Induction of phytoextraction, phytoprotection and growth promotion activities in Lupinus albus under mercury abiotic stress conditions by Peribacillus frigoritolerans subsp., mercuritolerans subsp. nov.

Strain SAICEUPBMT was isolated from soils of Almadén (Ciudad Real, Spain), subjected to a high mercury concentration. SAICEUPBMT significantly increased aerial plant weight, aerial plant length and the development of secondary roots under mercury stress; increased twice the absorption of mercury by the plant, while favoring its development in terms of biomass. Similarly, plants inoculated with SAICEUPBMT and grown in soils contaminated with mercury, express a lower activity of antioxidant enzymes; catalase enzymes (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) for defense against ROS (reactive oxygen species). Whole genome analysis showed that ANI (95. 96 %), dDDH (72.9 %), AAI (93.3 %) and TETRA (0.99) values were on the thresholds established for differentiation a subspecies. The fatty acids analysis related the strain with the Peribacillus frigoritolerans species. And the synapomorphic analysis reveals a common ancestor with analysis related the strain with the Peribacillus frigoritolerans species. Results from genomic analysis together with differences in phenotypic features and chemotaxonomic analysis support the proposal of strain SAICEUPBMT as the type strain of a novel subspecies for which the name Peribacillus frigoritolerans subps. mercuritolerans sp. nov is proposed. The absence of virulence genes and transmissible resistance mechanisms reveals its safety for agronomic uses, under mercury stress conditions. The ability of Peribacillus frigoritolerans subsp. mercuritolerans subsp. nov to improve plant development was tested in a Lupinus albus model, demonstrating a great potential for plant phytoprotection against mercury stress.

Bioremediation of environments contaminated with mercury. Present and perspectives.

Mercury is a highly toxic heavy metal whose emission sources can be both natural and the result of anthropic activity. Its polluting action on soils, and its ability to spread through the atmosphere and aquatic environments, constitutes a threat to human and environmental health; both for its bioaccumulation capacity and for biomagnification through the trophic chain. For this reason, there is a growing scientific and social interest in the reduction of this heavy metal in ecosystems. Bioremediation based on the use of microorganisms and/or plants is postulated as a sustainable alternative to traditional physicochemical methods. The main strategies used for this purpose (individually or in combination) are the volatilization of the contaminant, biosorption, phytoextraction and phytoremediation. All these tools are based on taking advantage of the natural and evolutionary capacity that different organisms have developed to adapt to the presence of various pollutants in the environment. Based on the consulted bibliography, these bioremediation methodologies focus on the use of microorganisms (freely or associated with plants) have been successfully applied in different ecosystems, postulating themselves as a respectful alternative for the future for the recovery of degraded environments. For these reasons there is a growing interest in the scientific community to design and use new techniques in a "One Health" context, which allow interpreting the positive impact of bioremediation. In this sense, the universalization of Omics techniques has allowed to abound in the knowledge of new bacterial taxa, and their biotechnological application. This study pretends to cover the present knowledge about mercury bioremediation techniques. In the same way, some new techniques and perspectives are presented in order to expand the frontiers of future research.

Reduced Antibiotic Resistance in the Rhizosphere of Lupinus albus in Mercury-Contaminated Soil Mediated by the Addition of PGPB.

The emergence of antibiotic resistance (AR) poses a threat to the "One Health" approach. Likewise, mercury (Hg) pollution is a serious environmental and public health problem. Its ability to biomagnify through trophic levels induces numerous pathologies in humans. As well, it is known that Hg-resistance genes and AR genes are co-selected. The use of plant-growth-promoting bacteria (PGPB) can improve plant adaptation, decontamination of toxic compounds and control of AR dispersal. The cenoantibiogram, a technique that allows estimating the minimum inhibitory concentration (MIC) of a microbial community, has been postulated as a tool to effectively evaluate the evolution of a soil. The present study uses the metagenomics of 16S rRNA gene amplicons to understand the distribution of the microbial soil community prior to bacterial inoculation, and the cenoantibiogram technique to evaluate the ability of four PGPB and their consortia to minimize antibiotic resistance in the rhizosphere of Lupinus albus var. Orden Dorado grown in Hg-contaminated soils. Results showed that the addition of A1 strain (Brevibacterium frigoritolerans) and its consortia with A2, B1 and B2 strains reduced the edaphic community´s MIC against cephalosporins, ertapenem and tigecycline. The metagenomic study revealed that the high MIC of non-inoculated soils could be explained by the bacteria which belong to the detected taxa,. showing a high prevalence of Proteobacteria, Cyanobacteria and Actinobacteria.

Evaluation of the oxidative stress alleviation in Lupinus albus var. orden Dorado by the inoculation of four plant growth-promoting bacteria and their mixtures in mercury-polluted soils.

Mercury (Hg) pollution is a serious environmental and public health problem. Hg has the ability to biomagnify through the trophic chain and generate various pathologies in humans. The exposure of plants to Hg affects normal plant growth and its stress levels, producing oxidative cell damage. Root inoculation with plant growth-promoting bacteria (PGPB) can help reduce the absorption of Hg, minimizing the harmful effects of this metal in the plant. This study evaluates the phytoprotective capacity of four bacterial strains selected for their PGPB capabilities, quantified by the calculation of the biomercuroremediator suitability index (IIBMR), and their consortia, in the Lupinus albus var. orden Dorado. The oxidative stress modulating capacity in the inoculated plant was analyzed by measuring the activity of the enzymes catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR). In turn, the phytoprotective capacity of these PGPBs against the bioaccumulation of Hg was studied in plants grown in soils highly contaminated by Hg vs. soils in the absence of Hg contamination. The results of the oxidative stress alleviation and Hg bioaccumulation were compared with the biometric data of Lupinus albus var. orden Dorado previously obtained under the same soil conditions of Hg concentration. The results show that the biological behavior of plants (biometrics, bioaccumulation of Hg, and activity of regulatory enzymes of reactive oxygen species [ROS]) is significantly improved by the inoculation of strains B1 (Pseudomonas moraviensis) and B2 (Pseudomonas baetica), as well as their corresponding consortium (CS5). In light of the conclusions of this work, the use of these strains, as well as their consortium, is postulated as good candidates for their subsequent use in phytostimulation and phytoprotection processes in areas contaminated with Hg.

Effect of Plant Growth-Promoting Bacteria on Biometrical Parameters and Antioxidant Enzymatic Activities of Lupinus albus var. Orden Dorado Under Mercury Stress.

Heavy metal contamination of soils is a large-scale environmental problem. It leads to significant disqualification of the territory, in addition to being a source of the potential risk to human health. The exposure of plants to mercury (Hg) generates responses in its growth and their oxidative metabolism. The impact of increasing concentrations of Hg on the development of Lupinus albus var. Orden Dorado seedlings has been studied, as well as the plant's response to the maximum concentration of Hg that allows its development (16 µg ml-1). The result shows that only the inoculum with plant growth promoting bacteria (PGPB) allows the biometric development of the seedling (root length, weight, and number of secondary roots) and prevents the toxic effects of the heavy metal from aborting the seedlings. Specifically, treatments with strains 11, 20 (Bacillus toyonensis), 48 (not determined), and 76 (Pseudomonas syringae) are interesting candidates for further PGPB-assisted phytoremediation trials as they promote root biomass development, through their PGPB activities. The plant antioxidant response has been analyzed by quantifying the catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) enzyme activity in the root, under 16 µg ml-1 of HgCl2 and different PGPB treatments. Results show that, although Hg stress generally induces enzyme activity, strains 31 and 69I (Pseudomonas corrugata) and 18 and 43 (Bacillus toyonensis) can keep SOD and APX levels close to those found in control without Hg (p < 0.01). Strain 18 also shows a significant reduction of GR to control levels without Hg. The present work demonstrates the benefit of PGPB treatments in situations of high Hg stress. These findings may be a good starting point to justify the role of PGPB naturally isolated from bulk soil and the rhizosphere of plants subjected to high Hg pressure in plant tolerance to such abiotic stress conditions. More studies will be needed to discover the molecular mechanisms behind the phytoprotective role of the strains with the best results, to understand the complex plant-microorganism relationships and to find effective and lasting symbioses useful in bioremediation processes.

Oxidative stress protection and growth promotion activity of Pseudomonas mercuritolerans sp. nov., in forage plants under mercury abiotic stress conditions.

SAICEUPSMT strain was isolated from soils in the mining district of Almadén (Ciudad Real, Spain), subjected to a high concentration of mercury. Using the plant model of lupinus, the strain was inoculated into the rhizosphere of the plant in a soil characterized by a high concentration of mercury (1,710 ppm) from an abandoned dump in the mining district of Almadén (Ciudad Real, Spain). As a control, a soil with a minimum natural concentration of mercury, from a surrounding area, was used. Under greenhouse conditions, the effect that the inoculum of the SAICEUPSMT strain had on the antioxidant capacity of the plant was studied, through the quantification of the enzymatic activity catalase (CAT), ascorbate peroxidase (APX), superoxide dismutase (SOD), and glutathione reductase (GR). Likewise, the capacity of the plant to bioaccumulate mercury in the presence of the inoculum was studied, as well as the effect on the biometric parameters total weight (g), shoot weight (g), root weight (g), shoot length (cm), root length (cm), total number of leaves (N), and total number of secondary roots (No). Finally, in view of the results, the SAICEUPSMT strain was identified from the phenotypic and genotypic point of view (housekeeping genes and complete genome sequencing). The inoculum with the SAICEUPSMT strain in the presence of mercury produced a significant reduction in the enzymatic response to oxidative stress (CAT, APX, and SOD). It can be considered that the strain exerts a phytoprotective effect on the plant. This led to a significant increase in the biometric parameters total plant weight, root weight and the number of leaves under mercury stress, compared to the control without abiotic stress. When analyzing the mercury content of the plant with and without bacterial inoculum, it was found that the incorporation of the SAICEUPSMT strain significantly reduced the uptake of mercury by the plant, while favoring its development in terms of biomass. Given the positive impact of the SAICEUPSMT strain on the integral development of the plant, it was identified, proving to be a Gram negative bacillus, in vitro producer of siderophores, auxins and molecules that inhibit stress precursors. The most represented fatty acids were C16:0 (33.29%), characteristic aggregate 3 (22.80%) comprising C16:1 ω7c and C16: 1ω6c, characteristic aggregate 8 (13.66%) comprising C18:1 ω7c, and C18: 1 cycle ω6c and C 17:0 (11.42%). From the genotypic point of view, the initial identification of the strain based on the 16S rRNA gene sequence classified it as Pseudomonas iranensis. However, genome-wide analysis showed that average nucleotide identity (ANI, 95.47%), DNA-DNA in silico hybridization (dDDH, 61.9%), average amino acid identity (AAI, 97.13%), TETRA (0.99%) and intergenic distance (0.04) values were below the established thresholds for differentiation. The results of the genomic analysis together with the differences in the phenotypic characteristics and the phylogenetic and chemotaxonomic analysis support the proposal of the SAICEUPSMT strain as the type strain of a new species for which the name Pseudomonas mercuritolerans sp. is proposed. No virulence genes or transmissible resistance mechanisms have been identified, which reveals its safety for agronomic uses, under mercury stress conditions.