Agronomic Efficiency of Signum Inoculant in Pre-inoculation of Soybean at 35 and 20 Days Before Sowing in Treated Seeds

Pre-inoculating soybean seeds can make sowing faster and provide additional benefits to farmers. However, it needs to guarantee the nitrogen supply to maintain the viability and sustainability of the technique. In this study, we evaluated the agronomic efficiency of SIGNUM ® inoculant in the pre-inoculation at 20 and 35 days before sowing chemically treated soybean seeds. Experiments were conducted in four field experiments located at Paraná, Brazil, with a history of soybean cultivation managed under no-tillage systems, with crop rotation according to regional edaphoclimatic conditions. Agronomic efficiency in fields were compared with standard inoculation with a registered product used by farmers. Chemical treatment of soybean seeds with Standak Top ® or Maxin XL ® + Cruiser ® associated with pre-inoculation of the inoculant SIGNUM ® for 25 and 30 days reduced the concentration of viable Bradyrhizobium cells recovered from seeds. However, no significant difference was observed regarding nodulation, biological nitrogen fixation, and yield between the soybean inoculated with standard inoculation on farm or pre-inoculation with SIGNUM ® in most studied areas.

Pre-inoculation allows farmers to dedicate themselves to the sowing operation, without having to deal with inoculation on the day of sowing, which is usually time-consuming and labor-intensive (Hungria et al., 2020). Some studies have reported the success of pre-inoculation practice in soybean. Anghinoni et al. (2017) and Silva et al. (2018) results indicated the storage of pre-inoculated seeds up to 10 days without affecting yield components; Hungria et al. (2020) reported the feasibility of pre-inoculation for a period of 15 days in soybean; and Machineski et al. (2018) reported the pre-inoculation of soybean seeds up to 60 days before sowing. However, the practice needs to guarantee the presence of viable bacteria at adequate concentrations in the seeds at the time of sowing, even after the necessary chemical treatment and seed storage. SIGNUM ® inoculant, according to manufacturer (Rizobacter of Brazil), is based on bioinducer technology, which stimulates bacteria to produce multiple determinants of nodulation, improving communication between bacteria and the root, and osmoprotection technology, that improves the physiological state and the resistance of bacteria, increasing their survival on the seed. Thus, this study aimed to evaluate the agronomic efficiency of SIGNUM ® inoculant in pre-inoculation at 20 and 35 days before sowing chemically treated soybean seeds.

Viability and Purity of Inoculants
The inoculant SIGNUM ® (Rizobacter do Brasil, Paraná, Brazil), which consists of a liquid inoculant composed of the strains Semia-5079 and Semia-5080, with a concentration of viable cells, according to the manufacturer's information, of 1.0 × 10 9 colony forming units (CFU) of Bradyrhizobium japonicum per mL of inoculant, was tested in pre-inoculation of soybean. The inoculant RIZOLIQ ® (Rizobacter do Brasil, Paraná, Brazil), consisting of Bradyrhizobium japonicum (Semia-5079 and Semia-5080 strains), with a concentration of viable cells of 5.0 × 10 9 CFU mL -1 of inoculant, according to the manufacturer's information, was used as a standard control inoculant.
The quality of inoculants was evaluated by counting viable cells by the droplet scattering method and counting Bradyrhizobium japonicum colony-forming units in YMA (yeast, mannitol, and agar) solid culture medium in Petri dishes. Analyses of pH and the presence of contaminants in the inoculants were also carried out by determining CFU of contaminants in two dilution series, as recommended in the Normative Instruction SDA/MAPA 30/2010, article 4 (DOU 11/17/2010) (MAPA, 2010).

Description, Characterization, and Preparation of Experimental Areas
Four experimental areas with different edaphoclimatic conditions but suitable for soybean cultivation in the 2017/2018 agricultural season were selected. The experimental areas are located in the municipalities of Londrina (23°21′26″S and 51°10′08″W, 565 m altitude), Santa Tereza do Oeste (25°05′19″S and 53°35′19″W, 750 m altitude), Pato Branco (26°07′32″S and 52°38′56″W, 720 m altitude), and Ponta Grossa (25°09′04″S and 50°09′14″W, 865 m altitude), and belong to the Instituto de Desenvolvimento Rural do Paraná IAPAR-EMATER (IDR-PR), with a history of soybean cultivation managed under the no-tillage system, with crop rotation according to regional edaphoclimatic conditions. Climatic data of all experimental areas are in Figure 1.  Vol. 14, No. 6; Soil chemical characterization of each location was carried out at the Laboratory of Soil and Tissue of IDR-PR, following the methodology of Pavan et al. (1992) (Table 1). Soil correction and fertilization were carried out based on the chemical analysis results and according to the technical recommendation for soybean fertilization (Moreira et al., 2017). Soil fertilization was performed with NPK fertilizer (0-20-20).   , 2010). One negative control, composed of uninoculated soybean seedlings, and one positive control, with inoculation of seedlings with the SEMIA 5080 strain, recommended for soybean inoculation, were added to these assays.

Treatments, Inoculation, Sowing, and Conduction of Experiments
The experimental design in all areas was randomized blocks with four replications and plots of 4.05 × 6 m (24.3 m 2 ) with nine sowing rows of 6 m in length, spaced at 0.45 m and density of 12 plants per linear meter. The treatments consisted of T-I Control without chemical seed treatment and without inoculation; T-II Standard inoculation with RIZOLIQ ® on the day of sowing in untreated seeds; T-III Standard inoculation with RIZOLIQ ® on the day of sowing in seeds treated with Standak Top ® ; T-IV Standard inoculation with RIZOLIQ® on the day of sowing in seeds treated with Maxin XL ® + Cruiser ® ; T-V Inoculation with SIGNUM ® at 35 days before sowing in seeds treated with Standak ® Top; T-VI Inoculation with SIGNUM ® at 35 days before sowing in seeds treated with Maxin XL ® + Cruiser ® ; T-VII Inoculation with SIGNUM ® at 20 days before sowing in seeds treated with Standak ® Top; and T-VIII Inoculation with SIGNUM ® at 20 days before sowing in seeds treated with Maxin XL ® + Cruiser ® .
The soybean varieties were BMX Potência RR in the assays carried out in Londrina and Pato Branco and BMX Apolo RR in the assays carried out in Santa Tereza do Oeste and Ponta Grossa, both indicated for cultivation in these regions of the Paraná State. The seeds showed a germination vigor above 85%. The seeds were chemically treated with the fungicide and insecticide Standak ® Top (pyraclostrobin, thiophanate methyl, and fipronil; 225, 250, and 25 g ai L -1 , respectively, SF, Basf) at a dose of 2 mL kg -1 seed; the fungicide Maxin XL ® (metalaxyl-M and fludioxonil; 10 and 25 g ai L -1 , respectively, FS, Syngenta) at a dose of 1 mL kg -1 seed; and the insecticide Cruiser ® 350FS (thiamethoxam; 350 g ai L -1 , FS, Syngenta) at a dose of 2 mL kg -1 seed. The seeds were inoculated according to the manufacturer's instructions described on the inoculant package. The treated seeds from treatments V to VIII were pre-inoculated at 20 and 35 days pre-sowing, as defined for each treatment, stored in paper bags, and maintained at room conditions (25±3 °C) protected from sunlight and humidity until the time of sowing.
The experiments were set up following the agroclimatic zoning of each region (MAPA, 2015) and meeting the soil moisture conditions suitable for sowing. Sowing was carried out on October 27 in Londrina, November 1 in Santa Tereza, November 6 in Pato Branco, and November 26, 2017, in Ponta Grossa. Cultural practices to manage weeds, pests, and diseases were carried out in accordance with agronomic practices recommended for soybean cultivation (Pas Campo, 2005).

Bradyrhizobium sp. Survival in Inoculated Seeds
The survival of Bradyrhizobium sp. inoculated to the seeds was determined at the time of sowing by evaluating the recovery of viable cells of Bradyrhizobium sp. on the seed surface through plating in semi-selective Ikuta culture medium (Ikuta, 1995) and CFU counting, as recommended (MAPA, 2010). The data were transformed into Log 10 for statistical analysis.

Assessments
Five soybean plants were collected at flowering (phenological stage R1) from each treatment and the number of nodules per plant, nodule dry mass per plant, root and shoot dry mass, and shoot nitrogen (N) concentration were evaluated according to the methodology described by Miyazawa et al. (1992). Grain production in the experimental plots and N concentration in grains  were evaluated after 50% of the plant population reached the R8 phenological stage. Yield and nitrogen exported by the crop in the grains were calculated using a population density of 200 thousand plants ha -1 . Grain moisture was corrected to 13% moisture and grain yield was expressed in kg ha -1 . The variables were analyzed according to MAPA Normative Instruction No. 30 of 2010 (DOU 11/17/2010) (MAPA, 2010).
The results were subjected to analysis of variance (p ≤ 0.05). The means of viable bacterial cells in the seeds, nodulation (number of nodules and nodule dry mass), biomass (root and shoot dry mass), N concentration in shoots and grains, and yield were compared pairwise with the treatment standard inoculation (treatment II: inoculation at seeding with RIZOLIQ ® without chemical treatment) by bilateral Dunnett's test (p ≤ 0.05) using SAS software (SAS Institute Inc. 2019).

Inoculant Quality
The evaluation of viable cells (CFU-colony forming units) of Bradyrhizobium japonicum in the inoculants used in the experiment is shown in Table 2. Note. *Bradyrhizobium japonicum (Semia 5079 and Semia 5080 strains).
The mean number of Bradyrhizobium japonicum cells was 2.83 × 10 10 CFU mL -1 of inoculant, meeting the requirement of Brazilian legislation, which establishes a minimum concentration of 1.0 ×10 9 (CFU) per gram or milliliter of product for inoculants with nitrogen-fixing bacteria for symbiosis with legumes, according to art. 1 of the Normative Instruction No. 13, of March 24, 2011. The inoculants RIZOLIQ ® (standard inoculation at sowing), SIGNUM ® (inoculation at 20 days pre-sowing), and SIGNUM ® (inoculation at 35 days pre-sowing) presented pH values of 7.6, 7.6, and 7.5, respectively. The inoculants did not present contaminants above 10 2 CFU mL -1 in two dilution series, meeting the standard described in art. 1, item IV, in Normative Instruction No. 13, of March 24, 2011, which requires inoculants free of unspecified microorganisms at a concentration of 1.0 × 10 5 mL -1 of inoculant.

Naturalized Population of Rhizobia in the Experimental Areas
All areas showed a high naturalized population of soybean-nodulating rhizobia, ranging from 1.08 to 8.37 × 10 6 bacteria g -1 soil ( Table 3). The history of regular cultivation of inoculated soybean in these areas may be related to this high population of rhizobia in the soil.

Survival of Bradyrhizobium sp. in Inoculated Seeds
The recovery of viable Bradyrhizobium sp. cells on the surface of inoculated seeds is shown in Table 4. Recovery of viable Bradyrhizobium sp. cells on the surface of seeds inoculated with RIZOLIQ ® on the day of sowing without treatment or with chemical treatments with Standak Top ® or Maxim XL ® + Cruiser ® ranged from 1.6 to 2.91 × 10 5 CFU seed -1 . Seeds pre-inoculated with SIGNUM ® at 20 and 35 days before sowing showed values of Bradyrhizobium japonicum cell recovery on seeds ranging from 0.95 to 4.25 × 10 3 CFU seed -1 , regardless of the chemical treatment, which is statistically lower than the values observed in seeds inoculated with RIZOLIQ ® in the sowing without treatment (Table 4).

Agronomic Efficiency in the Field
In the field experiment carried out in Londrina, the variables number of nodules, nodule dry mass, root dry mass, shoot dry mass, shoot nitrogen, nitrogen exported in the grains, and yield showed no significant difference between treatments compared with the standard inoculation (Table 5). Only the nitrogen content of soybean grains pre-inoculated with SIGNUM ® at 35 days before sowing and treated with Standak Top ® (T-V) was significantly higher than the content observed in the standard inoculation treatment (T-II).  Note. T-I: no seed treatment and no inoculation; T-II: standard inoculation at sowing with RIZOLIQ ® ; T-III: standard inoculation with RIZOLIQ ® and seed treatment with Standak Top ® ; T-IV: standard inoculation with RIZOLIQ ® and seed treatment with Maxin XL ® + Cruiser ® ; T-V: pre-inoculation with SIGNUM ® at 35 days before sowing and seed treatment with Standak Top ® ; T-VI: pre-inoculation with SIGNUM ® at 35 days before sowing and seed treatment with Maxin XL ® + Cruiser ® ; T-VII: pre-inoculation with SIGNUM ® at 20 days before sowing and seed treatment with Standak Top ® ; T-VIII: pre-inoculation with SIGNUM ® at 20 days before sowing and seed treatment with Maxin XL ® + Cruiser ® . Means followed by * differ significantly from treatment T-II (standard inoculation) by bilateral Dunnett's test at p ≤ 0.05.
Santa Tereza do Oeste also showed that the variables number of nodules, nodule dry mass, shoot dry mass, and nitrogen in grains (g kg -1 ) did not present significant differences between treatments compared to the standard inoculation (Table 6). Soybean inoculated with RIZOLIQ ® (standard inoculation T-III) in seeds treated with Standak Top ® showed a reduction in root dry mass compared to that which received standard inoculation in seeds without treatment (T-II). Treatments T-I, T-III, T-V, T-VII, and T-VIII resulted in lower shoot nitrogen contents compared to the values observed in T-II. However, treatments T-I, T-V, and T-VIII showed higher contents of N exported by grains (kg ha -1 ). Treatments T-I, T-III, T-V, T-VI, and T-VIII promoted higher grain yields compared to T-II (Table 6). Note. T-I: no seed treatment and no inoculation; T-II: standard inoculation at sowing with RIZOLIQ ® ; T-III: standard inoculation with RIZOLIQ ® and seed treatment with Standak Top ® ; T-IV: standard inoculation with RIZOLIQ ® and seed treatment with Maxin XL ® + Cruiser ® ; T-V: pre-inoculation with SIGNUM ® at 35 days before sowing and seed treatment with Standak Top ® ; T-VI: pre-inoculation with SIGNUM ® at 35 days before sowing and seed treatment with Maxin XL ® + Cruiser ® ; T-VII: pre-inoculation with SIGNUM ® at 20 days before sowing and seed treatment with Standak Top ® ; T-VIII: pre-inoculation with SIGNUM ® at 20 days before sowing and seed treatment with Maxin XL ® + Cruiser ® . Means followed by * differ significantly from treatment T-II (standard inoculation) by bilateral Dunnett's test at p ≤ 0.05.
In Ponta Grossa, the variables number of nodules, nodule dry mass, root dry mass, nitrogen in the shoot, nitrogen exported by the grains, and soybean yield showed no significant difference between treatments compared to the standard inoculation. Soybean presented lower shoot dry mass than in treatment T-II only in treatment T-VIII (Table 7).  Note. T-I: no seed treatment and no inoculation; T-II: standard inoculation at sowing with RIZOLIQ ® ; T-III: standard inoculation with RIZOLIQ ® and seed treatment with Standak Top ® ; T-IV: standard inoculation with RIZOLIQ ® and seed treatment with Maxin XL ® + Cruiser ® ; T-V: pre-inoculation with SIGNUM ® at 35 days before sowing and seed treatment with Standak Top ® ; T-VI: pre-inoculation with SIGNUM ® at 35 days before sowing and seed treatment with Maxin XL ® + Cruiser ® ; T-VII: pre-inoculation with SIGNUM ® at 20 days before sowing and seed treatment with Standak Top ® ; T-VIII: pre-inoculation with SIGNUM ® at 20 days before sowing and seed treatment with Maxin XL ® + Cruiser ® . Means followed by * differ significantly from treatment T-II (standard inoculation) by bilateral Dunnett's test at p ≤ 0.05.
In Pato Branco, the variables nodule dry mass, shoot dry mass, nitrogen in the shoot, nitrogen exported by the crop, and soybean yield did not present significant differences between treatments compared with the standard inoculation (T-II) ( Table 8). Soybean with seeds treated with Standak Top ® and inoculated with RIZOLIQ ® at sowing (T-III) had a higher number of nodules than the soybean under standard inoculation (T-II). Treatments T-IV, T-VI, and T-VII promoted a significant reduction in soybean root dry mass. Treatment T-VI significantly increased the N content of soybean grains. Note. T-I: no seed treatment and no inoculation; T-II: standard inoculation at sowing with RIZOLIQ ® ; T-III: standard inoculation with RIZOLIQ ® and seed treatment with Standak Top ® ; T-IV: standard inoculation with RIZOLIQ ® and seed treatment with Maxin XL ® + Cruiser ® ; T-V: pre-inoculation with SIGNUM ® at 35 days before sowing and seed treatment with Standak Top ® ; T-VI: pre-inoculation with SIGNUM ® at 35 days before sowing and seed treatment with Maxin XL ® + Cruiser ® ; T-VII: pre-inoculation with SIGNUM ® at 20 days before sowing and seed treatment with Standak Top ® ; T-VIII: pre-inoculation with SIGNUM ® at 20 days before sowing and seed treatment with Maxin XL ® + Cruiser ® . Means followed by * differ significantly from treatment T-II (standard inoculation) by bilateral Dunnett's test at p ≤ 0.05.

Discussion
The four studied areas had the establishment of a naturalized population of Bradyrhizobium sp. capable of nodulating soybean in the order of 10 6 bacteria g -1 soil (Table 3) due to the history of annual soybean production. Soybean is a species introduced in Brazil from Asia, hindering the occurrence of native rhizobia of species symbiotic to soybean (Lima et al., 1998). The symbiosis is the result of an evolutionary process lasting millions of years between the bacterium and the plant, and it is at its center of origin (Asia) that native bacteria are also found (Hungria et al., 2007).
However, intensive and continuous cultivation with inoculated seeds, as in the experimental areas used in this study, may allow bacteria from the inoculant to survive in the soil, establish and "naturalize," forming a population capable of competing for sites of root infection with those inoculated and nodulate soybean (Mendoza-Suárez et al., 2021), fixing nitrogen for the plant (Campos & Gnatta, 2006).
In this study, the control treatments (without inoculation) presented nodulation, BNF, and yields similar to the other inoculated treatments (Tables 4 to 7). These results are similar to other studies where soybean inoculation did not increased yields in sites where soybean was previously grown (Carciochi et al., 2019;De Bruim et al., 2010). Mendes et al. (2014) suggested that soybean selects a specific microbial community that inhabits the rhizosphere based on functional characteristics, which may be related to benefits to the plant, such as growth and nutrition promotion, such as rhizobia. Furthermore, the authors reported that long-term cultivation in an area strengthens this selective power of the crop for beneficial microorganisms for the plant.
below the recommended by the research (6.0 × 10 5 cells per seed) to guarantee the success of nodulation and BNF (Hungria et al., 2007;RELARE, 2014). Although, in Brazil there is no official definition of a minimum limit value of bacterial cells recovered from seeds established by the Ministry of Agriculture, Livestock, and Food Supply. The storage of seeds inoculated with SIGNUM ® technology and treated with Standak Top ® or Maxin XL ® + Cruiser ® in pre-inoculation treatments at 20 and 35 days before sowing reduced the survival of bacterial cells in the seeds. This reduction in the number of cells recovered from treated seeds is a concern that has been reported in other studies (Costa et al., 2013;Pereira et al., 2010). Some authors have reported that this reduction is related to the toxicity of phytosanitary products used in seed treatment for bacteria present in inoculants (A. S. F. D. Araújo & R. S. Araújo, 2006;Hartley et al., 2012;Marks et al., 2013), characterized as a challenge to be overcome to guarantee the efficiency of the soybean inoculation technology.
The low recovery of cells in seeds in all treatments did not reflect losses to nodulation. Soybean nodulation (number of nodules and nodule dry mass) was adequate in all locations (Tables 4 to 7). According to Hungria et al. (2007), a well-nodulated soybean plant at the time of flowering should have between 15 to 30 nodules or 100 to 200 mg of dry nodules per plant.
Although some treatments in all locations showed differences in the root or shoot dry mass compared to the standard inoculation, all treatments showed yield and nitrogen exported in the grains similar or even higher than the standard inoculation (Tables 4 to 7). These results indicate that pre-inoculation with SIGNUM ® technology promoted plant development, BNF, nodulation, and soybean production. Furthermore, all treatments in the experiments of Londrina, Ponta Grossa, and Santa Tereza do Oeste showed higher yields than the national mean for the 2017/18 growing season (i.e., 3,394 kg ha -1 ) and the mean for the Paraná State (i.e., 3,508 kg ha -1 ), which has the highest national yield. Although the yield in Pato Branco was above 2,000 kg ha -1 , as recommended to validate tests with soybean by MAPA Normative Instruction No. 30 of 2010 (DOU 11/17/2010) (MAPA, 2010), the values were below the national and state means. In this case, yield may have been affected by the 25-day drought period between December 2017 and January 2018, a critical period for soybean, as flowering and the beginning of grain filling occur during this period, which may have led to a reduction in production.
Pre-inoculation technologies have already been reported as successful in several studies, aiming to optimize the sowing process and stimulate and expand the use of inoculants in soybean cultivation (Gemell et al., 2005;Anghinoni et al., 2017;Machineski et al., 2018;Hungria et al., 2020). In this study, pre-inoculation with SIGNUM ® at 20 or 35 days before planting did not differ from the treatment with standard inoculation, regardless of the used seed treatment.