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Introduction
Understanding the complexity of the soil microbiome remains a challenge. Plant growth promoting rhizobacteria (PGPR) are among the most studied because they positively change the environment and roots of plants, improving nutrient uptake and their development (Vejan, Abdullah, Khadiran, Ismail and Nasrulhaq, 2016). Some of the ef fects that these bacteria originate in the host plant include the increase in the germination rate, in the foliar area and stem and root growth (Panwar, Tewari, Gulati and Nayyar, 2016).
The microbiota that is associated with the plant has a very important role since it participates in increasing stress tolerance, using dif ferent mechanisms such as the accumulation of osmolytes, exopolysaccharide phytohormones, production of volatile compounds, modulation of the expression of related genes with stress, as well as inhibition and elimination of phytopathogens from the soil, in addition to contributing to the improvement of crop yield (Ali and Xie, 2020).
Salinity is the environmental stress that has the most prejudicial ef fects on plant growth and crop yields (Mayak, Tirosh and Glick, 2004). The halophytes can react in presence of salinity, either stimulating or inhibiting (Flowers and Colmer, 2008). These plants show adaptability to saline enviroments thanks to their associated microbiome and their genotype. For these reasons, this type of plant grows easily in dry regions of the world (Alexander, Mishra and Jha, 2019). Some studies demonstrated, that halophytes have succulence mechanisms that help them to maintain cell turgor pressure (Lokhande and Suprasanna 2012; Nikalje, Srivastava, Pandey and Suprasanna, 2018). Such is the case of the Suaeda plant, of which a series of works have increased knowledge of the interaction with its microbiome (Hidri et al., 2022).
In the scientific community, Arabidopsis thaliana has been successfully used as a model plant, being ideal to work with it due to its small genome and its short life cycle (Goodman, Ecker and Dean, 1995). Dif ferent studies recognize their factibility for co-inoculation with beneficial bacteria in order to analyze the ef fects on plant development. Some recent results are reported by Ravelo-Ortega and López-Bucio (2022), García-Cárdenas, Ortiz, Ruiz, Valencia and López (2022) and Jiménez-Vázquez et al. (2020) who have described dif ferent mechanisms of interaction between microorganism-plant.
The use of PGPR is one of the ways towards food production with a more ecological and organic approach to environmental protection (Calvo, Nelson and Kloepper, 2014). Taking into account the great increase in the world population, the need for more sustainable agriculture that is capable of preserving our future becomes latent. Since today the world faces great challenges in relation to land degradation, the productivity of crops and the availability of fertile land are increasingly at stake. Just in Mexico 14% of the territory is dedicated to agriculture and in the coming years, friendly practices will play a crucial role in crop productivity and stability. The use and application of microbial inoculants over agricultural systems have been widely examined, such as the Pagnani et al. (2019) study demonstrates the positive influence on the growth, physiological and performance responses of wheat; which agrees with what was found by Cordero, Balaguer, Rincón and Pueyo (2018) confirming that PGPR can minimize the negative ef fects of abiotic stresses and arguing that this is the first step to guarantee sustainable agriculture worldwide.
Microorganism-plant interaction has long been of great interest since plants produce various compounds (sugars, organic acids, and vitamins) that microorganisms use as nutrients or signals. In turn, these microbial populations release phytohormones and volatile organic compounds that regulate plant growth and morphogenesis (Ortiz-Castro, Contreras, Macias and Lopez, 2009). It has previously been reported that dif ferent isolates belonging to the genus Bacillus have caused various ef fects on A. thaliana and favorably influenced its development (Palacio-Rodríguez et al., 2017; López-Bucio, Acevedo, Ramirez, Molina and Herrera, 2006). Similarly, although less studied is the case for Staphylococcus, reported by Mora-Ruíz et al. (2018) where they showed that bacteria of the Staphylococcus genus promoted growth activity in A. thaliana.
Therefore, in the present investigation, the isolation of bacteria from the root of Suaeda sp was carried out, taking into consideration that the Comarca Lagunera is a region of great agricultural demand, however, currently it presents serious desertification problems. Therefore, farmers have seen the need to make excessive use of inorganic fertilizers, further af fecting soil quality and crop yields. Consequently, it is important to evaluate and adopt best practices in relation to sustainable production. Therefore, the present study was developed where some biochemical characteristics that improve plant growth, the ef fect of its inoculation on the germination rate, and the growth promotion of Solanum lycopersicum L. (Saharan) were evaluated, selecting this crop by its great agricultural importance in the lagunera region, Mexico. Likewise, determine the identification of the strains by means of molecular techniques. Hoping that it leads to an increase in the development and exploitation of beneficial microbes to permit more sustainable agriculture. Based on the premise that the use of PGPR isolated from halophytes considerably increases plant development, the objective of this work was to evaluate the ef fect of inoculating three strains of PGPR on Arabidopsis thaliana and Solanum lycopersicum seedlings.
Materials and Methods
The bacteria used in the present work were isolated in 2016 from the rhizosphere of Suaeda sp., a native plant of the Poza Salada in the Sobaco Valley, coordinates 102° 58’ 58” W and 25° 45’ 32” N, at a height of 1090 altitude meters (Czaja, Estrada and Flores, 2014), located in the municipality of San Pedro de las Colonias Coahuila, Mexico (Figure 1a,b.
Figure 1: Prospecting the Chihuahuan Desert sampling area. (a) Geographic Location of the "Poza Salada" in the Sobaco Valley Coahuila, Mexico. (b) Sample collection site. (c) Halophyte Suaeda sp., grown near the "Poza Salada" body of water.
For the isolation of the bacteria, a solution was prepared with one gram of plant root and 9 ml of 0.5X phosphate buf fer saline (PBS), of which a serial dilution was made up to 10-3; 100 µL was taken from each dilution and were inoculated separately in Petri dishes with Luria Bertani (LB) solid medium (Bertani, 1951), the incubation was for 24 h at 32 ± 2 degrees Celsius.
Three colonies with dif ferent morphology were selected, to which the identification keys were assigned: Ecto10(6), Endo10(7), and Endo10(5); they were then stored in suspension with 30% glycerol at -70 °C (Hunter and Belt, 1996).
In vitro assays with Arabidopsis thaliana
Three in vitro experiments were carried out, with the objective of evaluating the growth promoting capacity of Ecto10(6), Endo10(7), and Endo10(5) in Arabidopsis thaliana (Col-0). Two of the tests were carried out to determine the ef fect of the inoculation distance of the bacteria in A. thaliana seedlings at 5 and 2 cm (distance and contact, respectively); and to analyze the ef ficacy of bacteria in promoting plant growth by means of volatile organic compounds (VOCs) a third test was developed in divided Petri dishes, inoculating the strain in a vertical line on one side, and the other side three A. thaliana seedlings were used.
Seeds of A. thaliana (Col-0) were disinfected for seven minutes with 96% ethanol and 20% sodium hypochlorite for five minutes; To remove the chlorine residues, they were washed five times with sterile distilled water, then they were kept for two days at 4 degrees Celsius.
The seeds were germinated in Petri dishes with Murashige & Skoog (MS) 0.2X growth medium [1% (w/v) agar, 0.75% (w/v) sucrose, pH 7.0], kept for six days in a bioclimatic chamber, with conditions of 25 ± 1 °C, 16 h light/8 h darkness, with an inclination of 70° allowing the gravitropic growth of the root (López-Bucio et al., 2007).
The preparation of the inoculum was carried out as follows; The isolates were grown individually in LB liquid medium and placed in an incubator for 24 h at 29 ± 2 °C, with shaking of 200 rpm (Scientific Precision 815®), and the bacterial suspensions were adjusted to a concentration of 1×108 CFU mL-1 with PBS 0.5X (Espinosa-Palomeque et al., 2017).
In the distance inoculation test, a series of six seedlings were placed per Petri dish with 0.2X MS medium, then the bacterial inoculum was applied horizontally, 5 cm below the tip of the Arabidopsis root. For the contact experiment, the bacterial suspension was first applied approximately 2 cm below the upper edge of the box, and six A. thaliana seedlings were placed on the inoculum (Gutiérrez-Luna et al., 2010).
To evaluate the VOCs, divided boxes were used, at one end the bacteria were inoculated and at the other, three A. thaliana seedlings were placed in parallel to the division of the box.
The experiments were carried out with three repetitions per treatment and were kept in the bioclimatic chamber (distributed by R.I.E.L. S.A. DE C.V.) with the conditions described above. The experimental unit consisted of each Petri dish. Twelve days af ter inoculation, the following parameters were evaluated: fresh weight, main root length, and number of secondary roots.
Trial with Solanum lycopersicum L. (Sahariana)
To determine the ef fect of the inoculation of the isolates in S. lycopersicum, two experiments were carried out in 2017: in the first, the stimulation or inhibition caused by the germination rate of the seeds was analyzed; the second was carried out to establish the influence of bacteria on tomato development in shade house conditions af ter 30 days of growing.
Solanum lycopersicum Var Sahariana seeds were used for the aforementioned experiments, which received a disinfection treatment with 96% ethanol for seven minutes and 20% sodium hypochlorite for five minutes, followed by five washes with sterile distilled water.
The suspensions of the bacteria were prepared and applied independently: Ecto10(6), Endo10(7), and Endo10(5) were grown in LB broth for 24 h and the concentrations were adjusted with PBS 0.5× to 1×108 CFU mL-1 (Espinosa-Palomeque et al., 2017).
To evaluate the germination rate, the seeds were immersed in the suspensions for 2 h at 29 ± 2 °C, in an incubator with shaking of 2 rpm (Scientific Precision 815®); Subsequently, they were sown in a tray for germination (200 cavities of 3.1 cm × 3.1 cm wide, 7.0 cm high, 5 mm drainage) with a mixture of substrate peat moss: perlite: vermiculite in proportions 1: 1: 1 and they were kept at room temperature in the dark for seven days. The test was carried out with ten repetitions per treatment and the germination percentage was calculated.
In the second experiment with tomato, the treatment of the seeds was carried out in the same way as in the previous one. Af ter germination, the seedlings were kept in a shade mesh for their development, with irrigation in the morning and at night, abundantly with running water. A second inoculation was carried out 20 days af ter sowing, before transplanting into the soil, the bacterial suspensions (prepared as described above) were applied (according to the treatment) in the area surrounding the roots of the plants, applying only morning watering. For its analysis, the following parameters were evaluated: plant height, root dry weight, number of lateral roots, foliar fresh weight, and foliar dry weight. The test was carried out with ten repetitions per treatment.
Mechanisms of rhizobacteria
Each of the bacterial isolates was evaluated to establish some characteristics that could promote plant growth, for which several biochemical tests were carried out such as the production of indole acetic acid (IAA) with the addition of L-tryptophan, determined with the Salkowsky technique modified and described by Bric, Bostock and Silverstone (1991). The IAA concentration was quantified with a colorimetric method and compared with a standard curve (5, 25, 50, 75, and 100 µg mL-1).
To test the production of siderophores, the Chrome azurol S (CAS) medium (Sigma-Aldrich, México DF) (Schwyn and Neilands, 1987) and the solubilization of phosphates with the selective culture medium NBRIP (National Botanical Research Institute Phosphate growth medium) (Nautiyal, 1999) were used. The positive confirmation for siderophore production was the formation of a yellow to orange halo surrounding the growth of the bacteria and the phosphate solubilization indicator was a clear halo. To measure the halos, a digital vernier (Carbon Fiber Composites Digital Caliper, Industrial & Scientific) was used and the results were evaluated by calculating the phosphate solubilization index (PSI) and siderophore production index (SPI) with the help of the PSI or SPI formula (as appropriate).
PSI or SPI = Diameter of the colony + solubilization halo / diameter of the colony (Edi-Premono, Moawad and Vleck, 1996).
The assay of all biochemical tests was made by three replications.
Statistical analysis
The data obtained from the tests with A. thaliana, S. lycopersicum and rhizobacterial mechanisms were analyzed for normal distribution with the Shapiro-Wilk test and then analyzed in a completely randomized experimental design and comparison of means with the Tukey test (P ≤ 0.05). The program used for the analysis was GraphPad Prism version 6 (Motolusky, 1989).
16S rRNA analysis
For their identification, the rhizobacteria were subjected to 16S rRNA sequence analysis. The extraction of chromosomal DNA was done from pure bacterial cultures, by means of the CTAB technique (Doyle and Doyle, 1990).
The purified PCR products were sent for sequencing at the Mc LAB laboratory in San Francisco, California, USA. Finally, the result was compared in the BLAST algorithm (Altschul et al., 1997) with the pre-established parameters in the NCBI database (NCBI, 1988).
Results and Discussion
In the present investigation, the ef fect of our isolates of the genera Bacillus and Staphylococcus on the growth modulation of A. thaliana and S. lycopersicum was characterized. Aneuirinibacillus migulanus, Staphylococcus sp. and Bacillus cereus were able to stimulate the development of A. thaliana, exhibiting PGPR abilities. We were able to observe a positive ef fects test (P ≤ 0.05) on A. thaliana in the in vitro assays in distance application. In the experiment carried out to evaluate the influence of the inoculation of Ecto10(6), Endo10(7), and Endo10(5) at a distance (5 cm), on the growth promotion of A. thaliana by dif fusible compounds, it was observed that the leaf weight of the plants that had treatment with the bacteria showed increases of 109.3, 70.8 and 48.4% respectively, more than those of the control (Figure 2a-d) and when analyzing the development of the main root, those that were treated with Endo 10(7) had 28% more length than the plants of the other treatments and control (Figure 2e) (P ≤ 0.05), in the case of the variables of the number of lateral roots and fresh weight, all the plants that had treatment with the bacteria were superior to those of the control however, Ecto10(6) benefited Arabidopsis more than other treatments (Figure 2f, g), (P ≤ 0.05). These results suggest that there are probably growth regulating substances that act according to their proximity to the root system of the plants (Figure 2).
Figure 2: Effect of confrontation with, Ecto10(6), Endo10(7) y Endo10(5), in Arabidopsis thaliana (Col-0), by diffusible compounds (distance experiment), grown for 12 days in agar medium 0.2X MS, in (a) without bacteria (Control)., (b-d) Seedlings inoculated with Aneurinibacillus migulanus, Bacillus cereus and Staphylococcus sp., respectively., (e) Root length., (f) Number of side roots., (g) Fresh weight. †Values represent the mean ± standard deviation of 24 seedlings grown on four independent plates. †Different letters in the same column indicate significant differences, according to Tukey's test (P ≤ 0.05).
While in investigations carried out by García-Cárdenas et al., (2022) and Zhou et al. (2016) have shown the ef fect of co-cultivating isolates of the Bacillus and Staphylococcus genus in direct contact. Persello-Cartieux et al. (2001) studied the close relationship of AIA with the promotion and inhibition of root growth. And similar to what was reported by Jiménez-Vázquez et al. (2020), the application of the inoculum at a distance of 2 cm showed repressive ef fects in the case of Aneurinibacillus migulanus, which, being in contact with the plant, increased its production of compounds until it is covered, inhibiting its growth and subsequently causing wilting (Figure 3b), contrary to Staphylococcus sp. and Bacillus cereus that had the ability to stimulate the development of A. thaliana in direct contact (Figure 3a, c, d). In the main root length parameters Endo10(7) and Endo10(5) were 47 and 60% respectively, higher than the control, the number of lateral roots and fresh weight were 116.6 and 100% respectively, more quantity than those that do not have treatment, the plants inoculated with the bacteria had more promotion in these parameters compared to the control (Figure 3e, f. g) (P ≤ 0.05). These results suggest that, although the direct application of A. migulanus is not suitable, it is possible that its promoting ef fect is regulated by bacterial metabolites. While probably in the case of Staphylococcus sp. and B. cereus could be the same or even some promotion by phytohormones.
Figure 3: Effect of the confrontation with Ecto10(6), Endo10(7) and Endo10(5), in A. thaliana (Col-0), by direct contact (contact experiment), grown for 12 days in agar medium 0.2X MS, in (a) without bacteria (Control)., (b-d) Seedlings inoculated with Aneurinibacillus migulanus, Bacillus cereus and Staphylococcus sp., respectively., (e) Root length., (f) Number of side roots., (g) Fresh weight. † Values represent the mean ± standard deviation of 24 seedlings grown on four independent plates. † Different letters in the same column indicate significant differences, according to Tukey's test (P ≤ 0.05).
Studies have been carried out in order to clarify the role of microbial VOCs during positive interactions between plants and microbes (Ortíz-Castro et al., 2009). Gutiérrez-Luna et al. (2010) reported isolates of Bacillus cereus, Bacillus simplex, and Bacillus sp., with the ability to increase plant and root development through VOCs. In our investigation it was shown that Aneurinibacillus migulanus, Staphylococcus sp. and Bacillus cereus favored the plant development of A. thaliana through the emission of VOCs (Figure 4a-d). Endo10(5) was better in the main root length parameter, increasing it by 63.5% (Figure 4e) (P ≤ 0.05), however, the three bacteria influenced a greater number of lateral roots than the control (Figure 4f) (P ≤ 0.05), increasing them 66.6% for Ecto10(6), 83.3% for Endo10(7) and 66.6% for Endo 10(5), finally, the inoculation with the latter increased the fresh weight of the plants 96.9, 90.0 and 106.9% respectively, compared to the control (Figure 4g) (P ≤ 0.05).
Previously, plant promotion of plants such as Arabidopsis by means of VOCs produced by pathogenic bacterial genera, such as Burkholderia cepacia, and Staphylococcus epidermidis, has been reported (Spinelli et al., 2011; Vespermann, Kai and Piechulla, 2007). In our study in the case of promoting plant growth due to the ef fect of volatile organic compounds (VOCs), both Bacillus and Staphylococcus were able to promote the development of A. thaliana. In most of the mechanisms that PGPR uses to interact with plants, the emission of VOCs plays a crucial role.
Figure 4: Effect of confrontation with Ecto10(6), Endo 10(7) and Endo10(5), in Arabidopsis thaliana (Col-0), by volatile organic compounds (VOC's), grown for 12 days in agar medium 0.2X MS, in (a) without bacteria (Control)., (b-d) Seedlings inoculated with Aneurinibacillus migulanus, Bacillus cereus and Staphylococcus sp., respectively., e) Root length., f) Number of side roots., (g) Fresh weight. † The values represent the mean ± standard deviation of 24 seedlings grown in four independent plates. † Different letters in the same column indicate significant differences, according to Tukey's test (P ≤ 0.05).
Germination of S. lycopersicum
In the test that was carried out to determine the ef fect of the inoculation of the isolates on S. lycopersicum seeds, it was observed that those that had received inoculation treatment increased the percentage of germination between 10 to 45%, in relation to the seeds of the control (P ≤ 0.05), which were the ones with the lowest proportion of germination 66%; followed by Endo10(7) with 73%; Endo10(5) with 86%, and the bacterium that provided the greatest stimulus to the seeds was Ecto10(6) with 96 percent.
Shade mesh trial with S. lycopersicum
Although no reference was found in tomato, the genus Staphylococcus has revealed the ability to promote growth through the regulation of ion balance and modulation of the corn response to salinity stress (Shahid et al., 2019); its application has also been related to increased yield in strawberry production (Ipek et al., 2014). Mukhtar et al. (2020) observed that the application of B. cereus in S. lycopersicum seedlings increased the length of its root, fresh and dry biomass and that they agree with those obtained in this study, in which the bacterium also shows promoting activity in these same parameters and our results also show that its inoculation has positively influenced the fresh and dry weight of tomato plants. In this trial (Figure 5a-d) it was observed that the ef ficacy of the inoculation of the isolates in S. lycopersicum was similar to that obtained in A. thaliana, relevant changes were produced in the morphology of the tomato seedlings, which could be due to the stimulation exerted by rhizobacteria, particularly in the variables: plant height, number of lateral roots, total fresh weight and total dry weight, which had higher development when compared to control plants. The height of the plants with the treatment of the isolates was notably higher, in 18.2% for Ecto10(6), 25.1% for Endo10(7), and 31.9% for Endo10(5) (Figure 5e) (P ≤ 0.05), in the case of the number of roots lateral Ecto10(6) increased 14.7% and Endo10(5) 17.6%, while Endo10(7) had similar results to the control (Figure 5f) (P ≤ 0.05); the variable dry weight of S. lycopersicum was increased 91.3% for Ecto10(6), 133% for Endo10(7), and 93.5% for Endo10(5) (Figure 5g, h). In the case of fresh weight, it was increased by 96.9% for Ecto10(6), 123.2% for Endo10(7), and 97.4% for Endo10(5) (Figure 5 i-j).
Figure 5: Effect of inoculation of Ecto10(6), Endo10(7) and Endo10(5), in seeds of S. lycopersicm (2 h), co-inoculated at 20 days of flooding and collected at 30 days. (a) Control without inoculation., (b-d). A. thaliana co-grown with Aneurinibacillus migulanus, Bacillus cereus and Staphylococcus sp., respectively., (e) Plant height., (f) Number of side roots., (g) Foliar dry weight., (h) Root dry weight., (i) Foliar fresh weight., (j) Root fresh weight. † The values represent the mean ± standard deviation of 40 seedlings. † Different letters in the same column indicate significant differences, according to Tukey's test (P ≤ 0.05).
Mechanisms of rhizobacteria
Among the most relevant characteristics of the isolates is the production of IAA from L-tryptophan. What was reported by Hassan and Bano (2015), where they analyzed an isolate of B. cereus with the addition of L-tryptophan, finding ef fectiveness with and without the addition of this, attributing it to the production of IAA and also agree with observed by Prakamhang et al., 2015, where its Bacillus sp. isolate was the largest producer of AIA. This is consistent with our Bacillus cereus isolate Endo10(5), which was able to obtain the highest amount of IAA with 15.12 µg ml-1, followed by Endo10(7) with 7.21 µg ml-1, the one with the lowest concentration was Ecto10(6) with 2.15 µg ml-1 (P ≤ 0.05). Vega, Rodriguez, Llamas, Bejar and Sampedro (2019) observed an increase in shoot length and root length of tomato seeds with the inoculation of Staphylococcus EN21 compared to the non-inoculated control and in our investigation, the co-inoculation of Endo10(7) in A. thaliana showed an increase in root length, which coincides with what was previously found. This indicates that the strains have the ability to produce the indole acetic acid phytohormone using the L-tryptophan metabolic pathway, as a property of PGPR, as observed in the biochemical test for IAA production.
The production of siderophores is another of the mechanisms evaluated and the presence of these compounds in the CAS medium was exposed by the formation of an orange halo around bacterial growth, which was measured to obtain the production index of each bacterium; Endo10(7) had the highest index with 6.5 mm, Ecto10(6) had 2.36 mm and Endo10(5) with the lowest value with 2.25 mm (P ≤ 0.05). The identification of Endo10(7) gave a similarity of 97.89% with Staphylococcus sp., and it was the one that showed a higher index in the production of siderophores, however, it has been little reported with this characteristic, an exception was in a group of bacteria isolated from barley and tomato, studied by Scagliola et al. (2016).
In the determination of phosphate solubilization Bacillus brevis has been previously reported among the potentially solubilizing species (Corrales-Ramírez, Sánchez, Arévalo and Moreno, 2014). The solubilization of phosphates determined through the formation of a clear halo surrounding the bacteria showed us that Ecto10(6), Endo10(5), and Endo10(7) had this ability, the values obtained were 1.67, 2.39, and 7.21 mm (P ≤ 0.05). Being our isolated strain Endo10(7) was identified as Aneurinibacillus migulanus, which showed the most solubilization capacity.
16S rRNA analysis
Using the NCBI database (NCBI, 1988), the sequences obtained were analyzed via BLAST, and by pairing it was determined that Ecto10(6) is similar to Aneurinibacillus migulanus, Endo10(5) is similar to Bacillus cereus and Endo10(7) is similar to Staphylococcus sp. (Table 1). Molecular identification will allow knowing the bacterial genera present in the collection sites and with the potential to be used as PGPR.
Table 1: Molecular identification using the 16S rDNA gene portion and BLAST sequence alignment from the NCBI database (NCBI, 1988).
| Molecular identification of rhizobacteria | ||||
| ID | Nearby species | Identity | Access number NCBI | Bp |
| % | ||||
| Ecto10(6) LB cs | Aneurinibacillus migulanus | 98.41 | KX083693.1 | 1385 |
| Endo10(7) LB cs | Staphylococcus sp. | 99.78 | MT225634.1 | 1395 |
| Endo10(5) LB cs | Bacillus cereus | 98.69 | MH985223.1 | 1377 |
Conclusions
The results of this study show that Bacillus cereus, Aneurinibacillus migulanus, and Staphylococcus sp. have characteristics that help promote plant growth by increasing root development, number of lateral roots and total weight, by activating biochemical mechanisms, such as phosphate solubilization, indole acetic acid production and siderophores, therefore, we can suggest their use as potential biological fertilizers capable of counteracting endogenous and exogenous limitations of plant growth. Although more tests should be done before being recommended as a fertilizer. These results also open the opportunity for future studies measuring the feasibility and ef fect of combining the characterized bacteria in order to demonstrate a broader coverage of response to plant stress.
