Enhanced legume growth and adaptation to degraded estuarine soils using Pseudomonas sp. nodule endophytes

Frontiers in Microbiology 01 frontiersin.org Enhanced legume growth and adaptation to degraded estuarine soils using Pseudomonas sp. nodule endophytes Noris J. Flores-Duarte 1, Sara Caballero-Delgado 1, Eloisa Pajuelo 1, Enrique Mateos-Naranjo 2, Susana Redondo-Gómez 2, Salvadora Navarro-Torre 1† and Ignacio D. Rodríguez-Llorente 1*† 1 Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain, 2 Department of Plant Biology and Ecology, Faculty of Biology, University of Sevilla, Sevilla, Spain The joint estuary of Tinto and Odiel rivers (SW Spain) is one of the most degraded and polluted areas in the world and its recovery is mandatory. Legumes and their associated bacteria are recommended sustainable tools to fight against soils degradation and loss of fertility due to their known positive impacts on soils. The aim of this work was to isolate and characterize plant growth promoting nodule endophytes (PGPNE) from inside nodules of Medicago spp. naturally growing in the estuary of the Tinto and Odiel Rivers and evaluate their ability to promote legume adaptation in degraded soils. The best rhizobia and non-rhizobia among 33 endophytes were selected based on their plant growth promoting properties and bacterial enzymatic activities. These strains, identified as Pseudomonas sp. N4, Pseudomonas sp. N8, Ensifer sp. N10 and Ensifer sp. N12, were used for in vitro studies using Medicago sativa plants. The effects of individual or combined inoculation on seed germination, plant growth and nodulation were studied, both on plates and pots containing nutrient-poor soils and moderately contaminated with metals/loids from the estuary. In general, inoculation with combinations of rhizobia and Pseudomonas increased plant biomass (up to 1.5-fold) and nodules number (up to 2-fold) compared to single inoculation with rhizobia, ameliorating the physiological state of the plants and helping to regulate plant stress mechanisms. The greatest benefits were observed in plants inoculated with the consortium containing the four strains. In addition, combined inoculation with Ensifer and Pseudomonas increased As and metals accumulation in plant roots, without significant differences in shoot metal accumulation. These results suggest that PGPNE are useful biotools to promote legume growth and phytostabilization potential in nutrient-poor and/or metals contaminated estuarine soils. KEYWORDS contaminated estuarine soils, enhanced nodulation, nodule endophytes, plant growth-promoting bacteria, phytostabilization TYPE Original Research PUBLISHED 20 October 2022 DOI 10.3389/fmicb.2022.1005458 OPEN ACCESS EDITED BY George Newcombe, University of Idaho, UnitedStates REVIEWED BY Parul Chaudhary, G. B. Pant University of Agriculture and Technology, India Julia W. Neilson, University of Arizona, UnitedStates *CORRESPONDENCE Ignacio D. Rodríguez-Llorente [email protected] †These authors have contributed equally to this work and share last authorship SPECIALTY SECTION This article was submitted to Microbiotechnology, a section of the journal Frontiers in Microbiology RECEIVED 28 July 2022 ACCEPTED 06 October 2022 PUBLISHED 20 October 2022 CITATION Flores-Duarte NJ, Caballero-Delgado S, Pajuelo E, Mateos-Naranjo E, Redondo-Gómez S, Navarro-Torre S and Rodríguez-Llorente ID (2022) Enhanced legume growth and adaptation to degraded estuarine soils using Pseudomonas sp. nodule endophytes. Front. Microbiol. 13:1005458. doi: 10.3389/fmicb.2022.1005458 COPYRIGHT © 2022 Flores-Duarte, Caballero-Delgado, Pajuelo, Mateos-Naranjo, Redondo- Gómez, Navarro-Torre and Rodríguez- Llorente. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Flores-Duarte et al. 10.3389/fmicb.2022.1005458 Frontiers in Microbiology 02 frontiersin.org Introduction Industrial and mining activity releases toxic metal/loids into the environment, such as As, Cd, Cu, and Pb, all of them very harmful to human health and most living beings (FAO and ITPS, 2015). In turn this has caused the degradation of soils, either by the modification of pH or by originating a decrease in the number of arable lands causing a problem at social, economic, and environmental levels (Singh etal., 2010; Chen etal., 2013). Heavy metals are dangerous because they are not degradable either chemically or biologically, being able to remain in the environment for hundreds or thousands of years (Herrera Marcano, 2011). These metals have also been repressing the enzymatic activity of the soils, causing a decrease in the growth and respiration of the populations of microorganisms, thus altering the diversity present in the rhizosphere (Abdu etal., 2017; Aponte etal., 2020). Marshes of river estuaries are particularly sensitive to heavy metals contamination by metals deposition. A good example is the combined estuary of Tinto and Odiel rivers (Huelva, SW Spain), known as one of the most contaminated regions in the world (Hudson-Edwards etal., 1999). Levels of toxic metal/loids as high as 125 ppm of As, 890 ppm of Cu, 275 ppm of Pb or 1,500 ppm of Zn in the Odiel river marshes, and 2 to 3-fold these levels in the Tinto River marshes, have been reported in the last decade (Mesa etal., 2015a; Navarro-Torre etal., 2017). These levels exceed those allowed by regional and national legislation in natural parks, agricultural and industrial soils (Junta de Andalucía and Consejeria de Medio Ambiente, 1999). One solution to this great environmental problem is the implantation of pioneering crops able to regenerate the soil and improve its yield, respecting the environment. This kind of strategies should include the use of leguminous plants (belonging to the Fabaceae family), due to their great benefits. First, legumes are an important product in the food industry both for human and animal consumption worldwide, since they are very rich in protein, fiber, vitamins, and minerals and also has a low cost, being highly consumed in underdeveloped countries (Ferreira et al., 2021). Second, legumes play a very important role in agriculture as cover crops, since they improve soil fertility increasing crops yield, help the constant movement of soil nutrients and release matter into the soil (Stagnari etal., 2017; Ferreira etal., 2021). Third, legumes are good candidates to adapt to degraded soils affected by biotic and abiotic factors, particularly these plants play an important role in the regeneration of soils degraded by heavy metals due to their ability to accumulate metals in the roots without affecting plant growth (Mesa etal., 2015b; Flores-Duarte etal., 2022a). Legumes are capable of fixing atmospheric nitrogen through symbiosis with many genera of soil nitrogen-fixing bacteria, called rhizobia, which penetrate the roots through the root hairs, forming nodules in which nitrogen fixation takes place (Poole etal., 2018). But nodules are not only occupied by rhizobia, within the legume nodules there are another great variety of bacteria called non-rhizobial nodule-associated bacteria (NAB; Rajendran etal., 2012), non-rhizobial endophytes (NRE; De Meyer etal., 2015), or just nodule endophytes (Velázquez etal., 2013). These bacteria do not induce nodule formation but can colonize nodules accompanying rhizobia forming a beneficial association and enhancing nodulation and plant growth. Within this group, bacteria of the genera, Acinetobacter, Agrobacterium, Bacillus, Burkholderia, Pseudomonas and Variovorax, among others, have been found (Shiraishi etal., 2010; Martínez-Hidalgo and Hirsch, 2017; Bessadok etal., 2020). These nodule endophytes usually behave as plant growth promoting bacteria (PGPB). Soil health and fertility are directly influenced by beneficial plant-microbe relationships that determine soil biodiversity (Vishwakarma etal., 2020). Plant-PGPB interactions have shown to provide benefits in plant growth and development by facilitating the acquisition of nutrients (Fasusi etal., 2021) and producing phytohormones related with plant and root lenth growth, and the formation of root hairs and lateral roots, such as cytokinin, gibberellins, abscisic acid, and IAA (Raza etal., 2019). Through atmospheric nitrogen fixation (Roy et al., 2020), phosphate mobilization/solubilization (Etesami etal., 2021) and siderophores production (Lurthy etal., 2020; Kang etal., 2021) bacteria provide N, P, Fe, and Zn to plants. PGPB also play a determinant role in plant adaptation and tolerance to biotic and abiotic stress (Chaudhary et al., 2022). For example, bacteria with ACC-deaminase activity are able to break the ethylene precursor ACC, altering plant stress perception (Penrose and Glick, 2003; Chandwani and Amaresan, 2022). Microorganisms have an important role in pollutant detoxification and heavy metal plant stress resistance (Caracciolo and Terenzi, 2021). Soil microorganisms have developed different resistance mechanisms, such as metal biosorption, bioaccumulation, modification of metal chemical state (methylation) or production of chelating compounds, particularly siderophores and biosurfactans, that cause a lowering in metal availability for plants (Verma and Kuila, 2019), then diminishing plant metal content (Caracciolo and Terenzi, 2021). PGPB are directly involved in metal detoxification through the production of secondary metabolites, such as siderophores, ACC or IAA (Caracciolo and Terenzi, 2021). Particularly in legumes, nitrogenfixing bacteria and endophytes inside nodules have probed to reduce metal translocation to aerial parts and increase plant nitrogen content and growth in metal contaminated soils (Navarro-Torre etal., 2020; Flores-Duarte etal., 2022b). In a recent work, a consortium of PGPB, including Pseudomonas, Chryseobacterium and Priestia genera, showed their ability to promote Medicago sativa growth and adaptation in poornutrient soils (Flores-Duarte etal., 2022b). These bacteria were isolated from the rhizosphere of different legumes growing in estuarine soils with low levels of minerals and organic matter. It is well accepted that endophytes maintain a much closer relationship with the plant and have lower competition with soil microorganisms than rizospheric bacteria (Adeleke and Babalola, 2021; Dwibedi et al., 2022). The present work is aimed to determine whether a consortium of endophytes isolated from Flores-Duarte et al. 10.3389/fmicb.2022.1005458 Frontiers in Microbiology 03 frontiersin.org nodules could enhance legume growth in degraded soils more efficiently than rhizosphere bacteria, introducing soil metal contamination as additional stress. The objectives that arise in this work are the following: (i) isolation and characterization of rhizobia and endophytes from nodules of Medicago spp. naturally growing in the Odiel river estuary (Huelva, Spain); (ii) selection of rhizobia and nodule endophytes based on their properties, enzymatic activities and ability to tolerate metal/loids; (iii) determine the ability of the selected bacteria to promote M. sativa growth, nodulation and metal accumulation in Odiel marshes soils under greenhouse conditions. Materials and methods Collection of samples and soil characterization Nodulated wild plants of Medicago spp. were collected from high marshes of the Odiel river estuary (Huelva, Spain; 37°150 N, 6°580 W; SW Spain) in February 2020. Soil samples (15–20 cm deep) were collected using a shovel, gloves and plastic bags. The samples were immediately transported to the laboratory and stored at 4°C. Three homogeneous soil samples were deposited in sterile bottles for chemical soil analysis, as described by Mateos- Naranjo etal. (2015). The soil texture (percentage of sand, silt, and clay) was determined using the Bouyoucos hydrometer method (Bouyoucos, 1936). Electrical conductivity was measured with a Crison-522 conductivity meter (Spain) and pH and redox potential with a Crison pH/mVp-506 portable meter (Spain). The concentration of nutrients in the soil was measured by inductively coupled plasma-optical emission spectroscopy (ICP-OES; ARLFisons3410, Thermo Scientific, Walthman, MA, UnitedStates). The amount of organic matter was determined by the method of Walkley and Black (1934). Results are presented in Table1. Isolation of endophytic bacteria from nodules of Medicago spp. Nodules were exscinded from roots with a scalpel, washed with water and placed on tubes. Samples were immersed in 70% (v/v) ethanol for 1 min with manual agitation to disinfect the surface. Then, they were washed again with sterile water and immersed in 3% (v/v) sodium hypochlorite for 15 min under continuous agitation and finally washed four times with sterile distilled water. Disinfected nodules were deposited in a mortar with 1 ml of sterile 0.9% (w/v) saline solution and grinded. Portions of 100 μl of the resulting mix were to spread and extended with a spatula in Petri dishes with TY medium (tryptone-yeast extract agar) and TSA (tryptone soya agar, Intron Biotechnology, Korea). Plates were incubated for 72 h at 28°C. Controls of nodule surface disinfection and solution sterility were performed. Bacteria were separated based on the different colony morphology and cell morphology was observed by Gram stain using an Olympus CX41 microscope with the 100x objective. Determination of plant growth promoting properties and enzymatic activities The ACC deaminase activity was assessed as described by Penrose and Glick (2003) and adapted by Mesa etal. (2015a). Activity was measured by monitoring α-ketobutyric acid at 540 nm in a spectrophotometer (Lambda25; PerkinElmer, Walthmam, MA, United States), and the amount of α-ketobutyric acid was determined using a standard curve with known concentrations. ACC deaminase activity was expressed in μmoles of α-ketobutyrate per mg of protein per hour. To verify nitrogen fixation, strains were plated in nitrogen free broth (NFB; Döebereiner, 1995) for 5 days at 28°C. Indole-3-acetic acid (IAA) production was determined using a colorimetric technique by incubating 3 ml of a liquid culture of TY or TSB supplemented with L-tryptophan (0.1 mg/ml) and incubated at 28°C for 72 h at 200 rpm. Cultures were then centrifuged for 5 min at 13,000 rpm and supernatants transferred to glass tubes. The appearance of a pink color after adding 4 ml of Salkowski’s reagent (Gordon and Weber, 1951) indicated that the test was positive. The amount of IAA was calculated by measuring the absorbance at 535 nm in a spectrophotometer (Lambda 25; Perkin Elmer, Waltham, MA, USA). Phosphate solubilization was carried out on plates with NBRIP medium (phosphate growth medium from the National Institute of Botanical Research) as TABLE1 Physicochemical properties and micronutrients concentrations of soil from high marshes of the Odiel River. Physicochemical properties Location Texture (%) *Organic material (%) Conductivity (μS·cm-1)pH High marsh 72/13/15 0.9±0.01 13.1±0.3 6.8±0.2 Metal/loid concentration (mg·kg−1) Location As Cd Cu Zn Mg Na Fe P High marsh 27.9±2.2 0.38±0.01 316.7±3.20 345.0±7.10 0.789±0.01 0.369±0.01 9345.56±9.35 0.056±0.01 Values are mean ± S.D. (n = 3). *Texture (sand/slit/clay percentage). Flores-Duarte et al. 10.3389/fmicb.2022.1005458 Frontiers in Microbiology 04 frontiersin.org described by Nautiyal (1999). The appearance of a transparent halo after 7 days of incubation at 28°C indicated that the bacteria had the capacity to solubilize phosphates. 100 μl of bacterial culture medium was added to each well. The formation of biofilms will be prolonged by checking the adhesion capacity of the bacteria in microplates with 96 wells in TY or liquid TSB at 28°C, incubating for 4 days, after incubation each well was stained with 200 μl of 0.01% crystal violet as described by del Castillo etal. (2012). Siderophores production was determined by the appearance of an orange halo around the well containing 100 μl of bacterial culture in CAS medium (Chromeazurol S), after incubation for 7 days at 28°C (Schwyn and Neilands, 1987). Enzymatic activities were determined on plates incubated at 28°C for 7 days. Pectinase and cellulase activities were examined as described by Elbeltagy etal. (2000). For pectinase activity, strains were plated on ammonium mineral agar (AMA). Plates were revealed with 2% CTAB and positive bacteria showed a halo around. For cellulase activity, strains were plated on solid M9 minimal medium supplemented with yeast extract (0.2%) and carboxymethylcellulose (1%). Plates were developed by covering the plate with 1 mg/ml Congo Red solution for 15 min and decolorizing with 1 M NaCl for 15 min. The appearance of a clear halo indicated positive result. Chitinase activity was performed as described by Mesa etal. (2015a). Amylase activity was performed on starch agar plates (Scharlab, Barcelona, Spain) and revealed with 10 ml lugol. The formation of a transparent halo indicated positive result. Lipase and protease activities were observed by the presence of halos around the bacteria after incubation in casein agar and Tween 80 mediums, respectively, as described by Prescott (2002). And finally, the DNAse activity was determined by cultures on DNA agar plates revealed with 1 M HCl. For PGP properties and enzyme activities, positive and negative controls from BIO-181 group collection were used (Supplementary Table1). Tolerance to metals/loids The isolated bacteria were plated in TY and TSA medium with increasing concentrations of As, Cd, Cu and Zn, from 1 M NaAsO 2 , 1 M CdCl 2 , 1 M CuSO 4 , 1 M ZnSO 4 stock solutions. Plates were incubated for 24–48 h at 28°C and tolerance expressed as the maximum tolerable concentration (MTC), that is the maximum concentration that allows visible bacterial growth. Analysis of diversity and identification of isolates The diversity of the isolated endophytes was analyzed by performing a Box-PCR. Bacterial genomic DNA was isolated using a G-spin™ Genomic DNA Extraction Kit for Bacteria (Intron Biotechnology, Gyeonggi-do, Korea) with the instructions determined by the manufacturer. Box-PCR was performed using 1 μl of DNA and Box A1R primer (5′-CTACGGCAAG GCGACGCTGACG-3′) using the Maxime™ PCR PreMix kit (i-Taq™; Intron Biotechnology, Gyeonggi-do, Korea) and following the PCR conditions: initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 20 s, annealing at 52°C for 20 s, extension at 72°C for 1 min, and final extension at 72°C for 5 min. Electrophoresis was performed in a 1.5% (w/v) agarose gel and a voltage of 70 V for 2 h. Representative bacteria of each different Box-PCR profile were identified by 16S rRNA gene amplification using 16F27 and 16R1488 primers (Navarro-Torre etal., 2016) and the Maxime™ PCR PreMix kit (i-Taq™; Intron Biotechnology, Gyeonggi-do, Korea) following the next PCR conditions: initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 20 s, annealing at 58°C for 10 s, extension at 72°C for 50 s, and final extension at 72°C for 5 min. Electrophoresis was performed in a 1% (w/v) agarose gel and a voltage of 120 V for 30 min. PCR products were purified with the enzyme ExoSAP IT (Affymetrix, Santa Clara, CA, USA), following the manufacturer instructions, and sequenced by the StabVida company (Caparica, Portugal). Then, 16S rRNA gene sequences were compared with those deposited in the EzBioCloud database (Yoon etal., 2017) using the Ez-Taxon e service (www.ezbiocloud. net/eztaxon; accessed July 2022). Finally, 16S rRNA gene sequences were deposited in the NCBI GenBank. Inoculant design Selected bacteria were grown together on TY plates to test growth compatibility as described in Navarro-Torre et al. (2016). The inoculants were prepared by cultivating selected strains separately in TSB or TY at 28°C for 24 h and 200 rpm. Afterwards, cultures containing 10 8 cells mL −1 were transferred to sterile Falcon tubes to becentrifuged at 8000 rpm for 10 min, washing twice with sterile 0.9% (w/v) saline solution, to remove traces of the culture medium (Navarro-Torre etal., 2016). At the end of the washes the cultures were resuspended in a sterile liquid nitrogen deficient buffered nodulation medium (BNM; Ehrhardt etal., 1992), or sterile water for experiments under greenhouse conditions. For co-inoculation and consortium inoculation designs, bacteria were mixed after resuspension. Inoculation conditions were defined as follow: C-: non inoculation; N4: inoculation with Pseudomonas sp. N4; N8: inoculation with Pseudomonas sp. N8; N10: inoculation with Ensifer sp. N10; N12: inoculation with Ensifer sp. N12; N4 + N10: co-inoculation with Pseudomonas sp. N4 and Ensifer sp. N10; N8 + N10: co-inoculation with Pseudomonas sp. N8 and Ensifer sp. N10; N4 + N12: co-inoculation with Pseudomonas sp. N4 and Ensifer sp. N12; N8 + N12: co-inoculation with Pseudomonas sp. N8 and Ensifer sp. N12; CSN: inoculation with Pseudomonas sp. N4, Pseudomonas sp., N8 Ensifer sp. N10 and Ensifer sp. N12. Flores-Duarte et al. 10.3389/fmicb.2022.1005458 Frontiers in Microbiology 05 frontiersin.org Labelling of Pseudomonas with fluorescence and microscopy Pseudomonas strains (Pseudomonas sp. N4 and Pseudomonas sp. N8) were marked with the fluorescent protein mCherry by triparental matting, mixing the donor Escherichia coli DH5α containing the plasmid pMP7604 (Lagendijk etal., 2010), the helper strain E. coli containing pRK600 (Finan etal., 1986) and Pseudomonas strains spontaneously resistant to rifampicin (100 μg/ml) as recipients. 100 μl aliquots of TSB overnight cultures were plated on TSA containing rifampicin (100 μg/ml) and tetracycline (10 μg/ml). Fluorescent Pseudomonas resistant to both antibiotics thus containing the plasmid were selected. M. sativa plants were co-inoculated with the rhizobia (Ensifer sp. N10 or Ensifer sp. N12) and labelled Pseudomonas in square plates (12 × 12 cm), 20 seedlings per condition, as described below. After 28 days, roots and nodules were cut with a sterile scalpel and 0.5 mm cuts observed (Mesa et al., 2015b). Fluorescent bacteria in plant tissues were visualized using a laser scanning spectral confocal optical microscope (Zeiss LSM 7 DUO, Zeiss, Jena, Germany) with an objective Plan-Apochromat 20X/0.8 M27, filters of 572–727, and a laser of 561 nm (5.3%). Images were processed with ZEN2011 software (Zeiss, Jena, Germany). Medicago sativa seeds germination and nodulation on plates Alfalfa (M. sativa) seeds were surface disinfected following the protocol described by Navarro-Torre etal. (2019) by immersing them for 10 min in 70% (v/v) ethanol, followed by 30 min in sodium hypochlorite at 3% (v/v) under gentle agitation, washing 6 times with sterile distilled water. The next step was to immerse 50 seeds in each inoculant for 1 h and for the non-inoculated control (C-) seeds were immersed for 1 h in 0.9% (w/v) NaCl. Seeds were transferred to plates containing 0.9% (w/v) water-agar (5 repetitions per condition and 10 seeds per plate) or water-agar plates containing a mixture of 7.5 μM As, Cd, Cu, and Zn, prepared from the stock solutions described above (paragraph 2.4), and finally incubated in the dark at 28°C. Germination was observed every 24 h for 7 days in absence of metals and for 21 days in presence of metals. For in vitro nodulation experiment, pre-germinated seeds were transferred to square plates (12×12 cm) with nitrogen-free BNM-agar medium (Ehrhardt etal., 1992), supplemented with sodium arsenite at a concentration of 30 μM. or in absence of arsenite. NH4NO3 was added to all plates at a concentration of 1 mM. 10 seeds were deposited in each plate and 5 replicates per treatment were performed. The seeds were inoculated or co-inoculated under the conditions described above using the selected bacteria, and non-inoculated seeds were used as controls. The plates were placed in a vertical position, incubated at 22°C with a cycle of 8 h of darkness and 16 h of light (120–130 μE m −2·s−1) in a growth chamber (AGP-700-HR ESP; Radiber, Barcelona, Spain). Plants were harvested after 21 days and nodules from each condition were counted. Experiments under greenhouse conditions Soil was collected from the upper marshes of the Odiel River and sterilized in an autoclave at 121°C and 1 atm of overpressure for 30 min. This sterilization was repeated twice. To confirm soil sterilization, 3 samples of 1 g of soil were washed by shaking in 10 ml of sterile water. After decanting for 10 min, three samples of 100 μl of supernatant per tube were plated on TSA and no growth was observed on plates incubated 48 h at 28°C. Plastic pots (11 cm squared pots with 12 cm height) were filled with 1 kg of the previously sterilized soil (3,1, three parts of the soil from the marshes and one part of sterile perlite) and 2 pre-germinated seeds were placed per pot, 8 pots per condition. The experiment lasted 60 days and plants were irrigated with sterile water once a week (50 ml) and weekly inoculated with their respective inoculum (50 ml of cultures). The greenhouse had controlled light and temperature conditions; natural light was supplemented with fluorescent/incandescent lamps to get a photoperiod of 16 h light: 8 h dark; and the temperature was adjusted to 25°C during the day and 15°C during the night. At the end, the length of roots and shoots were measured, the number of nodules and leaves were counted, the diameters of the leaves were determined, and the nitrogen content in the plant was evaluated using an InfrAlyzer 300 (Technicon, Tarrytown, NY, UnitedStates), as described by Carrasco etal. (2005). Finally, samples were placed in an oven at 80°C for 48 h to determine the dry weight of both shoots and roots separately. Determination of photosynthetic parameters and total chlorophyll content For the determination of gas exchange, leaves were randomly selected from M. sativa plants and measured with an infrared gas analyzer (IRGA) LI-6400 (LI-COR Biosciences, Lincoln, NE, USA) equipped with a Li-6,400-02B leaf light chamber. Measurements were made between 9 am and 2 pm under a photosynthetic photon flux density of 1,500 μmol m−2s−1, a vapor pressure deficit of 2–3 kPa, at a temperature of approximately 25°C and an environment of CO2 concentration of 400 μmol·mol−1 air. Gas exchange stabilization (120 s) was equilibrated, and measurements were recorded to determine the net photosynthetic rate (AN). In addition, a fluorometric analysis was performed to study the energy use efficiency of photosystem II (PSII). The maximum quantum efficiency of PSII photochemistry (Fv/Fm) and the quantum efficiency of PSII (ΦPSII) were determined using a Flores-Duarte et al. 10.3389/fmicb.2022.1005458 Frontiers in Microbiology 06 frontiersin.org saturation pulse method described in Genty et al. (1989). Selected leaves were light- and dark-adapted for 30 min, followed by a saturated actinic light pulse of 10,000 μmol m−2s−1 for 0.8 s at noon (1700 μmol photons m−2s−1) using a FMS-2 modulated fluorimeter (Hansatech Instruments Ltd., Pentney, UnitedKingdom). Details are described in Schreiber etal. (1986). With the recorded data, the electron transport rate (ETR) was calculated as described in Krall and Edwards (1992). Finally, the total content of chlorophyll described by Hiscox and Israelstam (1979) was determined. 50 mg of randomly selected leaves were ground using a mortar containing 100% acetone: 0.9% saline solution (4.1; v/v). The resulting extract was resuspended in acetone and measured at 652 nm using a spectrophotometer (Lambda 25; PerkinElmer, Walthmam, MA, United States) and the total chlorophyll content was determined using the formula described by Arnon (1949). Samples were run in duplicate. Antioxidant enzyme analysis Guaiacol peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzymatic activity were measured with leaves and roots randomly collected from each of the treatments at the end of the experiment. Immediately after the collection, the samples were deposited in nitrogen liquid and stored at −80°C until homogenized. 48 h after storage, 500 mg of plant material were homogenized in extraction buffer (50 mM sodium phosphate buffer at pH 7.6) and centrifuged at 4°C and 9,000 rpm for 20 min, and the plant extract was stored at −80°C. Total protein concentration was measured in the plant extract following the Bradford method (Bradford, 1976) using a BSA standard curve. In guaiacol peroxidase activity (GPX), guaiacol oxidation was measured at 470 nm. Catalase (CAT) was determined by measuring the disappearance of H 2 O 2 at 240 nm. Superoxide dismutase (SOD) activity was assayed using autoxidation of pyrogallol at 325 nm. Finally, for ascorbate peroxidase (APX), the oxidation of L-ascorbate was measured at 290 nm. For the determination of the autoxidation of substrates, control tests were carried out without the presence of the enzyme extract. Enzymatic activities were expressed as units per μg of protein and were measured in triplicate. Statistical analysis Statistical analyzes were determined using Statistical version 12.0 software (Statsoft Inc., Tulsa, OK, United States). The normality of the results was verified using the Kolmogorov Smirnov test. The results of each treatment were compared using one-way ANOVA, and Fisher test was performed to determine statistical differences. Results Isolation and characterization of endophytic bacteria from nodules Soil characterization Bacteria were isolated from nodules of Medicago spp. that grows naturally in the high marshes of the Odiel River. The soil was composed of 72% sand, 0.9% organic matter, had low electrical conductivity and nutrients content, as well as moderate amounts of As, Cd, Cu and Zn. The soil characteristics are presented in Table1. Isolation of endophytes A total of 33 endophytic bacteria were isolated from Medicago spp. nodules (13 strains grew in TY and 20in TSA). They were identified based on colony morphology (color and shape of the colonies), cellular morphology (cocci, bacilli, coccobacilli) and type of cell wall by Gram staining (Gram positive or Gram negative). Other aspects such as grouping, if any (clusters, chains), or the presence of spores (central or terminal) were also observed. 85% of the strains were Gram negative bacilli and 15% were Gram positive bacilli, and only one of them presented spores (Supplementary Table2). Subsequently, a Box-PCR of the 33 strains was performed in order to study the collection genotypically (Supplementary Figure1). Several of the isolates showed identical band profiles, so the study was reduced to 24 strains for which the 16S rRNA genes were partially sequenced. The sequences showed similarity with Achromobacter, Bacillus, Enterobacter, Ensifer, Lelliottia, Priestia and Pseudomonas genera, being Pseudomonas and Lelliottia the most represented (Table2). Characterization of PGP properties, enzymatic activities and metals tolerance PGP properties of the endophytes were determined, showing all of them four to five out of six analyzed properties, while two strains presented the six properties (Table 3; Supplementary Table3). All the bacteria were able to produce siderophores and most of them (30) solubilized phosphate (Supplementary Table3). N4 strain showed the highest production of siderophores according to the diameter of the halo in CAS medium (Table3; Supplementary Table3) and N15, N20, N30 and N31 were the best phosphate solubilizers (Supplementary Table3). A high number of strains were able to grow in N free medium (30 isolates) and/or produced auxins (27 isolates). N8 strain produced the highest concentration of IAA, close to 18 mg.L −1 (Table3; Supplementary Table3). 20 strains formed biofilm and 11 isolates showed ACC deaminase activity, presenting N8 strain the highest activity, close to 10 μmol α-ketobutyrate mg protein −1 h −1 (Table3; Supplementary Table3). The presence of enzymatic activities in the endophytes was studied (Supplementary Table4). Cellulase and protease activities were the most abundant, since they were found in 22 and 17 strains, respectively (Supplementary Table3). A low number of Flores-Duarte et al. 10.3389/fmicb.2022.1005458 Frontiers in Microbiology 07 frontiersin.org isolates (2 to 5) showed pectinase, lipase, chitinase or DNase activities (Supplementary Table4). Tolerance of the strains towards As, Cd, Cu, and Zn were determined and expressed as the maximum tolerable concentration (Table 4; Supplementary Table 5). Endophytes showed good levels of metal/loids tolerance. They tolerated concentrations as high as up to 20 mM As, 2 mM Cd, 6 mM Cu and Zn. TABLE2 Identification of cultivable entophytic bacteria isolated from nodules of Medicago spp. using the EzBiocloud database. Strain Sequence size (bp) Related species % ID Accession number N1 461 Achromobacter piechaudii 99.57 OP060607 N2 602 Lelliottia jeotgali 96.75 OP060608 N3 343 Lelliottia jeotgali 95.27 OP060609 N4 1,154 Pseudomonas mucoides 97.47 OP060610 N5 722 Lelliottia jeotgali 98.53 OP060611 N6 1,218 Achromobacter spanius 99.01 OP060612 N7 1,147 Lelliottia jeotgali 98.27 OP060613 N8 1,127 Pseudomonas mucoides 99.13 OP060614 N9 694 Lelliottia jeotgali 99.54 OP060615 N10 1,111 Ensifer meliloti 98.83 OP060616 N11 1,126 Pseudomonas mucoides 98.84 OP060617 N12 863 Ensifer meliloti 99.88 OP060618 N13 1,051 Lelliottia amnigena 97.71 OP060619 N14 865 Priestia megaterium 99.65 OP060620 N15 1,310 Pseudomonas lactis 97.10 OP060621 N16 1,035 Pseudomonas lactis 99.13 OP060622 N18 1,374 Bacillus velezensis 96.44 OP060623 N20 1,328 Pantoea vagans 95.41 OP060624 N21 678 Pseudomonas lactis 99.26 OP060625 N23 949 Enterobacter huaxiensis 96.63 OP060626 N25 1,257 Bacillus proteolyticus 95.83 OP060627 N30 1,314 Pseudomonas capeferrum 96.12 OP060628 N32 950 Bacillus tequilensis 99.58 OP060629 N33 1,209 Pseudomonas capeferrum 98.76 OP060630 Selected strains appear in bold. TABLE3 PGP properties and enzymatic activities showed by selected strains. PGP properties N4 N8 N10 N12 Phosphate solubilisation 11 12 15 14 Siderophores production 73 44 13 10.2 IAA production 1.562 17.939 1.266 1.00 Biofilm formation + + + + N fixation + + + + ACC deaminase activity 9.677 9.987 −− Enzymatic activities DNAse − − + + Amylase − − − − Cellulase −+ + + Lipase − − − − Pectinase − − − − Protease + + − − Chitinase − − − − N4: Pseudomonas sp. N4; N8: Pseudomonas sp. N8; N10: Ensifer sp. N10; N12: Ensifer sp. N12; +, presence of the activity; −, absence of the activity. Values of phosphate solubilisation and siderophores production express the diameter of the halo in mm. Values of IAA production are expressed in mg·L−1. Values of ACC deaminase activity are expressed in μmoles α-ketobutyrate·mg protein−1·h−1. Flores-Duarte et al. 10.3389/fmicb.2022.1005458 Frontiers in Microbiology 08 frontiersin.org AB CD FIGURE1 In vitro effects of inoculation of M. sativa with selected PGPNE. (A) Percentage of germinated seeds without metals and (B) with metals. Values are means ± S.D. (n = 50). (C) Number of nodules in plants without As and (D) with As. Values are means ± S.D. (n = 5). Different letters indicate statistical differences between means (One-way ANOVA, LSD test, p < 0.0001). C-: non inoculation; N4: inoculation with Pseudomonas sp. N4; N8: inoculation with Pseudomonas sp. N8; N10: inoculation with Ensifer sp. N10; N12: inoculation with Ensifer sp. N12; N4 + N10: co-inoculation with Pseudomonas sp. N4 and Ensifer sp. N10; N8 + N10: co-inoculation with Pseudomonas sp. N8 and Ensifer sp. N10; N4 + N12: co-inoculation with Pseudomonas sp. N4 and Ensifer sp. N12; N8 + N12: co-inoculation with Pseudomonas sp. N8 and Ensifer sp. N12; CSN: inoculation with Pseudomonas sp. N4, Pseudomonas sp. N8, Ensifer sp. N10 and Ensifer sp. N12. Selection of endophytes Among isolated endophytes, only N10 and N12 strains belonging to Ensifer genus were rhizobia able to nodulate M. sativa, so they were selected for assays in planta. N4 and N8 were selected as nodule enhancing bacteria (NEB) candidates based on their PGP properties and enzymatic activities, since they showed all the properties assayed and 2 enzymatic activities. They were also able to tolerate moderate levels of the assayed metal/ loids (Table4). In vitro effect of endophytes in Medicago sativa germination and nodulation The germination of M. sativa seeds was evaluated in the presence and absence of a mixture of metals/loids by inoculating them with the selected strains, performing individual inoculations (N4, N8, N10 and N12), co-inoculations combining each of the Pseudomonas and each of the rhizobia (N4 + N10, N8 + N10, N4 + N12, and N8 + N12) or inoculation with the four strains together (N4 + N8 + N10 + N12). Inoculation improved seed germination and the consortium with the four strains reported the greatest increase, showing the highest percentage of germination both in absence and presence of metals (Figures1A,B). The increase in the germination rate in absence or presence of metals, followed this pattern: CSN > N8 + N10 > N4 + N10 > N8 + N12 > N4 + N12 > N8 > N10 > N12 > N4 > C-. In absence of As, differences in germination among seeds inoculated with the four strains and those co-inoculated con Pseudomonas and Ensifer were not statistically significant (p < 0.001), but these germination rates were significantly higher than those recorded in seeds inoculated with a single strain. Seeds inoculated with single strains did not show significant differences in germination rates among them. The same results could beobserved in presence of As, with one single difference, since seeds inoculated with the consortium showed significantly higher germination rates than seeds co-inoculated with Pseudomonas TABLE4 Maximum tolerable concentration of metal/loid showed by selected strains. Strain Cd (mM) As (mM) Cu (mM) Zn (mM) N4 0.7 1 3 1.9 N8 0.4 2.5 1.9 1.8 N10 0.4 0.4 1.4 2.5 N12 0.1 0 1.8 1.4 N4: Pseudomonas sp. N4; N8: Pseudomonas sp. N8; N10: Ensifer sp. N10; N12: Ensifer sp. N12. Flores-Duarte et al. 10.3389/fmicb.2022.1005458 Frontiers in Microbiology 09 frontiersin.org and Ensifer (p < 0,001). Non-inoculated seeds showed the lowest rates of seed germination, with significant differences, both in absence and presence of As. These significant differences were recorded at the end of the experiment. To evaluate the ability of the Pseudomonas strains to improve M. sativa nodulation induced by Ensifer, plant seedlings were inoculated or co-inoculated with combinations of Pseudomonas and Ensifer strains in square plates with or without As. Co-inoculation with either of the Pseudomonas and a Ensifer strain significantly increased the number of nodules, both in absence and presence of As, compared with the single inoculation with Ensifer (Figures1D,E). The combination Pseudomonas sp. N8 and Ensifer sp. N10 induced more nodules than any other couple, although the plants inoculated with the combination of the four strains (CSN) showed the highest number of nodules in both conditions, with significant differences. Ensifer sp. N10 showed a better behavior in nodulation than N12 and Pseudomonas sp. N8 seemed to bebetter as nodule enhancing bacteria than N4. Pseudomonas behave as nodule endophytes in Medicago sativa Localization of Pseudomonas strains was studied using confocal laser scanning microscopy (CLSM) and mCherry-labeled Pseudomonas sp. N4 and N8. Cells were visualized from 0.5 mm sections of roots and nodules of M. sativa co-inoculated with Ensifer sp. N10 and labelled N4 or N8. Examples of the results obtained are presented in Figure 2. The presence of labelled bacteria with red fluorescence could beobserved both in root (Figures2A,C) and nodule cells (Figures2B,D). Groups of bacteria were clearly seen in the nitrogen fixation zone of M. sativa nodules, marked with a white square in the figures (Figures2B,D). Effect of inoculations under greenhouse conditions Inoculation increased plant biomass M. sativa plants were grown and inoculated under greenhouse conditions using soil from the marshes of the Odiel River (Huelva). Plant inoculation with any of the strains increased biomass, both shoots and roots, compared with non-inoculated control plants (Figure3A). Single inoculation with Pseudomonas sp. N8 reported higher plant shoot biomass than single inoculations with Ensifer or N4, that showed the lowest shoot and root biomasses among single inoculated plants. No significant differences in root biomass were found among plants inoculated with N8, N10 and N12. Co-inoculation with Pseudomonas-Ensifer couples produced higher values of root and shoot biomasses than any of the single inoculation treatments, except for plants inoculated with N4-N10 couple, that did not show significant differences in shoot biomass compared with plants inoculated with N8. N8 + N10 combination showed the highest value in plant shoot biomass among the Pseudomonas-Ensifer co-inoculation treatments, with significant differences (Figure3A). Nevertheless, AB CD FIGURE2 Bacterial colonization. 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