Stimulation of PGP Bacteria on the Development of Seeds, Plants and Cuttings of the Obligate Halophyte Arthrocaulon (Arthrocnemum) macrostachyum (Moric.) Piirainen & G. Kadereit

Citation: Barcia-Piedras, J.-M.; Pérez-Romero, J.-A.; Mateos-Naranjo, E.; Parra, R.; Rodríguez-Llorente, I.-D.; Camacho, M.; Redondo-Gómez, S. Stimulation of PGP Bacteria on the Development of Seeds, Plants and Cuttings of the Obligate Halophyte Arthrocaulon (Arthrocnemum) macrostachyum (Moric.) Piirainen & G. Kadereit. Plants 2023,12, 1436. https://doi.org/10.3390/plants 12071436 Academic Editor: Beatriz Ramos Solano Received: 20 February 2023 Revised: 14 March 2023 Accepted: 21 March 2023 Published: 24 March 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Stimulation of PGP Bacteria on the Development of Seeds, Plants and Cuttings of the Obligate Halophyte Arthrocaulon (Arthrocnemum) macrostachyum (Moric.) Piirainen & G. Kadereit José-María Barcia-Piedras 1,* , Jesús-Alberto Pérez-Romero 2, Enrique Mateos-Naranjo 3, Raquel Parra 3, Ignacio-David Rodríguez-Llorente 4, María Camacho 1and Susana Redondo-Gómez 3,* 1Centro Las Torres, Instituto de Investigación y Formación Agraria y Pesquera (IFAPA), Carretera, Sevilla-Cazalla de la Sierra Km 12.2, 41200 Alcaládel Río, Spain; [email protected] 2 Departamento de Biología, Instituto Universitario de Investigación Marina (INMAR), Universidad de Cádiz, 11510 Puerto Real, Spain 3Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes s/n, 41012 Sevilla, Spain 4Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain *Correspondence: [email protected] (J.-M.B.-P.); [email protected] (S.R.-G.) Abstract: The Earth is undergoing alterations at a high speed, which causes problems such as environmental pollution and difficulty in food production. This is where halophytes are interesting, due to their high potential in different fields, such as remediation of the environment and agriculture. For this reason, it is necessary to deepen the knowledge of the development of halophytes and how plant growth-promoting bacteria (PGP) can play a fundamental role in this process. Therefore, in this work were tested the effects of five PGP bacteria on its rhizosphere and other endophytic bacteria at different concentrations of NaCl on seed germination, plant growth (0 and 171 mM) and cutting growth (0 mM) of Arthrocaulon macrostachyum. The growth promotion in this strict halophyte is highlighted due to the presence of PGP bacteria and the fact that no salt is needed. Thus, without salt, the bacterial strains Kocuria polaris Hv16, Pseudarthrobacter psychrotolerans C58, and Rahnella aceris RTE9 enhanced the biomass production by more than 60% in both stems and roots. Furthermore, germination was encouraged by more than 30% in the presence of both R. aceris RTE9 and K. polaris Hv16 at 171 mM NaCl; the latter also had a biocontrol effect on the fungi that grew on the seeds. Additionally, for the first time in cuttings of this perennial species, the root biomass was improved thanks to the consortium of K. polaris Hv16 and P. psychrotolerans C58. Finally, this study demonstrates the potential of PGPs for optimising the development of halophytes, either for environmental or agronomic purposes. Keywords: Arthrocaulon (Arthrocnemum) macrostachyum; halophyte; salinity; inoculant; PGP; cutting 1. Introduction Halophytes are plants that are able to thrive in an environment with a concentration of NaCl greater than 200 mM [ 1 ]. In the past few decades, these plants have gained interest due to the potential that they present in different fields. In this way, thanks to their physiology, they can be used to desalinate soils degraded by excess salt [ 2 , 3 ], It has also been proven that they are effective phytoremediators against heavy metals [ 4 ]. In addition, halophytes are starting to be used as food for both livestock [5] and humans [6]. For these reasons, the ability of many halophytes to develop in both salt and salt-free conditions are well known [ 1 , 7 – 9 ]. Even multiplication by seeds is very well described for Plants 2023,12, 1436. https://doi.org/10.3390/plants12071436 https://www.mdpi.com/journal/plants Plants 2023,12, 1436 2 of 16 many of these halophilic species [ 10 , 11 ]. Despite this knowledge, no studies have been developed to optimise the propagation by asexual methods such as cuttings. This method of propagation is beneficial in the sense that plants are obtained faster than from seeds [12,13] . Furthermore, these plants are clones, thereby avoiding the variability that sexual reproduction introduces and could cause differences in the behaviour of the plant [14]. In addition, plants present a wide variety of relationships with other organisms. Among those interactions, those established with plant growth-promoting bacteria (or PGP bacteria) stand out from the rest due to their ability to enhance the plants’ conditions [ 15 – 17 ]. Specifically, there are many works that have used PGP bacteria to enhance the development of halophytes in different fields, such as for the restoration of saline ecosystems [ 18 ] or to increase the production of crops [ 19 ]. Among the bacterial properties that support plants are included those that provide nutrients, such as siderophore production for uptake iron [ 20 ] or phosphate solubilisation [ 21 ]. Other bacterial effects are related to the colonization of the root, e.g., acyl-homoserine lactone production [ 22 ] or the promotion of secondary roots via indole acetic acid production [23]. Arthrocaulon macrostachyum (Moric.) Piirainen & G. Kadereit was previously included in the genus Arthrocnemum as Arthrocnemum macrostachyum (Moric.) K. Koch, but the taxonomic nomenclature is a complex issue that is currently under evaluation [ 24 – 26 ]. This species is a perennial shoot succulent obligate halophyte that has been proposed to desalinate agricultural soils [ 27 ]; it is known that it is protected by its own endophytes at extreme concentrations of salt [ 28 ]. In this way, we hypothesised that PGP rhizobacteria isolated from different marsh plants could improve germination and growth, as well as improve the development of cuttings of A. macrostachyum. The propagation of A. macrostachyum has gained importance due to interest in the species as food [ 6 , 29 , 30 ] and for its nutritional and protective health benefits [ 31 ], in addition to its previously described bioremediation capacity. Thus, in this work, there were three proposed objectives: (a) to study the effect that different bacteria with PGP properties (especially auxin-producing bacteria) have on plant growth and the physiology of A. macrostachyum grown both at 0 mM and at 171 mM NaCl; (b) analyse the effect of inoculation on germination of A. macrostachyum in non-saline and saline conditions; and (c) determine the rooting capacity of cuttings of A. macrostachyum. 2. Materials and Methods 2.1. Isolation of Bacteria For bacterial isolates, soil samples of the rhizosphere of A. macrostachyum (5 cm deep) were collected from the Lebrija saltmarsh (36 ◦ 54 0 N–6 ◦ 12 0 W), characterised by having a loam-clay-sandy texture, a neutral pH, and an EC of 15 Sm −1 . The isolates were obtained after mixing 1 g of soil in 99 mL of mineral sales [ 32 ], homogenising, and making serial decimal dilutions up to 10 −5 –10 −6 , which were spread on the surface in plates containing Plate Count Agar (PCA) (Difco ® , Madrid, Spain) and 50% supplemented with cycloheximide (100 mg L −1 , Sigma-Aldrich ® , St. Louis, MO, USA) to prevent fungal growth. The same procedure was performed to add NaCl to the medium to a final concentration of 300 mM. The plates were incubated at 28 ◦ C for 24–48 h until the appearance of individual colonies. After isolating colonies, the plates were incubated for an additional 72 h, and the slow-growing colonies were isolated. The purified strains were stored in cryogenic tubes with a mixture of peptone (0.5%) and glycerol (15%) at − 80 ◦ C. The strain RTE9, which was loaned from the collection of the Microbiology Department of the University of Seville, was isolated from the interior of an undetermined plant from the Rio Tinto saltmarsh. The origin of the bacterial strains used in the study is described in Table 1. Plants 2023,12, 1436 3 of 16 Table 1. Strains and environments from which the bacteria have been isolated and closest species to bacteria used in this work based on 16S rRNA partial sequences. Strain Environment Related Species Identity (%) Accession Number Hv16 Rhizosphere of A. macrostachyum from Guadalquivir River saltmarsh. Kocuria polaris 98.6 LN794848 Hvs13 Rhizosphere of A. macrostachyum from Guadalquivir River saltmarsh (isolated at 350 mM NaCl). Brevibacterium frigoritolerans 100 OU596762 Hvs76 Rhizosphere of A. macrostachyum from Guadalquivir River saltmarsh (isolated at 350 mM NaCl). Pseudarthrobacter siccitolerans 99.4 OU596760 C58 Rhizosphere of A. macrostachyum from an irrigation canal close to Guadalquivir River saltmarsh. Pseudarthrobacter psychrotolerans 99.3 OU596763 C59 Rhizosphere of A. macrostachyum from an irrigation canal close to Guadalquivir River saltmarsh. Ensifer meliloti 99.9 OU596759 RTE9 Interior of an indeterminate plant of the Tinto River saltmarsh. Rahnella aceris 99.3 OU596761 For the inoculation in the different tests, each frozen strain was spread on nutrient agar plates (NA) composed of nutrient broth (NB) (Scharlau ® , Barcelona, Spain) with 16 g L−1 agar bacteriological (Difco ® , Madrid, Spain) and grown at 28 ◦ C for 48 h. For strains isolated in salt, NaCl was added to the culture medium to a final concentration of 300 mM. Next, a colony was taken and inoculated into tubes with 5 mL of NB that were incubated for 72 h at 28 ◦ C with continuous agitation (180 rpm), obtaining a final bacteria concentration of 109mL−1. For the inoculation of plant material (seed or plant), the bacterial cultures were centrifuged at 12,000 rpm for 5 min, and the cells were resuspended in 0.03 M magnesium sulphate buffer, until a bacteria concentration of 108mL−1was achieved. 2.2. Molecular Characterisation of Bacteria: Amplification, Sequencing, and Phylogenetic Analysis of 16S rDNA For the amplification of 16S rDNA, this work made use of the method described by Camacho et al. [ 33 ], in which 27F and 1492R primers were used [ 34 ]. The PCR products were purified with the Favorgen GEL/PCR Purification Kit (Biotech Corp ® , Doral, FL, USA) and sequenced at the Sequencing Service of the University of Alcaláde Henares. To compare similarities with known sequences, the EzBioCloud database (https://www.ezbiocloud.net (accessed on 19 February 2023)) was used [35] (Table 1). 2.3. Bacterial Tolerance to High Salinity For each strain, 10 µL of bacterial culture was placed on an NA plate with increasing concentrations of NaCl, between 0–25% (with 1% increments). Then, they were incubated for one week at 28 ◦C (n= 3). 2.4. Bacterial Tolerance to High Temperature For each strain, 10 µ L of bacterial culture was placed on an NA plate and incubated for two to seven days at 40 ◦C (n= 3). 2.5. Indole Acetic Acid Production (IAA) To determine the IAA produced by the strains, the colorimetric method described by [ 36 ] was followed. The bacteria grew as described previously, adding 100 mg L −1 of L-Tyr to the NB. After growing, the cultures were centrifuged at 12,000 rpm for 5 min, Plants 2023,12, 1436 4 of 16 and the supernatant was used to quantify the IAA concentration, adding the Salkowski reagent in a 1:4 (v:v). After 20 min at room temperature, the absorbance of the mixture was quantified (iMark Microplate Absorbance Reader, Bio-Rad ® , Hercules, CA, USA) at 535 nm (n= 3), and the results were extrapolated with a standard line made from a stock of IAA (Panreac ® , Barcelona, Spain) of 200 µ g g −1 dissolved in 10% ethanol (serial dilutions between 0 and 200 µg g−1with NB). 2.6. Acyl-Homoserine Lactone Production (AHL) The bacteria grew as previously described and was centrifuged to determine AHL production. Then, 100 µL of supernatant was used for a colorimetric assay (n= 3) [37]. 2.7. Siderophore Production For each bacterium, 10 µ L was added on a plate of CAS medium [ 38 ], which was incubated for 78 h at 28 ◦ C. Subsequently, the production of siderophores was quantified by measuring the diameter of the hydrolytic halo formed (n= 3). 2.8. Phosphate Solubilisation For phosphate solubilisation, 10 µ L of bacterial cultures was placed in a plate of PVK medium [ 39 ] and kept at 28 ◦ C for five days. Then, the activity was assessed according to the generated hydrolytic halo (n= 3). 2.9. Vegetal Material In this research, commercial seeds of rapeseed (Brassica napus L.) were used. In addition, seeds of A. macrostachyum were recollected from the saltmarsh of the Odiel River, SW of the Iberian Peninsula (37 ◦ 13 0 4.4” N–6 ◦ 57 0 35.3” W) in mid-September of 2014 for experiments 1 and 2, and 2015 for experiment 3. Once the seeds were collected, they were kept at 5 ◦C until use. For the disinfection of seeds of B. napus, the protocol specified by [ 40 ] was followed, and for A. macrostachyum, the seeds were immersed 30 s in 96% H 2 SO 4 , followed by 7 washes with sterile water, sodium hypochlorite 5% for 3 min, then finally another 7 washes with sterile water. 2.10. Inoculation A solution of MgSO 4 0.03 M was used as an inoculation control. For the seeds of B. napus and A. macrostachyum, the bacteria were cultivated as described previously (see the Indole Acetic Acid Production section). Then, the seeds of both species were inoculated as described by Patten and Glick [36]. In experiment 1, both seedlings and three-month-old plants of A. macrostachyum were inoculated with 200 µL and 1 mL of bacterial suspension (108cells ml−1), respectively. For the cuttings of A. macrostachyum (experiment 3), they were immersed in 100 mL of bacterial suspension (108cell ml−1) for one hour immediately after they had been cut. Throughout the experimental period, seedlings, plants, and cuttings from experiments 1 and 3 were reinoculated once per month (ensuring the presence of bacterial strain). 2.11. Rapeseed Root Elongation Test To select the strains that were applied in experiment 1, a rapeseed test was previously performed (Figure 1). This species is sensitive to IAA changes, which are reflected in the elongation of roots [ 41 ]. Therefore, after inoculating 40 seeds of B. napus for each treatment, they were distributed into four sterile growth bags (DIK-710A, Daiki ® , Kounosu, Japan) containing 12 mL Hewitt solution 0.5 × [ 42 ]. The bags were placed in a growth chamber (Sanyo Electric ® , Osaka, Japan) with a 12 h light and 12 h dark cycle, illuminated by a light source ( λ = 400–700 nm) with a flux density of 250 µ mol m −2 s −1 , and 80% relative humidity for seven days. After this period, the length of the radicle was measured. Plants 2023,12, 1436 5 of 16 Plants 2023, 12, x FOR PEER REVIEW 5 of 17 treatment, they were distributed into four sterile growth bags (DIK-710A, Daiki®, Kounosu, Japan) containing 12 mL Hewitt solution 0.5× [42]. The bags were placed in a growth chamber (Sanyo Electric®, Osaka, Japan) with a 12 h light and 12 h dark cycle, illuminated by a light source ( = 400–700 nm) with a flux density of 250 mol m−2 s−1, and 80% relative humidity for seven days. After this period, the length of the radicle was measured. Figure 1. Organization chart of experimental design indicating the order and correlations between the tests and experiments carried out in this research. 2.12. Experiment 1: Influence of Bacterial Strains and Salt on Plant of A. macrostachyum The strains selected in the rapeseed root elongation test were used to inoculate the seeds of A. macrostachyum (Figure 1). One hundred inoculated seeds per treatment were placed in 4 Petri dishes with 1% agar-water. After this, the plates were kept in a growth chamber (Sanyo Electric®, Osaka, Japan) with a cycle of 10 h of light at 20 °C and 14 h of darkness at 5 °C for one month. Then, the seedlings were transplanted into perforated seedbeds with a substrate composed of 2:1 perlite-vermiculite (v/v). In addition, the seedbeds were placed in trays that were filled with Hewitt 0.5× solution to a depth of 1 cm. Previously, all materials were sterilised by moist heat sterilisation (60 min at 121 °C). The seedbeds were placed in the greenhouse of the IFAPA Center Las Torres in Seville (37°30′42.4″ N–5°57′47.4″ W) with a cycle of 16 h of light at 30 °C and 8 h of darkness at 20 °C for three months. One day after transplanting, the seedlings were inoculated. Next, the length, central diameter, and surface area of the main branch and the number of secondary branches were quantified (n = 20). Then, plants were placed in sterile Leonard jars with a 2:1 perlite–vermiculite (v/v) ratio. In order to determine the effect of the presence of salt in A. macrostachyum with respect to the inoculated strains, 10 plants used 0.2 L of Hewitt 0.5× nutrient solution as the liquid phase; in 10 other plants, the same nutrient solution was enriched with 171 mM NaCl (keeping both the volume and concentration of the nutrient solution constant). The plants grew for three months under identical environmental conditions. Reinoculation was performed one day after the transplant. 2.13. Experiment 2: Effects of Bacterial Inoculation and Salt on the Germination of A. macrostachyum With the bacterial treatments from experiment 1 that promoted the most growth of A. macrostachyum, a new germination test was performed (Figure 1). This germination experiment was performed in the same manner as experiment 1, except for the number of seeds (fifty seeds were germinated without salt and another fifty were germinated with a salt concentration of 171 mM per treatment). The germinations were recorded daily to calculate the final germination percentage, number of days until the first and last germination, and mean time to germinate (MTG) [43]. Lower MTG values indicate faster germination. Furthermore, to determine whether the bacterial treatment could inhibit the growth of fungi associated with seeds, counts of the apparent colony forming units (CFUs) on the plates were made at the end of the experiment. 2.14. Experiment 3: Influence of Bacterial Inoculation on Cutting of A. macrostachyum Figure 1. Organization chart of experimental design indicating the order and correlations between the tests and experiments carried out in this research. 2.12. Experiment 1: Influence of Bacterial Strains and Salt on Plant of A. macrostachyum The strains selected in the rapeseed root elongation test were used to inoculate the seeds of A. macrostachyum (Figure 1). One hundred inoculated seeds per treatment were placed in 4 Petri dishes with 1% agar-water. After this, the plates were kept in a growth chamber (Sanyo Electric ® , Osaka, Japan) with a cycle of 10 h of light at 20 ◦ C and 14 h of darkness at 5 ◦C for one month. Then, the seedlings were transplanted into perforated seedbeds with a substrate composed of 2:1 perlite-vermiculite (v/v). In addition, the seedbeds were placed in trays that were filled with Hewitt 0.5 × solution to a depth of 1 cm. Previously, all materials were sterilised by moist heat sterilisation (60 min at 121 ◦ C). The seedbeds were placed in the greenhouse of the IFAPA Center Las Torres in Seville (37 ◦ 30 0 42.4 00 N–5 ◦ 57 0 47.4 00 W) with a cycle of 16 h of light at 30 ◦C and 8 h of darkness at 20 ◦C for three months. One day after transplanting, the seedlings were inoculated. Next, the length, central diameter, and surface area of the main branch and the number of secondary branches were quantified (n= 20). Then, plants were placed in sterile Leonard jars with a 2:1 perlite–vermiculite (v/v) ratio. In order to determine the effect of the presence of salt in A. macrostachyum with respect to the inoculated strains, 10 plants used 0.2 L of Hewitt 0.5 × nutrient solution as the liquid phase; in 10 other plants, the same nutrient solution was enriched with 171 mM NaCl (keeping both the volume and concentration of the nutrient solution constant). The plants grew for three months under identical environmental conditions. Reinoculation was performed one day after the transplant. 2.13. Experiment 2: Effects of Bacterial Inoculation and Salt on the Germination of A. macrostachyum With the bacterial treatments from experiment 1 that promoted the most growth of A. macrostachyum, a new germination test was performed (Figure 1). This germination experiment was performed in the same manner as experiment 1, except for the number of seeds (fifty seeds were germinated without salt and another fifty were germinated with a salt concentration of 171 mM per treatment). The germinations were recorded daily to calculate the final germination percentage, number of days until the first and last germination, and mean time to germinate (MTG) [ 43 ]. Lower MTG values indicate faster germination. Furthermore, to determine whether the bacterial treatment could inhibit the growth of fungi associated with seeds, counts of the apparent colony forming units (CFUs) on the plates were made at the end of the experiment. 2.14. Experiment 3: Influence of Bacterial Inoculation on Cutting of A. macrostachyum Cuttings of A. macrostachyum were obtained by sectioning the first 5 cm of the new tertiary branches from a 2-year-old plant grown in a greenhouse with Hoagland solution [ 44 ] 1% NaCl (Figure 1). In total, 200 cuttings were obtained (40 per treatment and 40 cuttings for the initial measurements). After inoculation, each cutting was placed 2 cm deep in a perforated seedbed filled with coconut fibre and perlite 5:1 (v/v). The seedbeds were placed in trays (n= 4) that were kept with a constant volume of Hoagland solution. The experiment was carried out in the greenhouse of the University of Seville ( 37◦21032.6” N–5◦59013.4” W ) at 21–25 ◦ C, a relative humidity of 40–60%, and 16 h of natural light ( 250 µmol m−2s−1 minimum and 1000 µmol m−2s−1maximum) for 60 days. Plants 2023,12, 1436 6 of 16 2.15. Gas Exchange Experiment 1: Just before harvesting the plants, photosynthetic gas exchange was measured (n= 5) using an infrared gas analyser (IRGA) in an open system (Lci portable photosynthesis, ADC ® , UK) [ 45 ]. The environmental parameters were: 30–35 ◦ C, 45% relative humidity, atmospheric CO 2 concentration 400 µ mol mol −1 , and photon flux density 1000 µmol m−2s−1. Experiment 3: Prior to harvest, gas exchange was measured in the same way as experiment 1, except that seven replicates per treatment were made with IRGA LI-6400 (LI-COR®, Lincoln, NE, USA). Additionally, the photosynthetic light had a 15% blue light (to maximise stomatal aperture), a vapour pressure deficit of 1.5 ± 0.1 kPa, and an air temperature of 24 ±0.5 ◦C. In both experiments, due to the branch of A. macrostachyum being cylindrical, the photosynthetic surface was considered to be half of the total photosynthetic surface of the branch and the photosynthetic parameters were calculated as described [45]. 2.16. Plant Growth Experiment 1: At the end of the experimental period, the plants were harvested, and the aerial parts and root were weighed (fresh weight). Subsequently, they were dried in an oven (at 60 ◦ C for 48 h), and they were weighed again (dry weight) to calculate the water content. Experiment 3: At the beginning of the assay, measurements of the length and diameter of all the cuttings were taken. Forty of them were dried and weighed (initial dry weight) as described in experiment 1. After two months, the number of alive cuttings, length and diameter of branches, and the fresh weight of both branches and roots were measured. Then, the cuttings were placed in plastic slider zip bags with distilled water at 5 ◦ C in the dark. After 24 h, the surfaces of the plants were dried and weighed to determine the turgid weight. Finally, they were dried to confirm the dry weight of both the branches and the roots. With these data, the relative water content (RWC) [ 46 ] and the relative growth rate (RGR) [47] were calculated. 2.17. Statistics Statistical analyses were developed using SPSS 21. The results correspond to the mean ± standard error (S.E.). For continuous variables, normality was tested with the Kolmogorov–Smirnov test and the Brown–Forsythe test to verify homoscedasticity. In order to test for differences, the Kruskal–Wallis test was used for the discrete variables, and an ANOVA was used for the continuous variables. For post-hoc tests, the Mann–Whitney U and Fisher’s LSD test were used, both with a significance threshold of p< 0.05. 3. Results 3.1. Bacterial Properties Regarding the physical properties, all of the strains grew in at least 1% NaCl, and the strain that withstood the highest salinity was Hv16 at 9% NaCl. Only Hvs13 had sufficient tolerance to grow at 40 ◦C (Table 2). Plants 2023,12, 1436 7 of 16 Table 2. Quantification of the properties of the bacterial strains: salinity range (SAL), tolerance to grow at 40 ◦ C (TEM), indole acetic acid production (IAA), relative acyl-homoserine lactone production (AHL), siderophore production (SID), and phosphate solubilization (PHO) (n= 3). Strain SAL (%) TEM IAA (µg g−1) AHL (%) SID (mm) PHO (mm) Hv16 0–9 −200 50 20 − Hvs13 0–6 + 50 75 10 − Hvs76 0–4 −150 100 10 − C58 0–4 −150 25 10 − C59 0–1 −100 50 - − RTE9 0–4 −150 0 - 30 In relation to PGP properties, the only property present in all strains was the production of IAA, with Hv16 producing the highest amount (Table 2). AHL production was only present in strains Hv16 and Hvs76, the former being the largest producer of AHL (Table 2). The siderophore production occurred in all strains except C59 and RTE9, and Hv16 repeated producing more siderophores than any other strain (Table 2). Finally, the phosphate solubilization was only present in RTE9 (Table 2). 3.2. Rapeseed Root Elongation Test The major root elongation values obtained for the treatments inoculated with C58 and Hv16 and were found to be approximately 30% longer than the non-inoculated control (Fisher’s LSD test, p< 0.05). In addition, the treatments C59 and RTE9 were also selected, which presented a greater root length than the control (14 and 19%, respectively), although they did not show significant statistical effects (Figure 2). Plants 2023, 12, x FOR PEER REVIEW 7 of 17 Table 2. Quantification of the properties of the bacterial strains: salinity range (SAL), tolerance to grow at 40 °C (TEM), indole acetic acid production (IAA), relative acyl-homoserine lactone production (AHL), siderophore production (SID), and phosphate solubilization (PHO) (n = 3). Strain SAL (%) TEM IAA (μg g−1) AHL (%) SID (mm) PHO (mm) Hv16 0–9 − 200 50 20 − Hvs13 0–6 + 50 75 10 − Hvs76 0–4 − 150 100 10 − C58 0–4 − 150 25 10 − C59 0–1 − 100 50 - − RTE9 0–4 − 150 0 - 30 In relation to PGP properties, the only property present in all strains was the production of IAA, with Hv16 producing the highest amount (Table 2). AHL production was only present in strains Hv16 and Hvs76, the former being the largest producer of AHL (Table 2). The siderophore production occurred in all strains except C59 and RTE9, and Hv16 repeated producing more siderophores than any other strain (Table 2). Finally, the phosphate solubilization was only present in RTE9 (Table 2). 3.2. Rapeseed Root Elongation Test The major root elongation values obtained for the treatments inoculated with C58 and Hv16 and were found to be approximately 30% longer than the non-inoculated control (Fisher’s LSD test, p < 0.05). In addition, the treatments C59 and RTE9 were also selected, which presented a greater root length than the control (14 and 19%, respectively), although they did not show significant statistical effects (Figure 2). Treatments non-inoc. Hv16 Hvs13 Hvs76 C58 C59 RTE9 Root length of rapeseed 0 1 2 3 4 5 6 7 8 9 10 11 bc a cc a ab ab Figure 2. Radicle length in rapeseed seedlings (Brassica napus L.) inoculated with six different bacteria, after 7 days. Values correspond to the mean ± S.E. (n = 4). Different letters indicate that the mean values are significantly different from each other (Fisher’s LSD test, p < 0.05). 3.3. Influence of Bacterial Strains and Salt on Plant of A. macrostachyum 3.3.1. Plant Growth After the first three months of growth of the seedlings of A. macrostachyum, it was observed that the branch length of the treatments Hv16 and RTE9 were 32% and 23% longer than the control, respectively. A similar trend was observed on the surface area. Figure 2. Radicle length in rapeseed seedlings (Brassica napus L.) inoculated with six different bacteria, after 7 days. Values correspond to the mean ± S.E. (n= 4). Different letters indicate that the mean values are significantly different from each other (Fisher’s LSD test, p< 0.05). 3.3. Influence of Bacterial Strains and Salt on Plant of A. macrostachyum 3.3.1. Plant Growth After the first three months of growth of the seedlings of A. macrostachyum, it was observed that the branch length of the treatments Hv16 and RTE9 were 32% and 23% longer than the control, respectively. A similar trend was observed on the surface area. Furthermore, regarding the number of secondary branches, the Hv16 treatment accelerated the branching of the seedlings. However, no significant difference was observed for the diameters (Table 3). At the end of experiment 1, strains Hv16, C58, and RTE9 promoted an Plants 2023,12, 1436 8 of 16 increase in both aboveand below-ground biomasses of more than 60% compared to the non-inoculated treatment (Fisher’s LSD test; p< 0.05) in the absence of salt (Table 4). In the treatments at 171 mM NaCl, growth stimulation was observed exclusively at the radical level, with the Hv16, C59, and RTE9 treatments presenting at least 50% more biomass than the non-inoculated treatment (Fisher’s LSD test, p< 0.05) (Table 4). On the other hand, no treatment increased the water content of the plants. Table 3. Length, diameter, surface area of main branch, and number of secondary branches of seedlings of A. macrostachyum inoculated with the different bacteria after 90 days. Values correspond to mean ± S.E. (n= 20). Different letters indicate that the mean values are significantly different from each other (Fisher’s LSD test for all variables except number of secondary branches, for which Mann–Whitney U test was used, both p< 0.05). Treatment Branch Length (cm) Branch Diameter (mm) Branch Surface Area (cm2) Secondary Branches Non-inoculated 2.2 ±0.2 b1.7 ±0.1 ab 1.2 ±0.1 c0±0c Hv16 2.9 ±0.2 a1.9 ±0.1 a1.7 ±0.1 a2±0a C58 2.1 ±0.2 bc 1.9 ±0.1 a1.4 ±0.1 ab 1±0bc C59 2.0 ±0.2 c1.6 ±0.1 bc 1.1 ±0.1 c1±0bc RTE9 2.7 ±0.2 a1.8 ±0.1 a1.7 ±0.2 a1±0bc Table 4. Dry weight of aerial part and roots and water content of aerial part of A. macrostachyum grown at 0 and 171 mM NaCl with and without inoculation with three different bacterial strains after 180 days. Values correspond to the mean ± S.E. (n= 10). Different letters indicate that the mean values are significantly different from each other (Fisher’s LSD test, p< 0.05). Treatments 0 and 171 mM are analysed independently. 0 mM NaCl Treatment Aerial Dried Weight (g) Root Dried Weight (g) Water Content (%) Non-inoculated 0.27 ±0.10 c0.19 ±0.06 c84.1 ±1.8 ab Hv16 0.59 ±0.12 a0.73 ±0.18 a85.3 ±0. 8 a C58 0.45 ±0.09 ab 0.81 ±0.23 a78.9 ±1.2 c C59 0.34 ±0.07 abc 0.40 ±0.11 bc 80.4 ±0.7 bc RTE9 0.57 ±0.13 ab 0.67 ±0.19 ab 81.4 ±0.5 abc 171 mM NaCl Non-inoculated 1.25 ±0.27 ab 0.75 ±0.18 c87.6 ±0.4 ab Hv16 1.66 ±0.30 a1.14 ±0.15 ab 88.0 ±0.2 ab C58 0.89 ±0.15 b0.77 ±0.13 bc 88.6 ±0.9 ab C59 1.84 ±0.32 a1.21 ±0.19 ab 87.4 ±0.6 b RTE9 1.93 ±0.23 a1.28 ±0.13 a87.7 ±0.5 ab 3.3.2. Gas Exchange At a concentration of 0 mM, all inoculated treatments showed higher values than the control in g s , and the Hv16 and RTE9 treatments also presented higher values in A and C i (Fisher’s LSD test, p< 0.05). Conversely, there were no differences at 171 mM NaCl (Fisher’s LSD test, p< 0.05). Regarding the iWUE, only the RTE9 171 mM treatment had an efficiency greater than 60% in comparison to the non-inoculated treatment (Fisher’s LSD test, p< 0.05) (Table 5). Plants 2023,12, 1436 9 of 16 Table 5. Net photosynthetic (A) and stomatal conductance (g s ) rates, intercellular CO 2 concentration (C i ) and intrinsic water use efficiency ( i WUE) of A. macrostachyum grown at 0 and 171 mM NaCl, with and without inoculation with four different bacterial strains after 180 days. Values correspond to the mean ± S.E. (n= 5). Different letters indicate that the mean values are significantly different from each other (Fisher’s LSD test, p< 0.05). Treatments 0 and 171 mM are analysed independently. Treatment 0 mM NaCl A (µmol m−2s−1) gs(mmol m−2s−1) Ci(µmol mol−1)iWUE (mmol mol−1) No-inoculated 7.5 ±1.4 c90 ±20 c215 ±20 bc 85 ±12 Hv16 14.1 ±1.0 a240 ±30 a250 ±6a70 ±14 C58 8.6 ±1.7 bc 120 ±40 b215 ±17 bc 82 ±12 C59 5.8 ±1.7 c90 ±20 c224 ±19 ab 78 ±12 RTE9 10.9 ±1.6 ab 150 ±30 b250 ±10 a76 ±16 171 mM NaCl No-inoculated 7.3 ±0.7 140 ±20 275 ±7a53 ±4b Hv16 5.8 ±1.1 100 ±30 252 ±9ab 61 ±7ab C58 6.8 ±1.2 90 ±20 232 ±19 ab 86 ±15 ab C59 5.5 ±0.6 90 ±10 246 ±19 ab 69 ±13 ab RTE9 5.6 ±0.9 90 ±40 215 ±13 b86 ±1a 3.4. Effects of Bacterial Inoculation and Salt on the Germination of A. macrostachyum In the treatments without salt, no germination parameters were affected by inoculation (Figure 3A and Table 6) (Fisher’s LSD test, p< 0.05). On the contrary, in presence of salt, the non-inoculated treatment had a deceleration in the curve of germinated seeds from the fifth day (Table 6). Meanwhile, the Hv16 and RTE9 treatments did not show that delay (Figure 3A) and, furthermore, reached the highest percentages of germinated seeds (Table 6). Plants 2023, 12, x FOR PEER REVIEW 10 of 17 Time (day) 0 2 4 6 8 10 12 14 Germination (%) 0 20 40 60 80 100 no-inoc. 0 mM Hv16 0 mM C58 0 mM RTE9 0 mM A Time (day) 0246810 12 14 Germination (%) 0 20 40 60 80 100 no-inoc. 171 mM Hv16 171 mM C58 171 mM RTE9 171 mM B * * * * * * *** * Figure 3. Cumulative germination percentage of A. macrostachyum grown at 0 (A) and 171 (B) mM NaCl non-inoculated (), or inoculated with Hv16 (), C58 () and RTE9 () bacterial strains over 14 days. Values correspond to the mean ± S.E. (n = 4). Asterisks indicate that there were significant differences between mean values are for that day (Mann–Whitney U test; p < 0.05). Treatments 0 and 171 mM were analysed independently. 3.5. Influence of Bacterial Inoculation on Cutting of A. macrostachyum 3.5.1. Plant Growth No significant differences were observed in the branches of cuttings, although a trend was observed in which the inoculated cuttings were slightly longer than the non-inoculated cuttings (Table 7). On the other hand, focusing on radical growth, it was observed that strain Hv16 promoted 20% more radical elongation than the control treatment. Furthermore, this treatment together with C58 stimulated the production of roots, so that the dry weight of both was about 20% higher than that of the treatment without inoculation (Table 7). Table 7. Length and dry weight of branch and roots, relative water content (RWC), and relative growth rate (RGR) of cuttings of A. macrostachyum inoculated with three different bacteria after 60 days. Values correspond to the mean ± S.E. (n = 4). Different letters indicate that the mean values are significantly different from each other (Fisher’s LSD test, p < 0.05). Branch Root Treatment Length (cm) Dry Weight (mg) RWC (%) RGR (mg g−1 day−1) Length (cm) Dry Weight (mg) Non-inoculated 82 ± 6 43 ± 3 89 ± 2 a 25 ± 3 44 ± 4 b 8.6 ± 0.6 b Hv16 88 ± 5 45 ± 3 79 ± 2 b 25 ± 3 52 ± 4 a 10.4 ± 0.6 a C58 84 ± 6 38 ± 3 87 ± 2 a 21 ± 3 48 ± 5 ab 10.4 ± 0.6 a RTE9 80 ± 5 39 ± 3 72 ± 2 c 27 ± 2 47 ± 5 ab 8.8 ± 0.7 ab Figure 3. Cumulative germination percentage of A. macrostachyum grown at 0 ( A ) and 171 ( B ) mM NaCl non-inoculated (o), or inoculated with Hv16 (  ), C58 ( ∇ ) and RTE9 ( ∆ ) bacterial strains over 14 days. Values correspond to the mean ± S.E. (n= 4). Asterisks indicate that there were significant differences between mean values are for that day (Mann–Whitney U test; p< 0.05). Treatments 0 and 171 mM were analysed independently. Plants 2023,12, 1436 16 of 16 59. Ramírez, E.; Chaâbene, Z.; Hernández-Apaolaza, L.; Rekik, M.; Elleuch, A.; de la Fuente, V. Seed priming to optimize germination in Arthrocnemum Moq. BMC Plant Biol. 2022,22, 527. 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