Copper (Cu), as an essential element, is added to animal feed to stimulate growth and prevent disease. The forage crop alfalfa (Medicago sativa L.) produced during Cu phytoextraction may be considered a biofortified crop to substitute the Cu feed additives for livestock production, beneficially alleviating Cu contamination in soils and reducing its input into agriculture systems. To assess this, alfalfa was grown in three similar soils with different Cu levels, i.e., 11, 439 and 779 mg kg⁻¹ for uncontaminated soil (A), moderately Cu-contaminated soil (B) and highly Cu-contaminated soil (C), respectively. EDDS (Ethylenediamine-N,N′-disuccinic acid) was applied to the soils seven days before the first cutting at four rates (0, 0.5, 2 and 5 mmol kg⁻¹) to enhance bioavailable Cu uptake. Alfalfa grew well in soils A and B but not in the highly Cu-contaminated soil. After applying EDDS, a significant biomass reduction of the first cutting shoot was only observed with 5 mmol kg⁻¹ EDDS in the highly Cu-contaminated soil, with a 45% (P < 0.05) decrease when compared to the control. Alfalfa grown in the three soils gradually wilted after the first cutting with 5 mmol kg⁻¹ EDDS, and Cu concentrations in the first cutting shoot were augmented strongly, by 250% (P < 0.05), 3500% (P < 0.05) and 6700% (P < 0.05) compared to the controls, respectively. Cu concentrations in alfalfa shoots were found to be higher in this study than in some fodder plants and further augmented in soils with higher Cu levels and with EDDS application. These findings suggest that alfalfa grown on clean soils or soils with up to 450 mg Cu kg⁻¹ (with appropriate EDDS dosages) has the potential to be considered as a partial Cu supplementation for livestock. This research laid the foundation for the integration between Cu-phytoextraction and Cu-biofortification for livestock.

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.

Chromium has been proven to be extremely phytotoxic. This study explored the impacts of increasing Cr(VI) exposure (up to 10 mg L⁻¹ K₂Cr₂O₇) on the growth and development of alfalfa plants and adaptation responses employed, in an environmentally relevant context. The threshold concentration of K₂Cr₂O₇ in irrigation water beyond which stress responses are initiated is 1 mg L⁻¹. Lower Cr(VI) exposure (0.5 mg L⁻¹ K₂Cr₂O₇) induced hormesis, evident through increased biomass and larger leaves, likely mediated by increased NO content (supported by elevated NR enzymatic activity and overexpression of NR and ndh genes). Elevated Cr(VI) exposure (5 and 10 mg L⁻¹ K₂Cr₂O₇) resulted in reduced biomass and smaller leaves, and lower levels of photosynthetic pigment (10 mg L⁻¹ K₂Cr₂O₇). Higher levels of lipid peroxidation, H₂O₂ and NO contents in these plants suggested nitro-oxidative stress. Stress responses included increased SOD and CAT enzymatic activities, further supported to some extent by MnSOD, FeSOD, Cu/ZnSOD and CAT transcripts levels. GST7 and GST17 gene expression patterns, as well as proline content, P5CS enzymatic activity and corresponding P5CS and P5CR gene expression levels emphasized the role of proline and GSTs in the adaptation responses. Results highlight the importance of managing Cr(VI) levels in irrigation water.

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 aim of this study was to develop a new experimental setup to determine parallel the emissions of greenhouse gases (GHG) and volatile organic compounds (VOCs) from silage during the opening as well as the subsequent aerobic storage phase of the complete bale without wrapping film. For this purpose, a special silage respiration chamber was used in which a silage bale could be examined. The gas analysis (CO₂, methanol, ethanol, ethyl acetate) of inlet, ambient and outlet air of the silage respiration chamber was carried out by photoacoustic spectroscopy. The gas samples taken inside the bale were analysed by gas chromatography for CO₂, O₂, CH₄, and N₂O. Three silage bales (grass and lucerne) as the smallest silage unit commonly used in practice were examined. The emission behaviour of the bales was recorded during experimental periods up to 55 days. The results allow a differentiation of the outgassing processes. On the one hand, gases produced during the anaerobic ensiling process (CO₂, CH₄, N₂O) are released once in a large amount during the first experimental hours after opening the silage. On the other hand, a continuous outgassing process takes place, which is particularly true for the VOCs ethanol, methanol, and ethyl acetate, whereby VOC emissions increase with rising ambient air temperatures. In this study, the emissions during the first 600 experimental hours from the grass silage bale and lucerne silage bale were 2313 g and 2612 g CO₂, 17.6 g and 145.2 g methanol, 132.3 g and 675.9 g ethanol, 55.1 g and 66.2 g ethyl acetate, respectively. Nevertheless, the focus of this study was on the technical recording of gas concentrations inside the silage bale itself and the emissions in the ambient air of the bale. For a better interpretation of the data, additional factors should be considered in further investigations.

The extensive application of nanomaterials has increased their levels in soil environments. Therefore, clarifying the process of environmental migration is important for environmental safety and human health. In this study, alfalfa was used to determine the effects of different doses of ZnO nanoparticles (NPs) on the growth of alfalfa and the soil environment. Results showed that the alfalfa biomass was inversely proportional to the exposure concentration of ZnO NPs. The Zn concentration in the alfalfa tissue and the exposure dose presented a significant positive correlation. A high concentration of ZnO NPs decreased the nitrogen-fixing area of root nodules while the number of bacteroids and root nodules, which in turn affected the nitrogen-fixing ability of alfalfa. At the same time, it caused different degrees of damage to the root nodules and root tip cells of alfalfa. A high dose of ZnO NPs decreased the relative abundance and diversity of the soil microorganisms. Therefore, short-term and high-dose exposure of ZnO NPs causes multiple toxicities in plants and soil environments.

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.

Plants, that naturally inhabit arsenic-contaminated areas may be used for effective arsenic-uptake from soil. The efficiency of this process may be increased by the reducing arsenic phytotoxicity and stimulating the activity of indigenous soil microbiota. As we showed, it can be achieved by the bioaugmenting of soil with arsenite-oxidizing bacteria (AOB). This study aimed to investigate the influence of soil bioaugmentation with AOB on the structure, quantity, and activity of the indigenous soil microbiota as well as to estimate the effect of such changes on the morphology, growth rate, and arsenic-uptake efficiency of plants. Plants-microbes interactions were investigated using the effective arsenites oxidizer Ensifer sp. M14 and the native plant alfalfa. The experiments were performed both in potted garden soil enriched with arsenic and in highly arsenic polluted, natural soil. The presence of M14 strain in soil contributed to the increase both in plants growth intensity and arsenic-uptake efficiency with regard to the soil without M14. After 40 days of plants culture, their average biomass increased by about 60% compared to non-bioaugmented soil, while the arsenic accumulation increased more than two times. The soil bioaugmentation contributed also to the increase in the quantity and activity of soil microorganisms without disturbing the natural microbial community structure. In the bioaugmented soil, the noticable increase in the quantity of heterotrophic, denitrifying, nitrifying and cellulolytic bacteria as well as in the activity of dehydrogenases and cellulases were observed. Soil bioaugmentation with M14 enables the application of native and commonly occurring plant species for enhancing the treatment of arsenic-contaminated soil. This in situ strategy may constitute a valuable alternative both to the chemical and physical methods of arsenic removal from soil and to the biological ways based on the arsenic hyperaccumulating plants and/or the arsenic mobilizing bacteria.

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.