Polymeric Formulations of Liquid Inoculants with Rhizobia Exopolysaccharides Increase Survival and Symbiotic Eciency of Bradyrhizobium Strains with Cowpea and Soybean

We studied the survival of four elite strains of Bradyrhizobium in liquid inoculants with three formulations with EPS extracted from other rhizobia genera, and their symbiotic eciency, with soybean and cowpea, in a greenhouse. For this purpose, we veried the utility of formulations for maintaining the cell viability of strains by counting the colony forming units (CFU) per milliliter of the liquid inoculants with formulations over 90 days. Survival of the soybean inoculant strains, 29W and CPAC15, in the PEPS formulation had the largest number of CFU (> 10 10 mL − 1 ) after 90 days. For the cowpea inoculant strains, INPA3-1B and UFLA3-84, the formulations REPS 1 had the largest number of CFU (> 10 10 mL − 1 ) after 90 days. Symbiotic eciency in soybean of the formulations PEPS and REPS 2 was higher than that shown by the commercial inoculant. For cowpea, the three formulations with EPS showed symbiotic eciency bigger than that of the commercial inoculant.


Introduction
The production systems of the soybean [Glycine max (L.) Merr.] and cowpea [Vigna unguiculata (L.) Walp.] crops require large amounts of nitrogen. Biological nitrogen xation (BNF) is a successful biotechnology responsible for a less expensive supply of N to these crops due to the use of inoculants with selected rhizobia strains.
Under laboratory conditions, a rhizobia strain may manifest considerable symbiotic e ciency, but to reproduce similar results under eld conditions, the use of vehicles capable of effectively transporting a maximum of viable bacterial cells from industry to the eld is necessary. Inoculants are formulations generally commercialized in powder, liquid, or granulated forms. However, most inoculants currently on the South-American market are liquid formulations. These formulations were developed to resolve problems associated with solid formulations (for example, the scarcity or limitation of natural peat deposits) or with their high cost of sterilization, as well as the di culty of application with planting equipment (Stephens and Rask 2000;Tittabutr et al. 2007; Albareda et al. 2008).
Liquid inoculant formulations have big advantages, such as easy handling and management to obtain speci c formulations, as well as greater shelf life and lower contamination. These advantages over peat formulations stimulate the development and application of liquid inoculant formulations, which has consolidated the market for this biotechnology (Sehrawat et al. 2017). Among the main characteristics that make for a good vehicle for liquid inoculants are low perishability, atoxicity, high water retention capacity, chemical and physical uniformity, and rapid release of rhizobia in the soil, as well as capacity for retaining sources of energy and nutrients able to support the survival and growth of the rhizobia (Sehrawat et al. 2017; Albareda et al. 2008). At the same time, these materials should be easy to obtain, abundant, and economically viable (Bashan et al. 2014). Liquid inoculants are microbial cultures or suspensions modi ed with chemical substances that are added to improve the rheological properties of aqueous systems, as well as to promote the formation of lms that assist in adherence and physical protection of Bacterial exopolysaccharides (EPS) are high molecular weight polymeric substances constituted by sugar monomers (Sutherland 2001;Donot et al. 2012) that are produced by several genera, including those belonging to N 2 -xing Leguminosae nodulating genera, collectively known as rhizobia. The EPS form an external layer that surrounds the bacterial cells, thus favoring cell interactions, retention of extracellular enzymes, and sorption of particles and nutrients dissolved in the aqueous phase, allowing them to be used as energy and nutrient sources. These natural, atoxic, and biodegradable polymers not only perform these functions, but also play an essential role in protection of cells against desiccation, predation, and toxic effects of chemical agents and secondary metabolites, as well as in formation of bio lms (Flemming and (Donot et al. 2012). They are also used as gelling agents to improve food quality and texture (Castellane et al. 2015). Therefore, rhizobia EPS may represent a viable vehicle in the formulation of liquid inoculants to substitute synthetic polymers.
In this study, we evaluated the survival of Bradyrhizobium elite strains in formulations with exopolysaccharides isolated from previously selected rhizobia strains as a vehicle. We also tested these formulations in the symbiosis of these elite strains with soybean and cowpea. The strains were cultivated, until log phase (±10 9 CFU mL -1 ), in 79-medium (Fred and Waksman 1928), also called YMA (Vincent 1970) (10 g L -1 of mannitol, 0.01 g L -1 of K 2 HPO 4 , 0.04 g L -1 of KH 2 PO 4 , 0.05 g L -1 of MgSO 4 .7H 2 O, 0.01 g L -1 of NaCl, 0.4 g L -1 of powdered yeast extract, and 15 g L -1 of agar, with pH adjusted to 6.8-7.0), at 28°C.

Exopolysaccharides used
In this study, we used three exopolysaccharides: PEPS, extracted from Paraburkholderia sp. -UFLA 04-269, isolated from Macroptilium atropurpureum in rocky eld soils (Araújo et al. 2017); REPS 2 , extracted from Rhizobium tropici -UFLA 05-16, isolated from Crotalaria spectabilis in gold mining soils (Rangel et al. 2017); and REPS 1 , extracted from Rhizobium tropici -CIAT 899 T , isolated from acid soils of South America (Graham et al. 1994). In previous studies (data not published), these three EPS exhibited physicochemical characteristics that make them excellent vehicles for the formulation of liquid inoculants.

EPS Extraction
After growth at liquid 79-medium for six days, bacterial cells were removed from the culture medium by centrifugation at 10,000 g for 10 minutes at 4°C, and cold 96% ethanol (4°C) was added to the cell-free supernatant mixture at a ratio of 3:1 (v/v) (Castellane et al., 2014). At this stage, the formation of a supernatant gel and a precipitate was immediately observed. The mixture was cooled to 4°C for 24 h. Ethanol was evaporated in a drying oven at 60°C. The precipitation solvent enabled partial puri cation of the polymer by eliminating the soluble components of the culture medium (Castellane & Lemos, 2007).
The precipitated product was dried to a constant weight using a Labconco FreeZone 2.5 lyophilizer to verify the number of EPS obtained (g of EPS per L of culture medium).

Antibacterial trial of the three EPS
We tested the possible antibacterial activity of the EPS to the elite inoculant strains 29 W, CPAC 15, INPA3-11B, and UFLA3-84 according to the procedures described by Bauer et al. (1966), with small modi cations. In short, each strain was grown in solid 79-medium in a plate and exposed to a sterilized disk impregnated with 200 µL of exopolysaccharide solution (10 mg mL -1 ) and to control treatments with sterile saline solution (disk imprenated with NaCl 0.85%, w/v) and two antibiotics (chloramphenicol and sulfazotrim). Antibiotics were made available in commercial disks at the concentration of 1 mg mL -1 . These antibiotics were used as positive controls of antibacterial activities. Each strain was tested in triplicate. After incubation of the dishes at 28°C over a period of 144 h, the inhibition zone diameter was measured.

Preparation of liquid polymeric formulations of an exopolysaccharide base
We used a modi ed 79-medium as a solution succinctly described as 40 g L -1 mannitol and 10 g L -1 glycerol as carbon sources, and 10 mg L -1 Fe-EDTA, 0.02 mg L -1 CuSO 4 .5H 2 O, and 0.01 mg L -1 H 2 MoO 4 .H 2 O as metal ions. After performing tests with different EPS-concentrations (2%, 1%, 0.5%, and 0.1%), we found the ideal concentration of each one of the EPS in the liquid formulations was 0.1% (w/v). We prepared the formulations from each one of the exopolysaccharides, which constituted three formulations of liquid inoculants: formulation 1 -REPS 1 ; formulation 2 -PEPS; formulation 3 -REPS 2 ; as well as a reference formulation with the original 79-medium (10 g L -1 mannitol) without the metal ions and the EPS. After preparation, the formulations were sterilized in an autoclave at 120°C at 1.0 kg cm 2 . Standardization of the concentration of the inocula was carried out as described above.
Aliquots of 0.1 mL of the culture of the strains 29 W, CPAC 15, INPA3-11B, and UFLA3-84 were inoculated in each one of the formulations. After that, the formulations were transferred to cone-bottom polypropylene centrifuge tubes, with a leak-proof elongated lid and 50 mL capacity, previously sterilized by gamma radiation. The samples were kept in an orbital shaker at 110 rpm and 28°C.

Evaluation of rhizobia growth and survival in the different liquid formulations
The growth and survival of the Bradyrhizobium strains in each liquid formulation described above was evaluated at 5, 10, 20, 30, 60, and 90 days of storage at ambient temperature (28°C). The number of viable cells was determined by counting of colony forming units, using the serial decimal dilution method, in which a 1.0 mL aliquot of each formulation was successively diluted from 10 -1 to 10 -8 in saline solution (0.85%, w/v). After that, 20 µL aliquots were inoculated on Petri dishes with 79-medium through the microdrop technique and then dried in a bacteriological chamber for 20 minutes before being inverted and then incubated at 28°C for 144 hours. Evaluations were made in quadruplicates. The number of colony forming units per milliliter was calculated using the the following formula:

Evaluation of EPS enriched inoculants in symbiosis with cowpea and soybean in a greenhouse
The studies were conducted in a greenhouse at the Instituto Federal do Maranhão, Campus São Luís -Maracanã, São Luís, MA, Brazil, from May to June 2017 to evaluate the performance of the liquid formulations of inoculants after one month of storage. The soil used, a Latossolo Amarelo Distró co típico (Oxisol) (Santos 2018), was collected at a depth of 0 to 20 cm. The soil was air dried, sieved in a four-millimeter mesh, homogenized, and placed in 8 kg pots. Soil samples were collected for chemical and textural characterization with the following results: pH of 4.8; organic matter of 1.97 dag kg -1 ; K, P, and S of 21.36, 2.78, and 4.5 mg dm -3 , respectively; Ca, Mg, Al, H+Al, SB, t, and T of 0.73, 0.15, 0.70, 4.37, 0.93, 1.63, and 5.30 cmol c dm -3 , respectively; V and m of 17.64 and 42.94 %, respectively; Zn, Fe, Mn, Cu, and B of 1.00, 232.74, 1.61, 0.19, and 0.07 mg dm -3 , respectively; and clay, silt, and sand fractions of 10, 3, and 87 dag ka -1 , respectively. Soil pH was determined in water at the soil:water proportion of 1:2.5; H+Al was determined by the Ca(OAc) 2 method in 0.5 mol L -1 ; pH was 7.0; exchangeable Ca 2+ , Mg 2+ , and Al 3+ were extracted with 1 mol L -1 KCl and determined by titration; P and K were extracted by Mehlich-1 and analyzed by colorimetry (P) and ame photometry (K); organic carbon was determined by oxidation with potassium dichromate; and Zn, Mn, and Cu were extracted by Mehlich-1 and determined by atomic absorption spectrophotometry. The

Statistical analysis
The data were analyzed using the statistical analysis software Sisvar® (Ferreira, 2019). Before analysis of variance (ANOVA), the presupposed requirements of normality and homogeneity were checked. Firstly, when data evidenced lack of normality, they were transformed (Y+0.5) 0.5 . The differences between the treatments were compared by the Scott Knott test (p < 0.01 or p < 0.05).

Results
Antibacterial trial of the three EPS The possible antibacterial activities of the PEPS, REPS1, and REPS2 against 29 W, CPAC 15, INPA3-11B, and UFLA3-84 were compared to chloramphenicol and sulfazotrim as positive controls with these same strains. The results show that the EPS do not exhibit antibacterial activity over the strains evaluated, i.e., no inhibition zone was detected. In contrast, the chloramphenicol exhibited antibacterial activities signi cantly (p < 0.05) greater than sulfazotrim in all the strains evaluated Evaluation of rhizobia growth and survival in the different liquid formulations As shown in Table 1, the PEPS formulation is the most important in maintaining survival of the strains 29 W and CPAC 15 soybean inoculants. The number of cells of 29 W and of CPAC 15 in the PEPS formulation is greater than that of the commercial inoculant and of the reference inoculant (Fig. 1). For cowpea, Table 2 shows that the formulations REPS 1 and PEPS are most important in maintaining survival of the strain INPA3-11B. For UFLA3-84, the best formulation is REPS 1 . The number of cells of the two strains in the formulations is greater than that of the commercial inoculant and of the reference inoculant (Fig. 2).
The results show that there was no decline in cell viability of the strains studied either for soybean or for cowpea in the most signi cant formulations. At 90 days of storage, growth still remains in the logarithmic phase ( Fig. 1 and  2). The three exopolysaccharides studied, PEPS extracted from Paraburkholderia sp. -UFLA 04-269, REPS 2 extracted from Rhizobium tropici -UFLA 05-16, and REPS 1 extracted from R. tropici -CIAT 899 T , signi cantly contributed to the maintenance of cells viability.

Study in a greenhouse
At 45 days after germination, in the study of formulations with the strain 29 W, soybean plants inoculated with the commercial inoculant exhibit the highest number of nodules and nodule dry matter (NN and NDM). For shoot dry matter and relative e ciency, the formulations PEPS, REPS 2 , and the commercial inoculant exhibit the second greatest mean value observed; the treatment with the highest mean value is the control with nitrogen. The PEPS formulation exhibits the highest shoot nitrogen concentration and accumulation (SNC and SNA) among the formulations, with values near the treatment with nitrogen (Table 3). For the study of formulations with the strain CPAC 15, the formulations BESP, REPS 2 , and the commercial inoculant exhibit a similar number of nodules (p < 0.05). For the other variables of symbiotic e ciency, the two formulations with EPS exhibit mean values higher than the other formulations, but with a mean value lower than the control with nitrogen (Table 3). Relative e ciency of the formulations REPS 2 and PEPS is equal to or greater than 90% for the two elite strains for soybean.
For cowpea, in the study of formulations with the strain INPA3-11B, the formulation REPS 1 exhibits the highest number of nodules and nodule dry matter (NN and NDM). For shoot dry matter (SDM) and shoot nitrogen concentration (SNC), the formulations with EPS and the commercial inoculant exhibit similar values (p < 0.05); nevertheless, the highest means observed are in the control with nitrogen. For shoot nitrogen accumulation (SNA), in the formulation with REPS 1 , the mean value observed is higher than the other formulations, and is below only the control treatment with nitrogen ( Table 4). The relative e ciency observed of the REPS 1 formulation is 85.5%. For the study of the formulations with the UFLA3-84 strain, the formulations PEPS and REPS 2 exhibit the highest number of nodules and nodule dry matter (NN and NDM). For the other variables, these two formulations have values below only the control treatment with nitrogen ( Table 4). The values observed for relative e ciency of the PEPS and REPS 2 formulations are 81.2 and 78.5 %, respectively.

Discussion
The EPS studied here can be used as carrier polymers in inoculant formulations, because they do not affect rhizobia survival. Studies show that exopolysaccharides can exhibit antibacterial activities, as shown by (Zhang et al. 2016) in EPS of the GST-6 strain studied in Escherichia coli, Shigella exneri, S. typhimurium, and S.aureus. Another study shows that EPS that have sulfate groups have antibacterial activity in E. coli -ATCC 25922 and in Staphylococcus aureus -CMCC 26003 (Li and Shah 2014). The exopolysaccharides REPS1, PEPS, and REPS2 studied here do not have chemical groups described as antibacterial in a previous study (data not published), and the in vivo tests con rmed these results. Probably due to the a nity with rhizobia strains because they were extracted from other rhizobia genera and species.
The success of a new formulation mainly depends on the use of adequate vehicles (Bashan et al. 2014). In this study, three potential formulations for Bradyrhizobium in soybean and cowpea are evaluated using bacterial exopolysaccharides as carriers. According to our results, the PEPS formulation, in B. elkanii -29 W and B. japonicum -CPAC 15, and REPS 1  In some of our formulations, we observed better responses in relation to all the growth parameters in comparison to the treatment inoculated with commercial inoculant (Table 4). Other studies with CMC, PVP, and sodium alginate in polymeric formulations of inoculants show a signi cant increase in most of the growth parameters (Sehrawat et al. 2017). Better responses in plant growth parameters (number of nodules, nodule dry matter, shoot dry matter, relative e ciency, and shoot N concentration and accumulation) are more pronounced in the PEPS and REPS 2 formulations. The EPS of the two R. tropici strains behaved differently probably due to different genotypic differences. Although CIAT899 genome is already available, UFLA5-16 has only its 16SrRNA partially sequenced, however, genome sequencing is being planned to detect among others, possible genetic differences related to this feature.
We concluded that exopolysaccharides can be used in formulations of liquid inoculants as a vehicle that can support rhizobia cell growth, as well as provide protection and stability during storage in the period and N2e ciency in soil conditions. It is also amazing that EPS from different genera (Paraburkholderia and Rhizobium) have a positive effect on Bradyhizobium strains. The results of the t test for variance among the means (±standard error) from observations of the study of the growth of inoculant strains in different formulations showed signi cant (p < 0.01) differences. Among lines, the values with different letters are signi cantly different. The results of the t test for variance among the means (± standard error) from observations of the study of growth of inoculant strains in different formulations showed signi cant (p < 0.01) differences. Among lines, the values with different letters are signi cantly different.   The number of cells of 29 W and of CPAC 15 in the PEPS formulation is greater than that of the commercial inoculant and of the reference inoculant (Fig. 1).