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.

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 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.

Anthelmintics, drugs against parasitic worms, are frequently used in livestock and might act as danger environmental microcontaminants. The present study was designed to monitor the possible circulation of common anthelmintic drug albendazole (ABZ) and its metabolites in the real agriculture conditions. The sheep were treated with the recommended dose of ABZ. Collected faeces were used for the fertilization of a field with fodder plants (alfalfa and clover) which served as feed for sheep from a different farm. The selective ultrasensitive mass spectrometry revealed surprisingly high concentrations of active ABZ metabolite (ABZ-sulphoxide) in all samples (dung, plants, ovine plasma, rumen content and faeces). Our results prove for the first time an undesirable permeation of ABZ metabolites from sheep excrement into plants (used as fodder) and subsequently to other sheep in real agricultural conditions. This circulation causes the permanent exposition of the ecosystems and food-chain to the drug and can promote the development of drug resistance in helminths.

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.

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.

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.

In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible.