremediation efficiency and soil quality during chelant assisted phytoremediation of metal-contaminated soils.

Application of signaling molecules in reducing metal accumulation in alfalfa and alleviating metal-induced phytotoxicity in Pb/Cd-contaminated soil.

The utilization of forages grown on metal-contaminated soil can increase the risk of heavy metals entering the food chain and affecting human health because of elevated toxic metal concentrations. Meanwhile, hydrogen sulfide (H2S) and nitric oxide (NO) as signaling molecules are known to promote plant growth in metal-contaminated soils. However, the regulatory mechanisms of such molecules in plant physiology and soil biochemistry have not been well-documented. Hence, we investigate the role of the exogenous application of H2S and NO on alfalfa growth in lead/cadmium (Pb/Cd)-contaminated soil. Our results indicate that the signaling molecules increase the alfalfa chlorophyll and biomass content and improve alfalfa growth. Further, H2S and NO reduce the translocation and bioconcentration factors of Pb and Cd, potentially reducing the risk of heavy metals entering the food chain. These signaling molecules reduce metal-induced oxidative damage to alfalfa by mitigating reactive oxygen species accumulation and increasing antioxidant enzyme activities. Their exogenous application increases soil enzymatic activities, particularly of catalase and polyphenol oxidase, without significantly changing the composition and structure of rhizosphere bacterial communities. Interestingly, H2S addition enriches the abundance of plant-growth-promoting rhizobacteria in soil, including Nocardioides, Rhizobium, and Glycomyces. H2S is more effective than NO in improving alfalfa growth and reducing heavy-metal contamination of the food chain. These results provide new insights into the exogenous application of signaling molecules in alleviating metal-induced phytotoxicity, including an efficient strategy for the safe use of forages.

Impact of Urea Addition and Rhizobium Inoculation on Plant Resistance in Metal Contaminated Soil.

Legume-rhizobium symbiosis has been heavily investigated for their potential to enhance plant metal resistance in contaminated soil. However, the extent to which plant resistance is associated with the nitrogen (N) supply in symbiont is still uncertain. This study investigates the effect of urea or/and rhizobium (Sinorhizobium meliloti) application on the growth of Medicago sativa and resistance in metals contaminated soil (mainly with Cu). The results show that Cu uptake in plant shoots increased by 41.7%, 69%, and 89.3% with urea treatment, rhizobium inoculation, and their combined treatment, respectively, compared to the control group level. In plant roots, the corresponding values were 1.9-, 1.7-, and 1.5-fold higher than the control group values, respectively. Statistical analysis identified that N content was the dominant variable contributing to Cu uptake in plants. Additionally, a negative correlation was observed between plant oxidative stress and N content, indicating that N plays a key role in plant resistance. Oxidative damage decreased after rhizobium inoculation as the activities of antioxidant enzymes (catalase and superoxide dismutase in roots and peroxidase in plant shoots) were stimulated, enhancing plant resistance and promoting plant growth. Our results suggest that individual rhizobium inoculation, without urea treatment, is the most recommended approach for effective phytoremediation of contaminated land.

Patterns of soil microbial nutrient limitations and their roles in the variation of soil organic carbon across a precipitation gradient in an arid and semi-arid region.

The effects of precipitation patterns on the metabolism of soil microbes are poorly understood, especially in water-limited ecosystems where soil microorganisms play crucial roles in the turnover of soil organic carbon (SOC) and nutrients. We investigated the influence of the gradient levels of mean annual precipitation (MAP from 300 to 900 mm) on soil microbial metabolism in an arid and semi-arid grassland region located in Loess Plateau, China and identified relationships between microbial metabolic limitations and the variation of soil organic matter (SOM). Microbial metabolism in this arid and semi-arid region was limited by soil C and phosphorus (P) or nitrogen (N). Microbial C and P limitations decreased with the increase of MAP. Microbial C and P limitations were lowest in the areas with MAPs of 700-900 mm, whereas N limitation was observed in the areas with MAPs >700 mm. The results of a variation-partitioning analysis and partial least squares path modeling indicated that the microbial C and N/P limitations on regional scales were mainly determined by climate factors (MAP and mean annual temperature (MAT)), followed by vegetation biomass and soil properties. The extents of soil drying-rewetting processing caused by different MAPs directly affected microbial nutrient limitation. Our results suggested that the influence of precipitation variation on microbial metabolic limitation strongly governed SOM stability and that an increase in the rate of SOM decomposition with increasing precipitation could be caused by increased microbial nutrient limitation. SOM may be most stable at a MAP of 700 mm in the arid and semi-arid regions (300-900 mm MAP) where microbial nutrient limitation was lowest. This study provided novel insights into the responses of soil microbial metabolism to precipitation change and is an important step toward understanding the mechanisms of SOM stability in an arid and semi-arid grassland ecosystem under scenarios of precipitation variation.

Natural grassland as the optimal pattern of vegetation restoration in arid and semi-arid regions: Evidence from nutrient limitation of soil microbes.

Soil microbial metabolism is vital for nutrient cycling and aboveground ecosystem stability. A general understanding of microbial metabolism and nutrient limitation under human disturbance in arid and semi-arid regions, which are the largest and most fragile oligotrophic ecosystems globally, however, is still limited. We quantified and compared the characteristics of nutrient limitation of soil microbes under natural/artificial grassland and shrubland, an ecological forest, an economic forest, and sloped cropland in typical arid and semi-arid ecosystems on the Loess Plateau, China. Vegetation restoration significantly affected the activities of extracellular enzymes and ecoenzymatic stoichiometry mainly by affecting soil nutrients and nutrient stoichiometry. A vector analysis of enzyme activity indicated that microbial communities were co-limited by carbon (C) and phosphorus (P) in all types of vegetation restoration. Linear regression indicated that microbial C and P limitations were significantly correlated with the stoichiometry of soil nutrient, suggesting that the balance of nutrient stoichiometry is an important factor maintaining microbial metabolism and elemental homeostasis. C and P limitations in the microbial communities were the lowest in the natural grassland. This implies that both vegetation and microbial communities under the restoration pattern of natural grassland were more stable under environmental stress, so the restoration of natural grassland should be recommended as the preferred option for ecosystem restoration in these arid and semi-arid regions.

Impact of co-inoculation with plant-growth-promoting rhizobacteria and rhizobium on the biochemical responses of alfalfa-soil system in copper contaminated soil.

The effects and regulatory mechanisms of co-inoculation of plant-growth-promoting rhizobacteria (PGPRs) and rhizobium in plant-soil systems remain unclear, despite numerous reports that PGPRs or rhizobium can alleviate metal toxicity. We used the co-inoculation of the PGPR Paenibacillus mucilaginosus and the metal-resistant rhizobium Sinorhizobium meliloti for exploring the physiological and biochemical responses of the plant-soil system in metal-contaminated soil. The co-inoculation with the PGPR and rhizobium significantly increased the nutrient (N, P, and K) contents in plant tissues and promoted plant growth in soil contaminated with copper (Cu). Stress from Cu-induced reactive oxygen species and lipid peroxidation were largely attenuated by the co-inoculation by increasing the activities of antioxidant enzymes. The contents and uptake of Cu in plant tissues increased significantly in the co-inoculation treatment compared with the uninoculated control and individual inoculation treatment. Co-inoculation with PGPR and rhizobium significantly increased soil microbial biomass, enzymatic activities, total nitrogen, available phosphorus, and soil organic matter contents compared with the uninoculated control. Interestingly, co-inoculation also affected the composition of the rhizospheric microbial community, and slightly increased rhizospheric microbial diversity. These improvements of the soil fertility and biological activity also had a beneficial impact on plant growth under Cu stress. Our results suggested that alfalfa co-inoculated with PGPR and rhizobium could increase plant growth and Cu uptake in metal-contaminated soil by alleviating plant Cu stress and improving soil biochemical properties. These results indicate that the co-application of PGPR and rhizobium can have a positive effect on the biochemical responses of alfalfa-soil systems in soil contaminated by heavy metals and can provide an efficient strategy for the phytomanagement of metal-contaminated land.