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

IntroductionThe 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).MethodsFor 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 discussionAfter 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

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

The microbial community in the rhizosphere determined by community-level physiological profiles (CLPP) and direct soil- and cfu-PLFA techniques

The composition of the microbial community in the rhizosphere was investigated using three different techniques: direct soil-phospholipid fatty acid (PLFA), cfu-PLFA (using culturable, bacteria only) and community-level physiological profiles (CLPP) using Biolog GN microtitre plates. All three techniques showed that the rhizosphere bacterial community differed from the bulk soil community, when pea plants were grown in pots. The two PLFA techniques also indicated that the effect of roots on microbial communities was greater than differences in the PLFA pattern between different bulk soils. The difference was most prominent using the direct soil-PLFA technique. Both PLFA techniques indicated that gram-negative bacteria were relatively more abundant in the rhizosphere, while PLFAs indicative of gram-positive bacteria were relatively more common in bulk soil samples. The results obtained with the direct soil-PLFA method did not differ between rhizosphere samples taken along a pea root, while this was the case with both the cfu-PLFA technique and the CUP method. According to both techniques, the differences in community composition were greatest near the root tip. Different plant species exhibited different microbial community composition in the rhizosphere, as judged from all three methods, when four different plant species were compared. In all cases, Trifolium differed most from the other species (three different grasses). Although all three methods were found to be suitable for rhizosphere studies, the CLPP method appeared less suitable than the two PLFA methods, since less of the variation in the data could usually be explained

Mercury and Antibiotic Resistance Co-Selection in Bacillus sp. Isolates from the Almadén Mining District

Antibiotic resistance (AR) in the environment is of great global concern and a threat to public health. Soil bacteria, including Bacillus spp., could act as recipients and reservoirs of AR genes of clinical, livestock, or agricultural origin. These genes can be shared between bacteria, some of which could be potentially human pathogens. This process can be favored in conditions of abiotic stress, such as heavy metal contamination. The Almadén mining district (Ciudad Real, Spain) is one of the environments with the highest mercury (Hg) contamination worldwide. The link between heavy metal contamination and increased AR in environmental bacteria seems clear, due to co-resistance and co-selection phenomena. In the present study, 53 strains were isolated from rhizospheric and bulk soil samples in Almadén. AR was tested using Vitek® 2 and minimum inhibitory concentration (MIC) values were obtained and interpreted based on the criteria of the Clinical and Laboratory Standards Institute (CLSI) guidelines. Based on the resistance profiles, five different antibiotypes were established. The Hg minimum bactericidal concentration (MBC) of each strain was obtained using the plating method with increasing concentrations of HgCl2. A total of 72% of Bacillus spp. showed resistance to two or more commonly used antibiotics. A total of 38 isolates expressed AR to cephalosporins. Finally, the environmental co-selection of AR to cephalosporins and tetracyclines by selective pressure of Hg has been statistically demonstrated

Selection of Mercury-Resistant PGPR Strains Using the BMRSI for Bioremediation Purposes

Heavy metal pollution of soil, particularly by mercury (Hg), is a problem that can seriously affect the environment and human health. For this reason, it is necessary to take steps to remediate these environments, prevent potential adverse effects, and restore these areas for subsequent use in agriculture, industry, ranching, and forestry. The present study has selected 40 bacterial strains from rhizosphere and bulk soil that grow naturally in high Hg-contaminated soils from the Almadén mining district in Ciudad Real, Spain. With the objective of evaluating the potential use of these strains in phyto-rhizoremediation, an evaluation and statistical analysis of their PGPR (Plant-Growth-Promoting Rhizobacteria) activity at different levels of Hg was carried out as the first condition of selection for their potential use in bioremediation. In addition, a Hg MBC (Maximum Bactericidal Concentration) was performed with the aim of selecting the strains with high Hg tolerance. Finally, strains with potential biotechnological use have been proposed according to the Bio-Mercury Remediation Suitability Index (BMRSI) criteria, which consider indole-3-acetic acid (IAA) production, acid 1- aminocyclopropane-1-carboxylic deaminase (ACCd) activity, phosphates solubilization, and siderophore production measured in the presence of Hg, as well as its MBC to Hg. The strains selected for further in vivo and in situ processes must reach at least an MBC (Hg) > 100 μg/mL and BMRSI ≥ 6.5

Bio-Mercury Remediation Suitability Index: A Novel Proposal That Compiles the PGPR Features of Bacterial Strains and Its Potential Use in Phytoremediation

Soil pollution from heavy metals, especially mercury, is an environmental problem for human health. Biological approaches offer interesting tools, which necessarily involve the selection of organisms capable of transforming the environment via bioremediation. To evaluate the potential use of microorganisms in phytorhizoremediation, bacterial strains were isolated from rhizospheric and bulk soil under conditions of chronic natural mercury, which were identified and characterized by studying the following: (i) their plant growth promoting rhizobacteria (PGPR) activities; and (ii) their maximum bactericide concentration of mercury. Information regarding auxin production, phosphate solubilization, siderophore synthesis and 1-aminocyclopropane-1-carboxylic acid deaminase (ACCd) capacity of the isolates was compiled in order to select the strains that fit potential biotechnological use. To achieve this objective, the present work proposes the Bio-Mercury Remediation Suitability Index (BMR-SI), which reflects the integral behavior of the strains for heavy metal polluted soil bioremediation. Only those strains that rigorously fulfilled all of the established criteria were selected for further assays

Evaluation of the PGPR Capacity of Four Bacterial Strains and Their Mixtures, Tested on Lupinus albus var. Dorado Seedlings, for the Bioremediation of Mercury-Polluted Soils

Soil contamination by mercury, which is one of the most toxic heavy metals due to its bioaccumulative capacity, poses a risk to the environment as well as health. The Almadén mining district in Ciudad Real, Spain is one of the most heavily-polluted sites in the world, making the soils unusable. Bioremediation, and more specifically phyto-rhizoremediation, based on the synergistic interaction established between plant and Plant Growth Promoting Rhizobacteria (PGPR), improves the plant’s ability to grow, mobilize, accumulate, and extract contaminants from the soil. The objective of this study is to evaluate the plant growth-promoting ability of four PGPR strains (and mixtures), isolated from the bulk soil and rhizosphere of naturally grown plants in the Almadén mining district, when they are inoculated in emerged seeds of Lupinus albus, var. Dorado in the presence of high concentrations of mercury. After 20 days of incubation and subsequent harvesting of the seedlings, biometric measurements were carried out at the root and aerial levels. The results obtained show that the seeds treatment with PGPR strains improves plants biometry in the presence of mercury. Specifically, strain B2 (Pseudomonas baetica) and B1 (Pseudomonas moraviensis) were those that contributed the most to plant growth, both individually and as part of mixtures (CS5 and CS3). Thus, these are postulated to be good candidates for further in situ phyto-rhizoremediation tests of mercury-contaminated soils

Changes in the bacterial communities structure of the rhizosphere of four wild Lupinus species from flowering to fruiting

Microbial rhizosphere communities of four different Lupinus species were studied at two plant growth stages in order to determine the dominance of r/K strategists and the pattern of taxa and diversity (Shannon index). Five hundred and seventy-six strains were isolated, determined and classified into eleven different bacterial genera and the Enterobacteriaceae family. Aureobacterium dominated on L. hispanicus and L. angustifolius at both sampling times (GS1: Growth Stage 1 (flowering); GS2: Growth Stage 2 (fruiting)), whereas it only dominated in the rhizosphere of L. albus at GS1 (58.45%). Cellulomonas dominated at GS2 (42.93%). In L. luteus, Bacillus dominated at GS1 (41.21%) and Aureobacterium at GS2 (39.74%). Principal Component Analysis (PCAs) showed that the microbial composition of the rhizosphere of L. albus is the only one that changed considerably from GS1 to GS2. These changes were due to a proportional increase of r strategists at GS2. Sample size (18 to 54 colonies, depending on lupin species) wasadequate according to the Shannon index. Despite the fact that diversity between GS1 and GS2 hardly varied in L. albus, L. luteus, and L. hispanicus, the PCAs revealed the largest differences in the microbial structure associated with L. albus. A tentative successional model for the lupin microbial population of the rhizosphere related to its biological cycle, is proposed.Se estudia la comunidad microbiana rizosférica de cuatro especies de Lupinus en dos momentos de su ciclo biológico, con el objeto de determinar la dominancia de r y K estrategas, el patrón de taxones y la diversidad biológica (índice de Shannon). Se aislaron 576 cepas, y se encontraron representantes de once géneros bacterianos y de la familia Enterobacteriaceae. Aureobacterium fue mayoritario en L. hispanicus y L. angustifolius en ambos momentos de muestreo (GS1: floración; GS2: fructificación), mientras que sólo dominó en L. albus en GS1 (58.45%). Cellulomonas dominó en GS2 (42.93%). En L. luteus fue Bacillus el mayoritario en GS1 (41.21%) y Aureobacterium en GS2 (39.73%). Los Análisis de Componentes Principales (ACPs) indican que sólo L. albus tiene cambios considerables en sus poblaciones microbianas rizosféricas de GS1 a GS2. Estos cambios se deben al incremento de estrategas de la r en GS2. El tamaño de la muestra (de 18 a 54 colonias, dependiendo de la especie de lupino) fue la adecuada, según indica el índice de Shannon. A pesar de que la diversidad apenas varía de GS1 a GS2 en L. albus, L. luteus y L. hispanicus, los ACPs reflejan cambios importantes en la estructura microbiana rizosférica en L. albus. Se propone un modelo sucesional tentativo de las poblaciones microbianas de la rizosfera del lupino, relacionándolo con su ciclo biológico

Data_Sheet_1_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.docx

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.</p