Rhizobia and arbuscular mycorrhiza fungi (AMF) are important symbiotic microbes that are advantageous to plants growing in metal-contaminated soil. However, it remains unclear how inoculated microbes affect rhizosphere microbial communities or whether subsequent changes in rhizosphere microbiomes contribute to improving plant resistance under metal stress. This study investigated the effects of rhizobia and AMF inoculation on alfalfa resistance to Cd stress. The response of rhizosphere microbial communities to inoculation and its role in increasing alfalfa’ ability to cope with stress were further analyzed using high-throughput sequencing of 16S and ITS rRNA genes. Results showed that single rhizobia or AMF inoculation significantly improved alfalfa resistance to Cd stress, while their co-inoculation resulted in the greatest overall improvement. Improved resistance was reflected by the significant mitigation of Cd-induced lipid peroxidation and reactive oxygen species (ROS) stress caused by increases in antioxidant enzyme activities along with co-inoculation. Furthermore, co-inoculation significantly altered the rhizosphere microbial community structure by decreasing fungal community diversity and increasing bacterial community diversity. Results of partial least squares path modeling (PLS-PM) and variation partitioning analysis (VPA) showed that the rhizosphere bacterial community predominated over the fungal community with respected to improvements in resistance to Cd stress under the co-inoculation treatments. This improvement was specifically seen in the enrichment of certain key bacterial taxa (including Proteobacteria, Actinobacteria, Acidobacteria, and Chloroflexi) induced by the rhizobia and AMF co-inoculation, enhancing alfalfa’ ability to uptake rhizosphere nutrients and reduce its release of photosynthetically-derived carbon (C) into soil. Our findings revealed that the co-inoculation of multiple symbiotic microbes can assist plants to effectively cope with Cd stress, providing a greater understanding of rhizosphere bacterial taxa in the microbe-induced phytomanagement.

Wastewater reclamation and reuse for agriculture have attracted a great deal of interest, due to water stress caused by rapid increase in human population and agricultural water demand as well as climate change. However, the application of treated wastewater for irrigation can lead to the accumulation of pharmaceuticals and personal care products (PPCPs) in the agricultural crops, grazing animals, and consequently to human dietary exposure. In this study, a model was developed to simulate the fate of five PPCPs; triclosan (TCS), carbamazepine (CBZ), naproxen (NPX), gemfibrozil (GFB), and fluoxetine (FXT) during wastewater reuse for agriculture, and potential human dietary exposure and health risk. In a reclaimed wastewater-irrigated grazing farm growing alfalfa, it took 100–535 days for PPCPs to achieve the steady-state concentrations of 1.43 × 10⁻⁶, 4.73 × 10⁻⁵, 1.17 × 10⁻⁶, 1.53 × 10⁻⁵, and 7.38 × 10⁻⁶ mg/kg for TCS, CBZ, NPX, GFB, and FXT in soils, respectively. The accumulated concentration of PPCPs in the plant (alfalfa) and grazing animals (beef) ranged 2.86 × 10⁻⁷− 4.02 × 10⁻³ and 4.39 × 10⁻¹⁵− 6.27 × 10⁻⁷ mg/kg, respectively. Human dietary exposure to these compounds through beef consumption was calculated to be 1.67 × 10⁻¹⁸− 1.74 × 10⁻¹⁰ mg/kg bodyweight/d, much lower than the acceptable daily intake (ADI). Similar results were obtained for a ‘typical’ reclaimed wastewater irrigated farm based on the typical setup using our model. Screening analysis showed that PPCPs with relatively high LogD value and lower ratios of degradation rate (in soils) to plant uptake have a greater potential to be transferred to humans and cause potential health risks. We established a modeling method for evaluating the fate and human health effects of PPCPs in reclaimed wastewater reuse for the agricultural system and developed an index for screening PPCPs with high potential to accumulate in agricultural products. The model and findings are valuable for managing water reuse for irrigation and mitigating the harmful effects of PPCPs.

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.

The long term vertical and horizontal mobility of mercury (Hg) in soils of agricultural areas of a historically contaminated Italian National Relevance Site (SIN Brescia-Caffaro) was investigated. The contamination resulted from the continuous discharge of Hg in irrigation waters by an industrial plant (Caffaro S.p.A), equipped with a mercury-cell chlor-alkali process. The contamination levels with depth ranged from about 20 mg/kg dry weight (d.w.) of soil in the top (plow) layer to less than 0.1 mg/kg d.w. at 1 m depth. The concentrations varied also spatially, up to one order of magnitude within the same field and showing a decreasing trend from the Hg source (i.e., irrigation ditches). The concentration profiles and gradients measured were explained considering Hg loading, soil properties, such as the texture, organic carbon content, pH and cation exchange capacity. A Selective Sequential Extraction (SSE) was also applied on soil samples from an ad hoc greenhouse experiment to investigate the role of different plant species in influencing Hg speciation in soils. Although most of the extracted Hg was included in scarcely mobile or immobile forms, some plant species (i.e., alfalfa) showed to importantly increase the soluble and exchangeable fractions with respect to the unplanted control soils, thus affecting mobility and potential bioavailability of Hg.

Hydrogen sulfide (H₂S) is well known as a gaseous signal in response to heavy metal stress, while methane (CH₄), the most prevalent greenhouse gas, confers cadmium (Cd) tolerance. In this report, the causal link between CH₄ and H₂S controlling Cd tolerance in alfalfa (Medicago sativa) plants was assessed. Our results observed that the administration of CH₄ not only intensifies H₂S metabolism, but also attenuates Cd-triggered growth inhibition in alfalfa seedlings, which were parallel to the alleviated roles in the redox imbalance and cell death in root tissues. Above results were not observed in roots after the removal of endogenous H₂S, either in the presence of either hypotaurine (HT; a H₂S scavenger) or DL-propargylglycine (PAG; a H₂S biosynthesis inhibitor). Using in situ noninvasive microtest technology (NMT) and inductively coupled plasma mass spectroscopy (ICP-MS), subsequent results confirmed the participation of H₂S in CH₄-inhibited Cd influx and accumulation in roots, which could be explained by reestablishing glutathione (GSH) pool (reduced/oxidized GSH and homoglutathione) homeostasis and promoting antioxidant defence. Overall, our results clearly revealed that H₂S operates downstream of CH₄ enhancing tolerance against Cd stress, which are significant for both fundamental and applied plant biology.

Molybdenum (Mo) contamination of agricultural soils around Mo-mining areas is of emerging environmental concern. This study evaluated potential practical techniques for chemical immobilization of three Mo contaminated agricultural soils via application of up to six amendments from four different types of materials including biosolids, biochar supported nanoscale zero-valent iron (BC-nZVI), drinking water treatment residues (WTR) and ferrous minerals (magnetite and ferrihydrite). The efficacy of the different amendments on soil Mo bioaccessibility and bioavailability was evaluated by monitoring Mo uptake in both monocotyledon (ryegrass) and dicotyledon (alfalfa) plants, soil extractable Mo, and Mo bioavailability as measured by Diffusive Gradient in Thin Films (DGT®). All amendments exhibited no immobilization effect and increased Mo extractability in the severely contaminated soil (264 mg Mo kg⁻¹). In contrast, in lightly and moderately contaminated soils (22 and 98 mg Mo kg⁻¹), biosolids, WTR and magnetite all reduced soil extractable Mo and decreased Mo uptake in both alfalfa and ryegrass shoots relative to controls (CK). Moreover, DGT showed that during incubation experiments while biosolids amendments increased Mo bioavailability from 115 to 378% compared to the CK treatments, all other amendments decreased Mo bioavailability insignificantly.

Bulk Cu compounds such as Cu(OH)₂ are extensively used as pesticides in agriculture. Recent investigations suggest that Cu-based nanomaterials can replace bulk materials reducing the environmental impacts of Cu. In this study, stress responses of alfalfa (Medicago sativa L.) seedlings to Cu(OH)₂ nanoparticle or compounds were evaluated. Seeds were immersed in suspension/solutions of a Cu(OH)₂ nanoform, bulk Cu(OH)₂, CuSO₄, and Cu(NO₃)₂ at 25 and 75 mg/L. Six days later, the germination, seedling growth, and the physiological and biochemical responses of sprouts were evaluated. All Cu treatments significantly reduced root elongation (average = 63%). The ionic compounds at 25 and 75 mg/L caused a reduction in all elements analyzed (Ca, K, Mg, P, Zn, and Mn), excepting for S, Fe and Mo. The bulk-Cu(OH)₂ treatment reduced K (48%) and P (52%) at 75 mg/L, but increased Zn at 25 (18%) and 75 (21%) mg/L. The nano-Cu(OH)₂ reduced K (46%) and P (48%) at 75 mg/L, and also P (37%) at 25 mg/L, compared with control. Confocal microscopy images showed that all Cu compounds, at 75 mg/L, significantly reduced nitric oxide, concurring with the reduction in root growth. Nano Cu(OH)₂ at 25 mg/L upregulated the expression of the Cu/Zn superoxide dismutase gene (1.92-fold), while ionic treatments at 75 mg/L upregulated (∼10-fold) metallothionein (MT) transcripts. Results demonstrated that nano and bulk Cu(OH)₂ compounds caused less physiological impairments in comparison to the ionic ones in alfalfa seedlings.

Efficiency and the preservation of soil functions are key requirements for sustainable remediation of contaminated soil. Microbial decomposition and conversion of substrates is a fundamental soil function. Pilot-scale EDTA-based soil washing recycled chelant generated no wastewater and removed 78% of Pb from acidic farmland soil with 860 mg kg⁻¹ Pb and 60% of Pb from calcareous garden soil with 1030 mg kg⁻¹ Pb. Remediation had an insignificant effect on microbial respiration in acidic soil induced by sequential additions of glucose, micro-cellulose, starch and alfa-alfa sprout powder (mimicking litter components, C-cycle). In contrast, remediation of calcareous soil reduced cumulative CO₂ production after glucose (simple) and alfalfa (complex substrate) addition, by up to 40%. Remediation reduced the nitrification rate (denoting the N-cycle) in acidic soil by 30% and halved nitrification in calcareous soil. Remediation in both soils slightly or positively affected dehydrogenase and β-glucosidase activity (associated with C-cycle), and decreased urease activity (N-cycle). Generally, EDTA remediation modestly interfered with substrate utilisation in acidic soil. A more prominent effect of remediation on the functioning of calcareous soil could largely be attributed to the use of a higher EDTA dose (30 vs. 100 mmol kg⁻¹, respectively).

The genus Enterobacter comprises a range of beneficial plant-associated bacteria showing plant growth promotion. Enterobacter ludwigii belongs to the Enterobacter cloacae complex and has been reported to include human pathogens but also plant-associated strains with plant beneficial capacities. To assess the role of Enterobacter endophytes in hydrocarbon degradation, plant colonization, abundance and expression of CYP153 genes in different plant compartments, three plant species (Italian ryegrass, birdsfoot trefoil and alfalfa) were grown in sterile soil spiked with 1% diesel and inoculated with three endophytic E. ludwigii strains. Results showed that all strains were capable of hydrocarbon degradation and efficiently colonized the rhizosphere and plant interior. Two strains, ISI10-3 and BRI10-9, showed highest degradation rates of diesel fuel up to 68% and performed best in combination with Italian ryegrass and alfalfa. All strains expressed the CYP153 gene in all plant compartments, indicating an active role in degradation of diesel in association with plants.

We investigated the effects of the agricultural Cross Compliance measures for European cultivated lands, focusing on nitrogen (N) fluxes from corn fields. Four scenarios have been designed according to some conservation farming practices, namely no-till, max manure, catch crop and N splitting. Results indicated that (1) in the no-till scenario the N₂O fluxes are decreased during the first simulated years, with a return to default fluxes in following years; no-till particularly decreased N₂O emission in the dryer and colder simulation spatial units (HSMUs); (2) the no-till and the N splitting scenarios slightly increased the N surplus because of a decrease in plant uptake; (3) introducing a rotation with alfalfa decreased the N leaching in the corn crops following the catch crops; and (4) the application of fertilizer and manure during the cold and wet seasons led to an increase of N leaching.