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.

Plant growth promotion of the forage plant Lupinus albus Var. Orden Dorado using Pseudomonas agronomica sp. nov. and Bacillus pretiosus sp. nov. added over a valorized agricultural biowaste.

Introduction: The overexploitation of natural ecosystems and the evolution of climate change currently force us to design new strategies for more sustainable agronomic uses. The recovery of plant residues, as an alternative to agrochemicals, can help alleviate these problems, for example, through its use for the synthesis of biofertilizers. In this work, the effect of the organic fertilizer matrix ORGAON® from the valorization of horticultural waste is tested, to which two strains of bacteria (and their consortium) are added (SAICEU11T identified as Bacillus pretiosus and SAICEU22T identified as Pseudomonas agronomica), selected for their demonstrated ability to promote plant growth (PGPB), on the lupine forage plant (Lupinus albus). Methods: For the synthesis of the biofertilizer, both strains were added to the ORGAON® organic matrix separately, until reaching a final optical density (OD) of 0.5 McFarland in each case in the irrigation matrix. As a control, sterile ORGAON® (ORGAON®st) was used, also supplemented with the PGPB strains and a chemical fertilizer widely used in agronomy (Chem-F). With these treatments, a 6-week experiment was started under controlled laboratory conditions and on agricultural substrate, to recreate field conditions as accurately as possible. All the tests were carried out with 9 repetitions and 3 replicates of each treatment. After harvest, the improvements on the following biometric variables were studied for each treatment: total weight (Weight_T, g), shoot weight (Weight_S, g), root weight (Weight_R, g), number of leaves (Leaves, No.), shoot length (Length_S), root length (Length_R) and number of secondary roots (Roots, No.). Likewise, the identification of the tested strains and their description as new species was carried out. For this, they were studied from the phenotypic point of view (Transmission electron microscopy (TEM), metabolic profile, PGP activities, fatty acid profile and Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)) and genotypic (sequencing of the main housekeeping genes and sequencing of the whole genome, genomic characteristics (dDDH and ANI) and phylogenetic analysis). Results and discussion: After the statistical analysis of the results, it is shown that the individual addition of both strains on the ORGAON® and ORGAON®st organic matrix improve certain biometric variables. In the case of the SAICEU11T (Bacillus pretiosus) strain, the variables root weight (Weight_R, g), total weight (Weight_T, g) and length of the plant, and number of secondary roots (Roots, No.) significantly improve, while in the case of the strain SAICEU22T (Pseudmonas agronomica), a significant improvement of root length (Length_R) and number of secondary roots (Roots, No.) is demonstrated. On the other hand, the genotaxonomic analysis showed that both species have not been described to date. The identification based on the main housekeeping genes, show that for the Bacillus strain (SAICEU11T) the sequence similarity of the 16S rRNA was 100%, gyrB 92.69%, rpoB 97.70% and rpoD 94.67%. For the Pseudomonas strain (SAICEU22T) the results were 100% for 16S rRNA, 98.43% for rpoD and 96.94% for gyrB. However, in both cases, the dDDH and ANI values, as well as the phylogenetic analysis, show that both species are below the species threshold, which would support the hypothesis that both are new species, in line with the chemotaxonomic results obtained by MALDI-TOF spectrometry and fatty acid profile. To verify the biosafety in their handling and release into the natural environment, we have ruled out the presence of genes that encode virulence factors or resistance to antibiotics, concluding that they are suitable for use in the field to improve the yield of crop plants. Type strains are SAICEU11T (= DSM 114702T = CECT30674T) for Bacillus pretiosus and SAICEU22T (= DSM 114959T = CECT30673T) for Pseudomonas agronomicae.

Valorization as a biofertilizer of an agricultural residue leachate: Metagenomic characterization and growth promotion test by PGPB in the forage plant Medicago sativa (alfalfa).

The abuse of chemical fertilizers in intensive agriculture has turned out in the contamination of ground and the soil on which they are applied. Likewise, the generation, storage, and destruction of plant residues from the agri-food industry poses a threat to the environment and human health. The current situation of growing demand for food implies the urgent need to find sustainable alternatives to chemical fertilizers and the management of agricultural waste. Valorization of this plant residue to produce natural biofertilizers using microbiological treatments is presented as a sustainable alternative. The microbial activity allows the transformation into simple molecules that are easily absorbed by plants, as well as the stimulation of plant growth. This double direct and indirect action induced significant increases against the variables of germination, viability, and biomass (dry weight). To guarantee biosafety, it is necessary to use new bio-technological tools, such as metagenomics, which allow the taxonomic analysis of microbial communities, detecting the absence of pathogens. In the present paper, a physicochemical and metagenomic characterization of a fertilizer obtained from agricultural plant waste valorization is carried out. Likewise, fertigation treatments were tested to which the Plant Growth Promoting Bacteria (PGPB) Pseudomonas agronomica and Bacillus pretiosus were added, both independently and in consortium. Metagenomic analysis has identified taxa belonging to the kingdoms Bacteria and Archaea; 10 phyla, 25 families, 32 genera and 34 species, none of them previously described as pathogenic. A 1/512 dilution of the fertilizer increased the germination rate of Medicago sativa (alfalfa) by 16% at 144 h, compared to the treatment without fertilizer. Both the fertilizer and the addition of PGPB in a double direct and indirect action induced significant increases against the variables of germination, viability, and biomass (dry weight). Therefore, the use of an agricultural residue is proposed, which after the addition of two new species is transformed into a biofertilizer that significantly induces plant growth in Mendicago sativa plants.