Silver (Ag) engineered nanomaterials (ENMs) are being released into waste streams and are being discharged, largely as Ag2S aged-ENMs (a-ENMs), into agroecosystems receiving biosolids amendments. Recent research has demonstrated that biosolids containing an environmentally relevant mixture of ZnO, TiO2, and Ag ENMs and their transformation products, including Ag2S a-ENMs, disrupted the symbiosis between nitrogen-fixing bacteria and legumes. However, this study was unable to unequivocally determine which ENM or combination of ENMs and a-ENMs was responsible for the observed inhibition. Here, we examined further the effects of polyvinylpyrollidone (PVP) coated pristine Ag ENMs (PVP-Ag), Ag2S a-ENMs, and soluble Ag (as AgSO4) at 1, 10, and 100 mg Ag kg−1 on the symbiosis between the legume Medicago truncatula and the nitrogen-fixing bacterium, Sinorhizobium melliloti in biosolids-amended soil. Nodulation frequency, nodule function, glutathione reductase production, and biomass were not significantly affected by any of the Ag treatments, even at 100 mg kg−1, a concentration analogous to a worst-case scenario resulting from long-term, repeated biosolids amendments. Our results provide additional evidence that the disruption of the symbiosis between nitrogen-fixing bacteria and legumes in response to a mixture of ENMs in biosolids-amended soil reported previously may not be attributable to Ag ENMs or their transformation end-products. We anticipate these findings will provide clarity to regulators and industry regarding potential unintended consequences to terrestrial ecosystems resulting from of the use of Ag ENMs in consumer products.

Globally, the legume–rhizobia symbiosis, contained within specialised organs called root nodules, is thought to add at least 30 Tg N annually to agricultural land. The growth and functioning of a modern white clover (Trifolium repens cv. Crusader) and red clover (T. pratense cv. Merviot) cultivar were investigated in current and future ozone scenarios in solardomes. Both cultivars developed leaf injury and had significant reductions in root biomass and root nodule number in response to ozone, with Crusader also displaying a reduced size and mass of nodules. In-situ measurements of N-fixation in Crusader by acetylene reduction assay revealed reduced N-fixation rates in a future scenario with an increased background and moderate peaks of ozone. The implications for the sustainability of temperate pasture are discussed.

Driven by human activities, air pollution and soil degradation are threatening food production systems. Rising ozone in the troposphere can affect several physiological processes in plants and their interaction with symbiotic microorganisms. Plant responses to ozone may depend on both soil fertility and the ontogenetic stage in which they are exposed. In this work, we studied the effects of ozone episodes and soil fertility on soybean plants. We analysed soybean plant responses in the production of aboveground and belowground biomass, structural and functional attributes of rhizobia, and seed production and quality. The experiment was performed with plants grown in two substrates with different fertility (commercial soil, and soil diluted (50%, v/v) with sand). Plants were exposed to acute episodes of ozone during vegetative and reproductive stages. We observed that ozone significantly reduced belowground biomass (≈25%), nodule biomass (≈30%), and biological nitrogen fixation (≈21%). Plants exposed to ozone during reproductive stage growing in soil with reduced fertility had lower seed production (≈10% lower) and seed protein (≈12% lower). These responses on yield and quality can be explained by the observed changes in belowground biomass and nitrogen fixation. The negative impact of ozone on the symbiotic interaction with rhizobia, seed production and quality in soybean plants were greater in soils with reduced fertility. Our results indicate that food security could be at risk in the future if trends in ozone concentration and soil degradation processes continue to increase.

The growth, development and functioning of legumes are often significantly affected by exposure to tropospheric ozone (O3) pollution. However, surprisingly little is known about how leguminous Nitrogen (N) fixation responds to ozone, with a scarcity of studies addressing this question in detail. In the last decade, ozone impacts on N-fixation in soybean, cowpea, mung bean, peanut and clover have been shown for concentrations which are now commonly recorded in ambient air or are likely to occur in the near future. We provide a synthesis of the existing literature addressing this issue, and also explore the effects that may occur on an agroecosystem scale by predicting reductions in Trifolium (clovers) root nodule biomass in United Kingdom (UK) pasture based on ozone concentration data for a “high” (2006) and “average” ozone year (2008). Median 8% and 5% reductions in clover root nodule biomass in pasture across the UK were predicted for 2006 and 2008 respectively. Seasonal exposure to elevated ozone, or short-term acute concentrations >100 ppb, are sufficient to reduce N-fixation and/or impact nodulation, in a range of globally-important legumes. However, an increasing global burden of CO2, the use of artificial fertiliser, and reactive N-pollution may partially mitigate impacts of ozone on N-fixation.

Microorganisms have developed copper-resistance mechanisms in order to survive in contaminated environments. The abundance of the copper-resistance genes cusA and copA, encoding respectively for a Resistance Cell Nodulation protein and for a P-type ATP-ase pump, was assessed in copper and non-copper-impacted Chilean marine sediment cores by the use of molecular tools. We demonstrated that number of copA and cusA genes per bacterial cell was higher in the contaminated sediment, and that copA gene was more abundant than cusA gene in the impacted sediment. The molecular phylogeny of the two copper-resistance genes was studied and reveals an impact of copper on the genetic composition of copA and cusA genes.

Saplings of alder (Alnus glutinosa), birch (Betula pendula), hazel (Corylus avellana), beech (Fagus sylvatica), ash (Fraxinus excelsior) and oak (Quercus robur) were exposed to five episodic ozone regimes in solardomes, with treatment means between 16 and 72 ppb. All trees were kept fully watered for the first 5 weeks of exposure, after which half the trees continued to be well-watered, whereas the other half were subjected to a moderate drought by applying approximately 45 % of the amount of water. Species-specific reductions in growth in response to both ozone and drought were found, which could result in reduced potential carbon sequestration in future ozone climates. In well watered conditions, the ozone treatments resulted in total biomass reductions for oak (18 %), alder (16 %), beech (15 %), ash (14 %), birch (14 %) and hazel (7 %) in the 72 ppb compared with the 32 ppb treatment. For beech, there was a reduction in growth in response to ozone in the well-watered treatment, but an increase in growth in response to ozone in the drought treatment, in contrast to the decreased growth that would occur as a result of stomatal closure in response to either the ozone or drought treatment, and therefore assumed to result from changes in hormonal signalling which could result in stomatal opening in combined ozone and drought conditions. For alder, in addition to a decrease in root biomass, there was reduced biomass of root nodules with high compared with low ozone for both drought-treated and well-watered trees. There was also a large reduction in the biomass of nodules from drought trees compared with well-watered. It is therefore possible that changes in the nitrogen dynamics of alder could occur due to reduced nodulation in both drought and elevated ozone conditions.

We highlighted some of the key problems associated with the use of beneficial microorganisms for improving adaptation of plants to soils, polluted with heavy metals (HMs), especially Cd. Inoculation of pea line SGE and its Cd-tolerant mutant SGECdᵗ with nodule bacteria Rhizobium leguminosarum bv. viciae demonstrated that nodulation process may be disturbed at Cd concentrations below threshold toxicity levels for each partner and the plant genotype plays a major role in nodulation under Cd stress. A comparative mathematical analysis of available information about Cd tolerance, accumulation of HMs (Cd, Cr, Cu, Ni, Pb, Sr and Zn), response to mycorrhizal fungus Glomus sp. and 15 phenotypic traits of 99 pea varieties revealed that (1) the Cd-sensitive varieties were more efficient in exploring the protective potential of symbiosis to compensate their deficit in Cd tolerance and (2) correlations between the studied traits exist and can be helpful for selection of plant-microbe systems adapted to polluted soils. In pot experiment with 11 varieties of Indian mustard, the plant growth-promoting effect of rhizobacterium Variovorax paradoxus 5C-2 negatively correlated with Cd tolerance and shoot Cd concentration of the plants grown in Cd-supplemented soil. In an outdoor pot experiment, inoculation of willow with the ectomycorrhizal fungus Pisolithus tinctorius and a cocktail of rhizobacteria stimulated root exudation, decreased soil pH and increased Cd mobilization in soil and Cd uptake by plants, but decreased plant growth at a moderate contamination level (25 mg Cd kg⁻¹). Opposite effects were observed in highly contaminated soil (77 mg Cd kg⁻¹). We propose a preliminary systematic framework of interactions between these factors that determine the success of microbial inoculation aimed at improving crop performance on HM-polluted soils or enhancing phytoremediation.

The increasing number of degraded soil areas caused by open cast mining activities has brought about a critical damage to the environment. The mine spoil must be ameliorated with anthropogenic interferences which consist of revegetating soils after organic matter amendment and provision of microbial diversity, to guarantee basic conditions for a sustainable soil biological activity. Five woody species, Acacia mangium Willd., Inga edulis Mart., Mimosa caesalpiniaefolia Benth, Parkia multijuga Benth., and Schinus terebinthifolia Schlecht. & Cham were cultivated under greenhouse conditions to evaluate the potential of plant establishment on cassiterite mining waste, considering the contribution of mycorrhizal fungi inoculation, organic compost, and thermophosphate amendment. The shoot height, dry weight, and nitrogen and phosphorus shoot contents were determined. Three species of nodulating legumes, A. mangium, M. caesalpiniaefolia, and S. terebinthifolia showed a great positive response to organic compost, termophosphate, and mycorrhizal inoculation, increasing the plant height and the shoot dry weight. Plants inoculated with arbuscular mycorrhizal fungi and fertilized with organic compost also increased their nitrogen and phosphorus shoot contents. The addition of organic compost and mycorrhizal fungi were essential for plant development and the reforestation of mining areas should be initiated with mycotrophic and nodulating legumes.

Excess copper (Cu) in soils has deleterious effects on plant growth and can pose a risk to human health. In the last decade, legume-rhizobium symbioses became attractive biotechnological tools for metal phytostabilization. For this technique being useful, metal-tolerant symbionts are required, which can be generated through genetic manipulation.In this work, a double symbiotic system was engineered for Cu phytostabilization: On the one hand, composite Medicago truncatula plants expressing the metallothionein gene mt4a from Arabidopsis thaliana in roots were obtained to improve plant Cu tolerance. On the other hand, a genetically modified Ensifer medicae strain, expressing copper resistance genes copAB from Pseudomonas fluorescens driven by a nodulation promoter, nifHp, was used for plant inoculation. Our results indicated that expression of mt4a in composite plants ameliorated plant growth and nodulation and enhanced Cu tolerance. Lower levels of ROS-scavenging enzymes and of thiobarbituric acid reactive substances (TBARS), such as malondialdehyde (a marker of lipid peroxidation), suggested reduced oxidative stress. Furthermore, inoculation with the genetically modified Ensifer further improved root Cu accumulation without altering metal loading to shoots, leading to diminished values of metal translocation from roots to shoots. The double modified partnership is proposed as a suitable tool for Cu rhizo-phytostabilization.