Isolation of pseudomonas strains with potential for protection of soybean plants against saline stress

agronomy Article Isolation of Pseudomonas Strains with Potential for Protection of Soybean Plants against Saline Stress Stefanie Bernardette Costa-Gutierrez 1,† , María Carolina del Valle Caram-Di Santo 1, Ana María Zenoff 1, Manuel Espinosa-Urgel 2, Ricardo Ezequiel de Cristóbal 1,* and Paula Andrea Vincent 1,*   Citation: Costa-Gutierrez, S.B.; Caram-Di Santo, M.C.d.V.; Zenoff, A.M.; Espinosa-Urgel, M.; de Cristóbal, R.E.; Vincent, P.A. Isolation of Pseudomonas Strains with Potential for Protection of Soybean Plants against Saline Stress. Agronomy 2021, 11, 2236. https://doi.org/10.3390/ agronomy11112236 Academic Editors: Othmane Merah, Purushothaman Chirakkuzhyil Abhilash, Magdi T. Abdelhamid, Hailin Zhang and Bachar ZEBIB Received: 25 August 2021 Accepted: 12 October 2021 Published: 4 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 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/). 1Facultad de Bioquímica, Química y Farmacia, Instituto Superior de Investigaciones Biológicas (INSIBIO, CONICET-UNT) e Instituto de Química Biológica “Dr. BernabéBloj”, Universidad Nacional de Tucumán, Chacabuco 461, San Miguel de Tucumán 4000, Tucumán, Argentina; [email protected] (S.B.C.-G.); [email protected] (M.C.d.V.C.-D.S.); [email protected] (A.M.Z.) 2Department of Environmental Protection, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, E-18008 Granada, Spain; [email protected] *Correspondence: [email protected] (R.E.d.C.); [email protected] (P.A.V.) † Stefanie Bernardette Costa-Gutierrez Present address: Planta Piloto de Procesos Industriales Microbiológicos (PROIMI-CONICET), Avenida Belgrano y Pasaje Caseros, San Miguel de Tucumán 4000, Tucumán, Argentina. Abstract: Salinity is a major detrimental factor for plant growth and crop productivity that could be alleviated by the use of plant growth promoting bacteria (PGPB) with a protective role in such stressful conditions. In this study, four native strains of the genus Pseudomonas were isolated from both a strongly saline soil and the rhizosphere of soybean plants grown in a slightly saline soil. These isolates were able to tolerate high NaCl concentration, showed efficient adhesion to biotic and abiotic surfaces and efficiently colonized the rhizosphere of soybean grown in slightly saline soil. In these conditions, the four strains outperformed Pseudomonas putida KT2440, a strain known as a good root colonizer of different plants. Inoculation with all the isolates improved seed germination and vigor index, particularly in saline conditions, and one of them also had a positive effect on shoot length and phenological state of soybean plants grown in slightly saline soil. Our results suggest that the search for classical plant growth promotion traits may not be mandatory for selecting putative PGPB. Instead, characteristics such as stress tolerance, adhesion, competitive colonization, and plant growth promotion should be tested using the soil types and crops in which the bacteria will be used. Keywords: plant-growth-promoting bacteria; salinity; rhizosphere; sustainable agriculture 1. Introduction Soybean (Glycine max (L.) Merrill) is the most widely produced and consumed oilseed crop worldwide. Soybeans and their by-products are used in human and livestock feed. The main constraint to its cultivation is drought, which is closely related to soil salinity; seedlings are considered a salt-sensitive to moderately salt-tolerant crop [ 1 ]. Salinity stress has detrimental effects on soybean, such as inhibition of seed germination, reduction of growth, nodulation, biomass accumulation, and yield [ 2 , 3 ]. Soil salinization, often caused by poor land management, is one of the main problems affecting crop productivity and is expected to cause the loss of almost 50% of the total agricultural land area by 2050 [ 4 , 5 ]. In a context of climate change, this situation, coupled with population increase, makes guaranteeing global food supply one of the greatest challenges to face in the near future [ 6 ]. In addition to education and training efforts to ensure proper land management by producers, alternatives strategies are required for improving crop productivity in salinity-affected lands. In 1978, Kloepper and Schroth popularized the term PGPR (plant growth-promoting rhizobacteria) to describe root-associated and rhizosphere bacteria capable of acting as biofertilizers or biocontrol agents [ 7 ]. Since not only rhizobacteria but also some soil bacteria and endophytes have been shown to have these capabilities, the broader term PGPB Agronomy 2021,11, 2236. https://doi.org/10.3390/agronomy11112236 https://www.mdpi.com/journal/agronomy Agronomy 2021,11, 2236 2 of 16 (plant growth-promoting bacteria) is now frequently used [ 8 ]. Among these beneficial bacteria, some can enhance plant growth under abiotic stress conditions such as salinity and drought, and have given rise to the term IST (induced systemic tolerance) [ 9 ]. Different bacterial genera have been proposed to alleviate salt stress in plants, such as Agrobacterium [ 10 ], Arthrobacter [ 11 ], Azotobacter [ 12 ], Azospirillum [ 13 ], Bacillus [ 14 ], Burkholderia [ 14 ], Klebsiella [15], Pseudomonas [16,17], Rhizobium [18], and Serratia [19]. Traditionally, the study of putative PGPB begins with the isolation of bacteria (from the soil or rhizosphere) and screening for plant growth promotion traits under in vitro conditions [ 15 – 17 ]. These traits include direct and indirect mechanisms, such as the synthesis of phytohormones [ 20 ], nutrient solubilization and uptake [ 21 , 22 ], biological nitrogen fixation [ 22 ], and siderophore production [ 23 ]. Subsequently, the bacteria with the highest number of desirable traits are selected to evaluate their plant growth promoting effect under axenic conditions [ 24 ]. So far, the development of alternative screening methods that allow the identification of putative PGPB without resorting to classical PGP traits has received very little attention [ 25 , 26 ]. Some reports have suggested that the potentially useful activities of PGPBs must be accompanied by efficient rhizosphere colonization and persistence of the bacteria under field conditions for effective use of these microorganisms as bioinoculants [ 26 , 27 ]. Thus, traits related to bacterial fitness in the environmental settings in which they will be applied must be considered an intrinsic part of the selection process for PGPBs. In the present work we have isolated native salt-tolerant bacteria of the genus Pseudomonas from a strongly saline soil and from the rhizosphere of soybean plants grown in a slightly saline soil. Strains were identified by 16S rRNA sequencing and further evaluated in terms of their salt tolerance, adherence to biotic and abiotic surfaces, fitness in the soybean rhizosphere, and plant growth promotion in slightly saline soils. This approach shows promising results for the identification and further exploitation of PGPBs for the development of bioinoculants. 2. Materials and Methods 2.1. Soil Sampling and Bacterial Isolation Soil samples were collected from an agricultural area of soybean monoculture in Leales, Eastern Tucumán, Argentina (27 ◦ 16 0 52 00 S 65 ◦ 01 0 08 00 W). Two saline soil samples were collected at a depth of 10 cm. Soil sample 1 was collected from an elevated zone of the field, while soil sample 2 was collected from a lower zone of the same field. A non-saline soil sample was also collected from the same area. The physical and chemical characteristics of the soil samples were analyzed by Laboratory PH7 Diagnóstico Agrícola (Tucumán, Argentina) and are summarized in Table 1. Table 1. Physicochemical characteristics of soil samples. Properties Slightly Saline Soil Strongly Saline Soil Non-Saline Soil Silt a(%) 66.8 61.2 63.7 Clay a(%) 5.7 14.5 5.0 Sand a(%) 27.5 24.3 31.3 pH b8.4 8.8 8.1 EC c(dS/m) 1.5 29.8 0.5 Sum of cations d (cmol/Kg) 26.0 66.8 17.7 Na+(ppm) 498.9 6496.9 124.1 K+(ppm) 1049.1 1450.8 624.0 Ca++ (ppm) 3834.0 1342.0 2750.0 Mg++ (ppm) 230.4 50.4 219.6 EC: Electrical conductivity. a Soil texture analysis by hydrometer: modification of the Bouyoucos method. b Soil to distilled water ratio of 1:2.5. cExtraction method with acetate NH4, pH7. dIn saturation paste of soil. Agronomy 2021,11, 2236 3 of 16 Bacterial isolation was carried out from saline soil samples and from the rhizosphere of soybean plants. To isolate soil bacteria, 0.5 g of each sample was suspended in 1.5 mL saline solution (NaCl 0.9% w/v) and 100 µ L of 10-fold serial dilutions were plated on the media described below. Soybean-associated rhizosphere bacteria were isolated as follows. Seeds (Glycine max, variety A8000) were kindly provided by Dr. Mariano Pardo (Obispo Colombres Agroindustrial Experimental Station, Tucumán, Argentina). The seeds were surface disinfected by immersion in ethanol (70% v/v) for 30 s, followed by rapid rinsing with sterile distilled water, immersion in sodium hypochlorite (20% v/v) for 30 s, and finally rinsing with abundant distilled water [ 28 ]. All trials requiring soybean cultivation used the saline soil with the lowest EC value (Table 1), since it allows soybean growth despite salinity. Thus, disinfected seeds were sown in plastic pots containing 0.5 kg of non-sterile slightly saline soil. Plants were grown under controlled conditions in a Panasonic Versatile Environmental Test Chamber (MLR-352H), at 24 ◦ C and 70% humidity with a daily light period of 16 h. Plants were irrigated periodically with sterile distilled water for 15 days. Subsequently, they were carefully removed and their roots placed in sterile 50 mL tubes containing 4 g of glass beads and 3 mL of saline solution. Tubes were vortexed for 2 min to remove bacteria attached to the root and 100 µ L of 10-fold serial dilutions of the suspension were plated on Petri dishes with different culture media with and without 0.8 M NaCl: Luria-Bertani (LB) 1 × and 0.1 × (10-fold diluted); M9 medium supplemented with glucose, sodium citrate, or sodium benzoate (final concentration 0.2% w/v); and a modified S1 medium for Pseudomonas, lacking trimethoprim [ 29 ]. Plates were incubated at 30 ◦ C for 48 h, the number of colonies was quantified and fluorescence was observed under UV light. Then, Pseudomonas strains were isolated using modified S1 medium. 2.2. Taxonomical Identification of the Isolates The isolated bacteria were identified by 16S rDNA analysis. Universal primers 27F (5 0 - AGAGTTTGATCMTGGCTCAG-3 0 ) and 1492R (5 0 -GGTTACCTTGTTACGACTT-3 0 ) were used for PCR amplification and sequencing in the conditions previously described [ 30 ]. Sequences were analyzed with DNAMAN and the MOLE-BLAST tool of the National Center for Biotechnology Information (NCBI; https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 4 October 2021). Sequences have been deposited in GenBank with accession numbers MZ020813 (EFY1), MZ020969 (EFY2), MZ020970 (EFY3), and MZ020971 (EFY4). The evolutionary history was inferred using the neighbor-joining method [ 31 ]. The optimal tree with the sum of branch length = 0.14652826 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method [ 32 ] and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. There were a total of 1372 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [33]. 2.3. Salinity Tolerance To test the salinity tolerance of the isolates, overnight cultures were diluted to an optical density (OD 600 ) of 0.5 and 50 µ L of the bacterial suspensions were inoculated in Erlenmeyer flasks containing 20 mL of LB medium with and without 0.8 M NaCl. Flasks were incubated at 30 ◦ C with constant agitation and OD 600 was measured every hour for 10 h and a final point at 24 h, using a Perkin-Elmer Lambda 20 spectrophotometer. Flasks with LB + 0.8 M NaCl medium were incubated up to 72 h. Alternatively, overnight cultures were diluted to an optical density (OD 600 ) of 1.0 and 10-fold serial dilutions (10 µ L) were spotted on LB plates with and without 0.8 M NaCl. The plates were incubated at 30 ◦ C for 24 h and growth was visually inspected. Pseudomonas putida KT2440 was used as a positive control in these experiments. Additionally, bacterial survival was evaluated in a solution obtained from the strongly saline soil sample. The solution was prepared following the protocol provided by Walter Agronomy 2021,11, 2236 4 of 16 and colleagues [ 34 ] with modifications. Briefly, 25 g of the strongly saline soil sample was suspended in 100 mL of sterile distilled water. The suspension was filtered through sterile gauze and centrifuged at 8000 rpm for 15 min. Then, the solution was filtered under vacuum through a 0.22 µ m filter. The resulting solution had an EC value of 4.2 dS/m and a pH of 7.4 and was used to test long-term survival of the bacteria. Erlenmeyer flasks containing 20 mL of the soil solution were inoculated with bacterial suspensions (6 × 10 6 CFU/mL) and incubated at 30 ◦ C with constant agitation for 12 days, after which 10-fold serial dilutions were plated in LB and CFU/mL were quantified. 2.4. Exopolysaccharide (EPS) Production, Bacterial Surface Adhesion, Colonization and Competition in Rhizosphere EPS production was tested using Congo red staining. Overnight cultures were diluted to an OD 600 = 0.05 and 5 µ L were spotted on LB plates supplemented with Congo red (40 µ g/mL), with and without 0.5 M NaCl [ 16 ]. The plates were incubated at 30 ◦ C for 48 h. Colonies were evaluated according to morphology and color [35]. Biofilm formation was evaluated during growth in 96-well polystyrene microtiter plates (Sorfa Life Science Research), using the protocol described by O’Toole and Kolter [ 36 ]. Biofilm formation was tested in LB and LB + 0.3 M NaCl [ 16 ]. Overnight cultures grown in the evaluated media were diluted to an OD 600 of 0.1 and placed in the wells. The plates were incubated at 30 ◦ C without agitation and biofilm formation was quantified at the indicated times by staining with crystal violet (0.4%), solubilizing the dye with 30% glacial acetic acid and measuring absorbance at 540 nm. Bacterial adhesion to soybean seeds was also tested, as previously described [ 16 ]. Bacteria were cultured in M9 medium supplemented with glucose and diluted to an OD 600 of 1 (equivalent to 10 9 CFU/mL). Bacterial suspensions were used to inoculate disinfected soybean seeds by immersion for 2 min. Unattached bacteria were removed from the surface of the inoculated seeds by brief rinsing with distilled water. Seeds (N = 10) were placed into sterile 50 mL tubes containing 10 mL of saline solution and 4 g of glass beads (diameter, 3 mm). Attached bacteria were removed by vortexing the tubes for 2 min. The suspensions were serially diluted and plated on M9 medium supplemented with sodium citrate. The plates were incubated at 30 ◦C for 24 h and the number of cells was quantified. A second group of inoculated seeds (N = 5) was sown in pots (0.5 kg) containing sterile sand and pots containing sterile slightly saline soil samples. The soil was sterilized by three successive sterilizations in autoclave (121 ◦ C for 20 min) every 24 h. Seedlings were periodically watered with sterile 80 mM NaCl saline solution (pots with sand) or sterile distilled water (pots with slightly saline soil). The pots were maintained under controlled conditions as described above. Ten days after sowing, the soybean seedlings were gently removed from the soil. Roots were cut and transferred to 50 mL tubes containing 10 mL of saline solution and 4 g of glass beads. Root-associated bacteria were removed and evaluated as above. Additionally, competitive colonization between each isolate against P. putida KT2440 was evaluated in the soybean rhizosphere. For this, tetracycline resistance mutants of the isolates were used. The mutants were performed by triparental mating using mini-Tn7-Tc transposon carrying a tetracycline resistance marker [ 37 ] following the protocol previously described [ 38 ]. A rifampicin-resistant strain of P. putida KT2440 was also used [ 16 ]. Disinfected soybean seeds were co-inoculated with 10 9 CFU/mL of each tetracycline-resistant isolate and the P. putida KT2440-Rif r strain, both in the same proportion (1:1 v/v) [ 16 ]. The soybean seedlings were grown in the two different substrates described above. Ten days after sowing, the seedlings were removed from the soil, roots were cut and processed as previously described. The bacterial suspensions obtained were serially diluted and plated on M9 medium supplemented with sodium citrate with Rif or Tc (Sigma-Aldrich, Shanghai, China), as appropriate. Agronomy 2021,11, 2236 5 of 16 2.5. Plant Growth Promotion in Slightly Saline Soil Plant growth promotion assays were performed in slightly saline and non-saline soils (Table 1). Seeds were disinfected and inoculated with each isolate or with P. putida KT2440 as described above. Inoculated and non-inoculated (control) seeds (N = 25) were sown in pots containing 1.5 kg of non-sterile soil and plants were grown under the controlled conditions described above for 21 days, irrigated with distilled water on demand. After that time, the total length of the plants was measured, their phenological state was determined based on leaf development, the percentage of germinated seeds (germination rate) was quantified, and the vigor index (VI) was calculated using the following equation [ 39 ]: VI =TL ×G 10 , where G is the germination rate and TL the total plant length. Two independent experiments were performed for each treatment. 2.6. Statistical Analysis All results correspond to three independent experiments, with three technical replicates each, unless otherwise indicated, and are expressed as mean values ± standard error. One-way ANOVA (Tukey’s test, p< 0.05) was applied, using MINITAB statistical software (version 17 for Windows), to detect significant differences among the bacteria evaluated, taking into account that a pvalue < 0.05 was considered statistically significant. 3. Results and Discussion 3.1. Soil Characteristics, Bacterial Isolation, and Selection of Isolates Table 1provides the physicochemical characteristics of the soil samples (two saline and one non-saline) used in this work. The texture of the evaluated soils was silty loam and their pH values were 8.4, 8.8, and 8.1 for slightly saline soil, strongly saline soil, and non-saline soil, respectively. The electrical conductivity (EC) values of the saline soils is extremely different (1.5 and 29.8 dS/m), due to the natural slope of the field which favors the accumulation of soluble salts after irrigation and rainfall. According to FAO classification, the soil sample with the highest EC value can be considered as strongly saline soil, where agricultural activity is not suitable [ 40 ]. Although soils with EC ≤ 2 dS/m are not classified as saline, the saline soil with EC = 1.5 was considered a slightly saline soil due to the high Na + content compared to the non-saline soil (Table 1), and the fact that many plant species decrease their productivity in such soil. Bacterial isolation was performed from both saline soil samples and from the rhizosphere of soybean grown in slightly saline soil. The number of culturable bacteria obtained on each culture medium for each soil is presented in Table 2. The number of CFU per gram of slightly saline soil is in accordance with the value obtained in other silty loam soil [ 41 ]. As expected, the values obtained from the rhizosphere samples were in general higher than those from soil samples, the strongly saline soil sample showed the lowest number of culturable cells. This is in line with the idea that microbial abundance is higher in the rhizosphere due to nutrients provided by root exudates, but it may also indicate that the root offers a protective environment against saline stress. Interestingly, the number of culturable cells in the modified S1 selective medium was three and four orders of magnitude higher in the soybean rhizosphere sample than in the slightly and strongly saline soil samples, respectively. This suggests that fluorescent Pseudomonads are favored in the plant environment, in agreement with previous studies [42]. Using the modified S1 medium, four isolates were selected for further analysis, three obtained from the rhizosphere of soybean grown in the slightly saline soil (EFY1, EFY2, and EFY4) and one from the strongly saline soil (EFY3). Agronomy 2021,11, 2236 6 of 16 Table 2. Number of culturable cells in soil and rhizosphere samples. Medium Soybean Rhizosphere Slightly Saline Soil Sample Strongly Saline Soil Sample LB 1×1.6 ×107±5.0 ×106 a 3.0 ×106±3.0 ×105 b 3.6 ×105±8.0 ×104 b LB 1×+ NaCl 6.0 ×105±4.0 ×104 a 6.6 ×105±1.2 ×105 a 1.0 ×105±1.5 ×104 b LB 0.1×1.8 ×107±2.4 ×106 a 4.2 ×106±3.0 ×105 b 5.7 ×105±3.0 ×104 c LB 0.1×+ NaCl 3.0 ×105±8.0 ×104 a 4.5 ×105±7.0 ×104 a 1.4 ×105±1.2 ×104 b M9-glucose 9.9 ×106±7.0 ×105 a 1.5 ×106±3.1 ×105 b 3.0 ×105±4.0 ×104 b M9-glucose + NaCl 5.4 ×105±6.0 ×104 a 4.2 ×105±5.0 ×104 b 4.5 ×104±1.5 ×104 c M9-citrate 9.0 ×106±1.0 ×106 a 1.4 ×106±2.0 ×105 b 1.8 ×105±1.3 ×104 b M9-citrate + NaCl 1.5 ×106±2.0 ×105 a 4.2 ×105±7.0 ×104 b 6.0 ×104±4.0 ×103 c M9-benzoate 6.5 ×105±5.0 ×104 a 3.9 ×105±2.0 ×104 b 4.5 ×105±5.0 ×104 b M9-benzoate + NaCl 6.0 ×106±8.0 ×105 a 4.8 ×105±3.0 ×104 b 2.0 ×103±5.0 ×101 c Modified S1 1.2 ×106±8.0 ×105 a 6.0 ×103±3.0 ×102 b 4.2 ×102±1.1 ×101 c Data correspond to CFU/g of soil or fresh weight of soybean root, as appropriate, and are mean values ± standard errors. NaCl was supplied at 0.8 M (final). Values within rows followed by different letters indicate significant differences (p< 0.05, Tukey’s test). 3.2. Identification of Pseudomonas Isolates Amplification, sequencing and analysis of the 16S rDNA indicated that the four isolates belong to the genus Pseudomonas. The phylogenetic tree is shown in Figure 1. Results indicate that isolates EFY1, EFY2, and EFY4 are closely related to Pseudomonas putida KT2440, while EFY3 is the most divergent isolate of the four, being closely related to P. alkylphenolica KL25. Colony morphology and Congo red binding assays also evidenced the divergence between EFY3 and the other three isolates (Supplementary Material 2). Isolates EFY1, EFY2, and EFY4 showed similar colony morphology as P. putida KT2440, whereas EFY3 exhibited wrinkled colonies with higher color intensity, indicative of different composition of the extracellular polymeric matrix. 3.3. Salt Tolerance Assays The growth of isolates was evaluated in solid and liquid media under saline and nonsaline conditions. P. putida KT2440 was used as a control, since it can tolerate a saline stress concentration up to 0.8 M NaCl [ 43 ]. The evaluated bacteria showed similar growth in solid medium under non-saline conditions (Figure 2a), whereas in the presence of 0.8 M NaCl, EFY1, EFY2, and EFY4 grew notably better than P. putida KT2440 (Figure 2b). Surprisingly, EFY3 showed the lowest tolerance to 0.8 M NaCl (Figure 2b), despite being isolated from a highly saline environment. Experiments done in liquid LB medium with 0.8 M NaCl also showed a slightly reduced growth rate of this strain with respect to the others, an effect that was less evident in non-saline conditions (Figure 3a,b). One possible reason could be that EFY3 needs an "adaptation period" to a condition (rich medium) very different from its original one (strongly saline soil). With this in mind, survival of P. putida KT2440 and isolates EFY3 and EFY4 was evaluated using a sterile solution obtained from the strongly saline soil as described in Materials and Methods section. As shown in Figure 3c, a decrease in the number of recovered cells of EFY3 was observed until day 3, followed by an increase in growth so that after 12 days there were no significant differences between the evaluated strains. Agronomy 2021,11, 2236 7 of 16 Agronomy 2021, 11, x FOR PEER REVIEW 7 of 17 Figure 1. Comparative 16S rRNA gene sequence analysis of Pseudomonas sp. strains EFY1, EFY2, EFY3, and EFY4, and representative related strains from GenBank. The significance of each branch is indicated by a bootstrap for 1000 subsets. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Figure 1. Comparative 16S rRNA gene sequence analysis of Pseudomonas sp. strains EFY1, EFY2, EFY3, and EFY4, and representative related strains from GenBank. The significance of each branch is indicated by a bootstrap for 1000 subsets. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Agronomy 2021, 11, x FOR PEER REVIEW 8 of 17 Figure 2. Growth of P. putida KT2440 and strains EFY1, EFY2, EFY3, and EFY4 on solid medium (a) LB and (b) LB + 0.8 M NaCl. Ten microliters of 10-fold serial dilutions of the bacterial cultures were plated on the plates. Images were taken after incubating the plates at 30 °C for 24 h. 10010-2 10-4 10-6 10-7 10-8 KT2440 EFY1 EFY2 EFY3 EFY4 LB LB + NaCl 0.8 M 10010-2 10-3 10-4 10-5 10-6 (a) (b) (a) Figure 2. Growth of P. putida KT2440 and strains EFY1, EFY2, EFY3, and EFY4 on solid medium ( a ) LB and ( b ) LB + 0.8 M NaCl. Ten microliters of 10-fold serial dilutions of the bacterial cultures were plated on the plates. Images were taken after incubating the plates at 30 ◦C for 24 h. Agronomy 2021,11, 2236 8 of 16 Agronomy 2021, 11, x FOR PEER REVIEW 8 of 17 Figure 2. Growth of P. putida KT2440 and strains EFY1, EFY2, EFY3, and EFY4 on solid medium (a) LB and (b) LB + 0.8 M NaCl. Ten microliters of 10-fold serial dilutions of the bacterial cultures were plated on the plates. Images were taken after incubating the plates at 30 °C for 24 h. 10010-2 10-4 10-6 10-7 10-8 KT2440 EFY1 EFY2 EFY3 EFY4 LB LB + NaCl 0.8 M 10010-2 10-3 10-4 10-5 10-6 (a) (b) (a) Agronomy 2021, 11, x FOR PEER REVIEW 9 of 17 Figure 3. Growth curves in: (a) LB, (b) LB + 0.8 M NaCl, and (c) strongly saline soil solution. Evaluated strains: (■) P. putida KT2440, (□) EFY1, (●) EFY2, (○) EFY3, and (▲) EFY4. Data are plotted as mean values ± standard error. Different letters indicate significant differences among the kinetics of the growth curves (p < 0.05, Tukey’s test). 3.4. EPS Production, Surface Adhesion, Colonization, and Competition in Rhizosphere Adhesion of the isolates to biotic and abiotic surfaces was evaluated and P. putida KT2440 strain was used as a positive control. Biofilm formation on the surface of polystyrene microtiter plates was tested under saline (0.3 M NaCl) and non-saline conditions. As shown in Figure 4a, under non-saline conditions, the isolate EFY3 had delayed biofilm formation dynamics compared to the other bacteria, showing a lower attached biomass at early times and a higher biomass after 24 h, while the other strains have almost completely detached by then. Salinity caused delayed biofilm formation, the maximum being reached at 6h (Figure 4b), with higher biomass attached compared to non-saline conditions. In the presence of high salinity, however, the isolate EFY3 showed the lowest attached biomass. The quantitative results of biofilm formation are in agreement with the qualitative results obtained for Congo red binding, in which isolate EFY3 showed a higher presence of Congo red-related EPS under 0.01 0.1 1 10 0 6 12 18 24 30 36 42 48 54 60 66 72 OD600 Time (h) LB + 0.8 M NaCl KT2440 b EFY1 a EFY2 a EFY3 b EFY4 a (b) (c) Figure 3. Growth curves in: ( a ) LB, ( b ) LB + 0.8 M NaCl, and ( c ) strongly saline soil solution. Evaluated strains: (  )P. putida KT2440, (  ) EFY1, ( • ) EFY2, ( # ) EFY3, and ( N ) EFY4. Data are plotted as mean values ± standard error. Different letters indicate significant differences among the kinetics of the growth curves (p< 0.05, Tukey’s test). Agronomy 2021,11, 2236 9 of 16 3.4. EPS Production, Surface Adhesion, Colonization, and Competition in Rhizosphere Adhesion of the isolates to biotic and abiotic surfaces was evaluated and P. putida KT2440 strain was used as a positive control. Biofilm formation on the surface of polystyrene microtiter plates was tested under saline (0.3 M NaCl) and non-saline conditions. As shown in Figure 4a, under non-saline conditions, the isolate EFY3 had delayed biofilm formation dynamics compared to the other bacteria, showing a lower attached biomass at early times and a higher biomass after 24 h, while the other strains have almost completely detached by then. Salinity caused delayed biofilm formation, the maximum being reached at 6h (Figure 4b), with higher biomass attached compared to non-saline conditions. In the presence of high salinity, however, the isolate EFY3 showed the lowest attached biomass. The quantitative results of biofilm formation are in agreement with the qualitative results obtained for Congo red binding, in which isolate EFY3 showed a higher presence of Congo red-related EPS under non-saline conditions. While in saline conditions, isolate EFY3 showed a slightly lower presence of EPS than the other bacteria evaluated. In a recent work, P. putida KT2440 mutants, which overproduce EPS but have low bacterial adhesion to abiotic surfaces, were shown to be able to enhance the growth of soybean and corn under saline stress [44]. Agronomy 2021, 11, x FOR PEER REVIEW 10 of 17 non-saline conditions. While in saline conditions, isolate EFY3 showed a slightly lower presence of EPS than the other bacteria evaluated. In a recent work, P. putida KT2440 mutants, which overproduce EPS but have low bacterial adhesion to abiotic surfaces, were shown to be able to enhance the growth of soybean and corn under saline stress [44]. Figure 4. Bacterial adhesion to the surface of polystyrene microtiter plates of P. putida KT2440 and the isolates EFY1, EFY2, EFY3, and EFY4, grown in (a) LB and (b) LB + 0.3 M NaCl. Data are plotted as mean values ± standard error. Different letters indicate significant differences among the strains in the same media (p < 0.05, Tukey’s test). Bacterial adherence to seeds is a key step for PGPB to be successful in plant growth promotion. Bacterial adhesion facilitates the subsequent proliferation of microorganisms attached to the seed and favors the colonization of the root system once the plant begins Time (h) Strains 24 6 3 EFY4 EFY3 EFY2 EFY1 KT2440 EFY4 EFY3 EFY2 EFY1 KT2440 EFY4 EFY3 EFY2 EFY1 KT2440 4 3 2 1 0 OD540nm LB Time (h) Strains 24 6 3 EFY4 EFY3 EFY2 EFY1 KT2440 EFY4 EFY3 EFY2 EFY1 KT2440 EFY4 EFY3 EFY2 EFY1 KT2440 4 3 2 1 0 OD540nm LB + 0.3 M NaCl a c b,c b c c c c c c c a,b b,c e e e e e e d,e d,e e (a) (b) a b,c c b b,c d,e c,d d,e Figure 4. Bacterial adhesion to the surface of polystyrene microtiter plates of P. putida KT2440 and the isolates EFY1, EFY2, EFY3, and EFY4, grown in ( a ) LB and ( b ) LB + 0.3 M NaCl. Data are plotted as mean values ± standard error. Different letters indicate significant differences among the strains in the same media (p< 0.05, Tukey’s test). Agronomy 2021,11, 2236 16 of 16 36. O’Toole, G.A.; Kolter, R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: A genetic analysis. Mol. Microbiol. 1998,28, 449–461. [CrossRef] [PubMed] 37. de Lorenzo, V.; Herrero, M.; Jakubzik, U.; Timmis, K.N. Mini-Tn5transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 1990,172, 6568–6572. [CrossRef] 38. Espinosa-Urgel, M.; Salido, A.; Ramos, J.L. Genetic analysis offunctions involved in adhesion of Pseudomonas putida to seeds. J. Bacteriol. 2000,182, 2363–2369. [CrossRef] 39. Ajithkumar, P.V.; Gangadhara, K.P.; Manilal, P.; Kunhi, A.A.M. Soil inoculation with Pseudomonas aeruginosa 3MT eliminates the inhibitory effect of 3-chloroand 4-chlorobenzoate on tomato seed germination. Soil Biol. Biochem. 1998 ,30, 1053–1059. [CrossRef] 40. Safdar, H.; Amin, A.; Shafiq, Y.; Ali, A.; Yasin, R. A review: Impact of salinity on plant growth. Nat. Sci. 2019 ,17, 34–40. [CrossRef] 41. Raimondo, E.E.; Aparicio, J.D.; Briceño, G.E.; Fuentes, M.S.; Benimeli, C.S. Lindane bioremediation in soils of different textural classes by an Actinobacteria consortium. J. Soil Sci. Plant Nutr. 2019,19, 29–41. [CrossRef] 42. Marrero, M.A.; Agaras, B.; Wall, L.G.; Valverde, C. Enriquecimiento diferencial de Pseudomonas spp. en el rizoplano de distintas especies cultivadas. Rev. Argent. Microbiol. 2015,47, 132–137. [CrossRef] 43. Costa-Gutierrez, S.B. Microflora of the Soybean Rhizosphere in Conditions of High Salinity: ISOLATION and Study of Pseudomonas putida Strains Benefits Against Saline Stress. Ph.D. Thesis, National University of Tucumán, Tucumán, Argentina, 2019. 44. Costa-Gutierrez, S.B.; Raimondo, E.E.; Lami, M.J.; Vincent, P.A.; Espinosa-Urgel, M.; de Cristóbal, R.E. Inoculation of Pseudomonas mutant strains can improve growth of soybean and corn plants in soils under salt stress. Rhizosphere 2020 ,16, 100255. [CrossRef] 45. Compant, S.; Clément, C.; Sessitsch, A. Plant growth-promoting bacteria in the rhizoand endosphere of plants: Their role, colonization, mechanisms involved and prospects for utilization. Soil Biol. Biochem. 2010,42, 669–678. [CrossRef] 46. Ansari, F.A.; Ahmad, I. Biofilm development, plant growth promoting traits and rhizosphere colonization by Pseudomonas entomophila FAP1: A promising PGPR. Adv. Microbiol. 2018,8, 235–251. [CrossRef] 47. Molina, L.Á.; Ramos, C.; Duque, E.; Ronchel, M.C.; García, J.M.; Wyke, L.; Ramos, J.L. Survival of Pseudomonas putida KT2440 in soil and in the rhizosphere of plants under greenhouse and environmental conditions. Soil Biol. Biochem. 2000 ,32, 315–321. [CrossRef] 48. Bashan, Y. Inoculants of plant growth-promoting bacteria for use in agriculture. Biotechnol. Adv. 1998,16, 729–770. [CrossRef] 49. Adhikari, A.; Khan, M.A.; Lee, K.E.; Kang, S.M.; Dhungana, S.K.; Bhusal, N.; Lee, I.J. The halotolerant rhizobacterium— Pseudomonas koreensis MU2 enhances inorganic silicon and phosphorus use efficiency and augments salt stress tolerance in soybean (Glycine max L.). Microorganisms 2020,8, 1256. [CrossRef] [PubMed] 50. Kasotia, A.; Varma, A.; Tuteja, N.; Choudhary, D.K. Amelioration of soybean plant from saline-induced condition by exopolysaccharide producing Pseudomonas-mediated expression of high affinity K+-transporter (HKT1) gene. Curr. Sci. 2016 , 1961–1967. [CrossRef] 51. Lami, M.J. Rhizospheric Bacteria for Plant Growth Promotion and Salt Stress Tolerance in Soybean Crops. Ph.D. Thesis, National University of Tucumán, Tucumán, Argentina, 2018.