Aluminum (Al) toxicity is a serious environmental constraint facing crop production in acidic soils, primarily due to the oxidative damage it causes to plant tissues. Alfalfa (Medicago sativa), a globally important forage crop, is highly susceptible to Al-induced stress, necessitating the development of Al-tolerant cultivars for sustainable forage production. In this study, we investigated the regulatory role of miR156 in Al stress response in alfalfa. Transcript analysis revealed significant downregulation of miR156 in alfalfa roots after 8 h of Al exposure, suggesting a negative role for miR156 in response to Al. To further investigate the role of miR156 in regulating agronomic traits and alfalfa's Al tolerance, we utilized the short tandem target mimic (STTM) method to silence miR156 in alfalfa (MsSTTM156), which led to an upregulation of SQUAMOSA PROMOTER BINDING-LIKE (SPL) target genes, albeit with variable miR156 dose-dependent effects across different transgenic genotypes. Morphological characterization of MsSTTM156 plants revealed significant negative changes in root architecture, root and shoot biomass, as well as flowering time. Under Al stress, overexpression of miR156 in alfalfa (MsmiR156OE) resulted in stunted growth and reduced biomass, whereas moderate MsmiR156 silencing enhanced root dry weight and increased stem basal diameter. In contrast, MsmiR156OE reduced plant height, stem basal diameter, shoot branching, and overall biomass under Al stress conditions. At the molecular level, silencing miR156 modulated the transcription of cell wall-related genes linked to Al tolerance, such as polygalacturonase 1(MsPG1) and polygalacturonase 4 (MsPG4). Furthermore, miR156 influenced the expression of indole-3-acetic acid (IAA) transport-related genes auxin transporter-like protein (MsAUX1) and auxin efflux carrier components 2 (MsPIN2), with MsSTTM156 and MsmiR156OE plants showing lower and higher transcript levels, respectively, upon Al exposure. These findings reveal the multi-layered role of miR156 in mediating Al tolerance, providing valuable insights into the genetic strategies that regulate response to Al stress in alfalfa.

Landfilling is common in developing countries since it is the easiest and cheapest way of waste disposal, however, it leads to serious environmental problems such as soil, water, and air pollution. A landfill has a life span of fifteen years after which it is closed leaving the site unusable, as a result, effective methods are needed for restoring and reclaiming the closed landfill site for future use. Phytoremediation has emerged as a viable and environmentally friendly method, which uses green plants to remove pollutants from soil, air, and water. In this study, Medicago sativa (alfalfa) and Trifolium repens (white clover) were planted in a pot trial as monocropped and intercropped in polluted soil collected from a landfill site to investigate stress tolerance and the extent of bioaccumulation of Cr, Mn, Ni, and Zn. All the plants remained healthy throughout the trial, with no signs of phytotoxicity except for monocropped white clover plants that showed stunted growth and eventually died. Intercropping resulted in the reduction of metals and their toxic effects in the soil which in turn limited the uptake of metals by both plants as a defence strategy against metal stress which resulted in lower amounts of metals in the intercropped plants compared to monocropped plants. The roots absorbed a significant amount of Zinc (Zn), Nickel (Ni), and Manganese (Mn) in the roots than the leaves. The concentration of Chromium (Cr) was significantly higher than the other metals in all the plants and there was no significant difference in the concentration of Cr in the roots and leaves. The Scanning Electron Microscopy (SEM) chromatographs, revealed greater damage in the tissues of monocropped plants than the intercropped plants, demonstrating that inter- cropping enhances plant growth and development by reducing the toxic effects of biotic stress such as metals in the soil than monocropping. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) revealed flavonoids as the main secondary metabolites that promoted resilience to biotic and abiotic stressors in Trifolium repens while saponins were found to play a similar major role in Medicago sativa.

Drought may impact plant-soil biotic interactions in ways that modify aboveground herbivore performance, but the outcomes of such biotic interactions under future climate are not yet clear. We performed a growth chamber experiment to assess how long-term, drought-driven changes in belowground communities influence plant growth and herbivore performance using a plant-soil feedback experimental framework. We focussed on two common pasture legumes-lucerne, Medicago sativa L., and white clover, Trifolium repens L. (both Fabaceae)-and foliar herbivores-cotton bollworm, Helicoverpa armigera (H & uuml;bner) (Lepidoptera: Noctuidae), and two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Soil was collected from a field facility where rainfall had been manipulated for 6 years, focussing on treatments representing ambient rainfall and prolonged drought (50% reduction relative to ambient), to consider the effects of biological legacies mediated by the prolonged drought. All soils were sterilized and re-inoculated to establish the respective home (i.e. where a given plant is cultivated in its own soil) and away (i.e. where a given plant is cultivated in another species' soil) treatments in addition to a sterile control. We found that the relative growth rate (RGR) and relative consumption of larvae were significantly lower on lucerne grown in soil with ambient rainfall legacies conditioned by white clover. Conversely, the RGR of insect larvae was lower on white clover grown in soil with prolonged drought legacies conditioned by lucerne. Two-spotted spider mite populations and area damage (mm2) were significantly reduced on white clover grown in lucerne-conditioned soil in drought legacies. The higher number of nodules found on white clover in lucerne-conditioned soil suggests that root-rhizobia associations may have reduced foliar herbivore performance. Our study provides evidence that foliar herbivores are affected by plant-soil biotic interactions and that prolonged drought may influence aboveground-belowground linkages with potential broader ecosystem impacts.

We report for the first time that larvae of the genus Bothynus Hope (Coleoptera: Melolonthidae) caused economic damage to lucerne (Medicago sativa L., Fabaceae) crops in General Acha, province of La Pampa, Argentina. In two consecutive years (2021 and 2022), this insect infested 150 ha of lucerne, causing seedling losses of 80 ha. Based on soil sampling and laboratory insect rearing, the species was identified as Bothynus striatellus (Faimaire) (Coleoptera: Melolonthidae). Information is given here on the damage recorded in this forage crop, the density of the larvae observed at the time of damage assessment and the morphological characteristics of the third instar larvae reported in this crop in Argentina.

Plant-beneficial bacteria (PBB) have emerged as a promising approach for assisting phytoremediation of heavy metal (HM)-contaminated soils. However, their colonization efficiency is often challenged by complex soil environments. In this study, we screened one rhizobacterium (Klebsiella variicola Y38) and one endophytic bacterium (Serratia surfactantfaciens Y15) isolated from HM-contaminated soils and plants for their high resistance to Cd and strong growth-promoting abilities. These strains were encapsulated individually or in combination with alginate and applied with Medicago sativa in Cd-contaminated soil pot experiments. The effectiveness of different bacterial formulations in promoting plant growth and enhancing Cd bioconcentration in M. sativa was evaluated. Results showed that PBB application enhanced plant growth and antioxidant capacity while reducing oxidative damage. Encapsulated formulations outperformed unencapsulated ones, with combined formulations yielding superior results to individual applications. Quantitative PCR indicated enhanced PBB colonization in Cdcontaminated soils with alginate encapsulation, potentially explaining the higher efficacy of alginateencapsulated PBB. Additionally, the bacterial agents modified Cd speciation in soils, resulting in increased Cd bioaccumulation in M. sativa by 217-337 %. The alginate-encapsulated mixed bacterial agent demonstrated optimal effectiveness, increasing the Cd transfer coefficient by 3.2-fold. Structural equation modeling and correlation analysis elucidated that K. variicola Y38 promoted Cd bioaccumulation in M. sativa roots by reducing oxidative damage and enhancing root growth, while S. surfactantfaciens Y15 facilitated Cd translocation to shoots, promoting shoot growth. The combined application of these bacteria leveraged the benefits of both strains. These findings contribute to diversifying strategies for effectively and sustainably remediating Cdcontaminated soils, while laying a foundation for future investigations into bacteria-assisted phytoremediation.

The aim of this study is to evaluate the seasonal changes in leachate compositions, and their impact on germination tests and alpha-amylase activity. Throughout the four seasons of the year 2022, leachate samples were collected in autumn, winter, spring, and summer directly from the collection conduit of the untreated leachate pond at Mediouna landfill (Casablanca, Morocco). The parameters analyzed in the leachate samples included pH, electrical conductivity (EC), chemical oxygen demand (COD), 5-day biochemical oxygen demand (BOD5), nitrate (NO3-), ammonium (NH4+), and orthophosphate (PO43-). The present study involved the execution of germination tests on municipal solid waste leachate. Lens culinaris and Medicago sativa seeds were exposed to leachate at different dilutions of 1%, 3%, 5%, 7%, and 10%, with tap water as control, for about 72 h in the dark at room temperature. Severe toxicity was observed for the 7% and 10% concentrations, observing that L. culinaris and M. sativa showed a mean value of germination index inferior to 50%. These results can be explained by the presence of inhibitor elements in the leachate such as heavy metals (Pb and Hg) and sodium. Lead (Pb) and mercury (Hg) have mean concentrations of 0.2276 ppm and 0.0159 ppm, respectively, while sodium (Na) exhibits an average concentration of 359.942 ppm. In addition, for the biochemical parameters, we noted a decrease in alpha-amylase activity proportionally to the germination index. In conclusion, this work highlights the significant metabolic disturbances induced by leachate, harming the germination of L. culinaris and M. sativa seeds. The present results highlight the potential deleterious effects of leachate pollution on agricultural activities and the ecosystem. However, it may also be possible to take advantage of the leachate's richness in organic matter and nutrient salts to fertilize agricultural land. Our next investigations will aim to verify whether the use of leachate as fertilizer will cause damage to crops and soil.

Biostimulants such as ascorbic acid, known as vitamin C, have been reported to have numerous positive roles in plant tolerance to abiotic stresses. However, little is known about the biostimulant effects of ascorbic acid on alfalfa (Medicago sativa). Accordingly, a pot experiment was conducted to investigate the effects of 1 mM ascorbic acid, applied as foliar spray, on the salt tolerance of a Moroccan alfalfa population Demnate 201. One month-old M. sativa seedlings were exposed to 200 mM NaCl for four weeks with or without 1 mM of exogenous ascorbic acid treatment. The results showed that salinity stress significantly (p < 0.001) reduced plant biomass, disturbed photosynthesis-related parameters and induced oxidative stress. However, ascorbic acid foliar spray counteracted the observed negative effects of salinity. It significantly (p < 0.001) improved plant growth and photosynthetic parameters. Besides, stress indicators, including Na+ in shoot and root, hydrogen peroxide and electrolyte leakage, were significantly reduced by 42%, 29%, 12% and 34%, respectively, in treated and salt-stressed alfalfa plants. Interestingly, the decrease in oxidative stress markers was positively correlated to the ability of ascorbic acid to induce the accumulation of flavonoids and to increase the antioxidant activity of guaiacol peroxidase. Furthermore, compatible solutes, such as proline and soluble sugars, were found higher especially in salt-stressed alfalfa plants treated with 1 mM ascorbic acid. Our findings showed that ascorbic acid supply could be an eco-friendly and sustainable technique to mitigate the toxic effect of salt and could improve alfalfa forage production when grown in salt-affected soils.