Influence of Bacillus sp. on soil chemical and microbiological attributes and development of soybean and maize

Plant growth-promoting rhizobacteria (PGPR) inhabit the rhizosphere of several cultivated plants. Bacteria of the genus Bacillus have great functional importance in plant development. The objective of this research was to evaluate different doses of Bacillus sp. in the development of soybean and corn crops and in the chemical and microbiological attributes of the soil. The essays were conducted in the field in the 2016/2017 harvest. The soybean and corn seeds were treated with two doses of Bacillus sp. at a concentration of 10 9 CFU ml -1 , with 10 and 20 ml of 100 kg of seed used in soybeans and 80 and 100 ml of 100 kg of seed in corn. The experiments were completely randomized in block with four repetitions. Chemical and microbiological attributes and plant development were evaluated. The results showed that the microbial activity was achieved at the dose of 20 ml of Bacillus sp. and positively influenced the weight of the grains and soybean productivity. In corn crops, the dose of 80 ml of Bacillus sp. showed better performance on chemical and microbiological attributes, resulting in increased shoot and root mass, and a significant increase in productivity. From the results obtained, it can be concluded that the use of Bacillus sp. it is a viable alternative for sustainable agriculture, and the dose of 20 ml and 80 ml of 100 kg of soybean and corn seeds, respectively, presented better results in microbial activity and in the development of plants.


Introduction
Soybeans and corn cultivation are growing agricultural activities in the world and Brazil stands out with record production of 120.9 million tons of soybeans and 100.9 million tons of corn in the 2019/2020 harvest (CONAB, 2020). These crops are important both for the production of animal feed, oils and fuel, and for human food (CONAB, 2020). Due to population growth, the demand for food production requires the development of new technologies applied to crops aiming to improve the quality and increase of production of grains (Ratz et al., 2017;Buchelt et al., 2019).
Success in establishing a crop depends on the soil and the environment, which must be suitable for seed germination, seedling emergence and development (Modolo et al., 2011). In addition, the cultivation of soybeans and corn depends on the propagation material, which is correctly selected, with high germination power, excellent vigor and without contamination with pests and diseases (Migliorini et al., 2017).
To increase productivity and decrease costs for the producers, alternatives to reduce the use of inputs and pesticides have been sought. An effective alternative is the use of plant growth promoting rhizobacteria (PGPR), which is a promising biological option in the control of phytopathogens, plant development and in increasing crop productivity (Hernández-Hernández et al., 2018). PGPR are bacteria that are found in the rhizosphere, root surface and in association with roots and are often isolated from the rhizosphere of several cultivated plants, to promote growth (Ratz et al., 2017). Bacillus, Pseudomonas, Azospirillum, Rhizobium, Serratia, Azotobacter among others are among the most studied genera (Araújo and Guerreiro, 2010). The benefits observed in several cultures with the inoculation of bacteria of the genus Bacillus, show its great potential as biological control agents, production of natural antibiotics and protective effect against soil phytopathogens. These organisms support growth and development of the root system, greater absorption of water and nutrients, favoring the development of the crop, leading to a quality production of its seeds (Ferreira et al., 2018;Machado and Costa, 2018). The use of Bacillus, or its metabolites, can contribute with phytohormones and molecular signals, increasing root growth, nodulation and N2 fixation rates, and reducing susceptibility to water stress (Ferreira et al., 2018;Machado and Costa, 2018;Buchelt et al., 2019).
The use of Bacillus spp., species as growth promoters for several agricultural crops provides an attractive, efficient and less aggressive method compared to pesticides and chemical fertilizers This makes the practice of using bioagents a more sustainable alternative from an economic and environmental standpoint (Shafi et al., 2017). Several species of PGPRs have been increasingly studied and their application intensified to increase productivity. However, any and all anthropic activities, whether cultural management or the introduction of products and organisms into the soil, can cause impact or stress to the soil microbial community responsible for nutrient mineralization and decomposition. Thus, the use of soil quality indicators (bioindicators) is sensitive to any change or management of the soil (Simão et al., 2020).
In this study, two field trials were carried out in order to evaluate the influence of seed treatments with two doses of Bacillus spp., on the chemical and microbiological attributes of the soil and on the development of soybean and corn crops.

Material and methods
The tests were conducted during the 2016/2017 crop year, at the farm school of the State University of Northern Paraná (S23º 06' 24.7" and W50º 21' 37.36" and 439 m altitude), Campus Luiz Meneghel (CLM), located in the municipality of Bandeirantes, Paraná, Brazil. The soil in the experimental area is classified as Eutropheric RED Latosol (EMBRAPA, 2006). The climate of the region is classified as Cfa type (Koppen and Geiger), humid subtropical. During the months of the experiments (October 2016 to February 2017) the maximum average temperature was 28 ºC and the minimum 18 ºC, and the average monthly rainfall was 200 mm (Center for Weather Forecast and Climate Studies-CPTEC). The tests were conducted with soybean (Glycine max L.) variety BMX Potencia and corn (Zea mays L.) variety KWS 9004 in randomized block with four replications.

Inoculated bacteria
The bacterium Bacillus sp., evaluated in this study was isolated from soil samples collected at Farm School of the State University of Northern Paraná-CLM. Planting area for agricultural crops with application of biological fertilizer for three consecutive years.

Cultivation and inoculation of Bacillus sp. in soy and corn seeds
Bacillus sp., was isolated and grown in nutrient broth and kept in BOD at 28 ºC until the bacterial population reached the concentration of 1x10 9 CFU ml -1 determined by a spectrophotometer at 530 nm, according to Normative Instruction No. 13 of the Agricultural Defense Department of March 24 of 2011. The soybean and corn seeds were inoculated approximately ten minutes before sowing. The seeds were inoculated with two doses of the bacterial suspension, the proposed commercial dose and double the dose (Table 1). For both cultures, 2 kg of seed were treated with Bacillus sp. in a concentration of 1x10 9 CFU ml -1 homogenized in plastic bags, transferred in paper bags and kept in the shade until sowing.

Experiment's installation
The cultivation of soybeans and corn were installed in the field in the no-tillage system and the seeds were sown with the aid of a bicycle planter. The soybean was sown at a density of 13 seeds per meter and 45 cm spacing between rows. Together with the sowing, a basic fertilization was carried out with 300 kg ha -1 of the formulated fertilizer (NPK) 0-10-10. The corn was sown at the density of three seeds per linear meter and with a spacing of 50 cm between the lines. Basic fertilization was carried out with 250 kg ha -1 of the formulated fertilizer (NPK) 10-18-18.
The experimental design was a randomized block, with three treatments and four replications for each crop (soybean and corn), each plot measuring 5 m wide and 6 m long, making an area of 30 m 2 . The treatments consisted of control and two doses of Bacillus sp., at a concentration of 10 9 CFU ml -1 (Table 1). Soil collection for chemical and microbiological analysis was carried out postharvest at a depth of 0-10 cm, with 7 samples being collected to compose a sample made up of a plot, which were homogenized, packed in plastic bags and transported in a thermal box to the Soil Microbiology Laboratory, in the State University of Northern Paraná, Campus Luiz Meneghel, where they were separated from plant and animal waste and sieved in a 2 mm mesh.

Soil chemical analysis
After air drying the soil samples, the pH was determined in 0.01 M CaCl2, P, Ca 2+ , Mg 2+ , K + and Al 3+ . The contents of Ca 2+ , Mg 2+ , K + and Al 3+ were extracted with 1 M KCl and determined by atomic absorption (Ca 2+ and Mg 2+ ) and titrated with 0.025 M NaOH (Al 3+ ); P and K + were extracted with Mehlich-1 extractor and determined by flame ionization spectrophotometry (K + ) and by the molybdenum blue method (P).

Soil microbiological analysis
Microbial biomass Carbon (MBC). The carbon content from microbial biomass of the soil was determined by the fumigation-indirect extraction (FIE) method (Vance et al., 1987). Basal respiration (BRS) and soil metabolic quotient (qCO2) determination. Basal soil respiration and qCO2 were determined according to the methodology proposed by Silva et al. (2010).
Total organic carbon (TOC) and microbial quotient (qMIC). TOC determination was carried out in combustion of organic matter via wet, using 0.5 g of sample, according to Walkley and Black (1934-modified), without external heating in the plate. The qMIC was determined by the MBC/TOC ratio.

Agronomic analysis
The root height and length evaluation was determined from 10 plants chosen at random. The dry shoot and root mass, being sampled 5 plants per plot. The soybean yield was determined by harvesting four linear meters per plot and the weight of 1 000 grains was also obtained. Corn productivity was determined by harvesting two linear meters per plot, obtaining a weight of 200 grains. At the time of harvest, the grains (soybeans and corn) were at 13% moisture. The productivity results were determined in kg ha -1 .
All statistical analyses were carried out using Sisvar (version 5.7, DEX/UFLA). The averages were submitted to Anova. When confirming a statistically significant p value, the Tukey test (p< 0.05) was applied for comparison purposes (Ferreira, 2019).

Results and discussion
In soybean cultures, chemical analysis showed that inoculation of Bacillus sp., regardless of the dose, resulted in a significant reduction in pH, organic matter and potassium content compared to the control (Table 2). This may be related to the degradation of organic matter by bacteria, which can oxidize a wide range of organic and fermentative compounds (Stamford et al., 2005). Plant growth-promoting rhizobacteria can help accelerate the decomposition of organic matter as in the mineralization process (Persello-Cartineaux et al., 2003). Organic matter serves as a source of carbon and energy for microorganisms, because a high content of organic material is associated with a great microbial diversity (Wetler-Tonini et al., 2010).
In T2, a slight increase in phosphorus content was observed. Studies carried out with Bacillus subtilis have shown greater availability of nutrients in the soil, indicating greater absorption of P and N, in plant inoculated with PGPRs in the seeds (Araújo, 2008). The increase may also be related to the no-tillage method, caused by less soil mobilization, in addition to the formation of phosphorus complexes with organic matter, greater biological activity and recycling of nutrients by the roots and crop residues. In corn, chemical analyzes showed less change in the parameters evaluated, with only a drop in the content of P and K observed with the inoculation of Bacillus sp. (Table 3). This is likely related to rhizobacteria promoting the mineralization of nutrients, solubilization of phosphates, nitrogen fixation and increased absorption of nutrients by the roots (Lazarovits and Nowak, 1997).
In soybean cultures, microbiological parameters showed a decrease in TOC content in treatments inoculated with the bacteria (T2 and T3). This may be related to the increase in microbial biomass, being significantly higher in T3 (154.66) when compared to the others treatments (Table 3). In addition to the significant increase in MBC in T3, there was a better rate of decomposition and mineralization of organic matter in the soil (qMIC), as well as lower respiration (SBR) and lower metabolic stress (qCO2) ( Table 3). In the corn crops, the best values for microbiological parameters were observed in the T2 treatment, with significantly higher microbial biomass (175.87) and higher qMIC (1.25). Consequently, less metabolic stress was observed (2.26) ( Table 4). Microbial biomass (BM) is the living part of the soil, and it acts on the decomposition of organic matter in the soil. The amount and composition of microbial biomass can be influenced by several factors, including the cultivation system, crop rotation and soil texture (Venzke-Filho et al., 2008). The microbial quotient (qMIC) is equivalent to the percentage of reserve of total organic carbon in the soil, considered as equilibrium soil values between 1.8 to 2.2% (Jakelaitis et al., 2008). Areas with low microbial activity present lower values, demonstrating loss of carbon in the soil, which indicates a lower availability of organic compounds to plants (Simão et al., 2020). Variations in qMIC values reflect the pattern of soil organic matter input, the efficiency of microbial C conversion, soil C losses and the stabilization of organic C by soil mineral fractions (Cunha et al., 2011).
As the microbial biomass becomes more efficient in the use of ecosystem resources, less CO2 is lost through respiration and the greater the proportion of C incorporated into microbial tissues, resulting in a decrease in qCO2 in the soil (Cunha et al., 2011). Estimates of microbial biomass have been used in studies of C and N flow, nutrient cycling and plant productivity in different terrestrial ecosystems, also enabling the association of the amount immobilized nutrients and the activity of microbial biomass with fertility and the potential for soil productivity (Gama- Rodrigues et al., 2008). In the study by Chagas et al. (2017), with soybeans and cowpea, the plants showed superior results in terms of biomass accumulation, demonstrating the potential of Bacillus sp., as a growth promoter for both cultures.
The agronomic attributes evaluated in the soybean culture did not show significant differences between the treatments, however, there was a trend of greater plant height, greater root length and, consequently, greater productivity in the T3 treatment (Table 6). This can be related to the increase in microbial biomass and microbial quotient (Table 5). (qCO2)= soil metabolic quotient; T1= control; T2= Bacillus sp., 10 ml 100 kg -1 seed; T3= Bacillus sp., 20 ml 100 kg -1 seed. Averages followed by the same lowercase letter in the column, do not differ by Tukey's test at 5% probability. Bacteria in natural habitats colonize the interior and exterior of plant organs and that can beneficial, neutral or harmful to their growth (Mariano et al., 2004). Domenech et al. (2006) demonstrated that Bacillus sp., auxin producers improved the development of the plant, increasing the height and fresh weight of the aerial part. This increase allowed a better photosynthetic rate and, consequently, a better efficiency in the use of water and an increase in production (Szilagyi-Zecchin et al., 2015).
Plants that have microorganisms associated with their roots or in the rhizosphere tend to have a better ability to survive and consequently better absorption of nutrients. Thus, plants that are assured of beneficial factors by microorganisms have a productive advantage over those that do not (Chagas et al., 2017).
In corn, the benefits of inoculation with Bacillus sp., was more evident, showing greater plant height and dry matter, both in shoot and root. However, productivity was significantly higher in the T2 treatment (6.026 kg ha -1 ) ( Table 7). Moreira (2014) highlighted the specificity of the association between the bacterium Bacillus spp., and the plant since the plants genotype is a determining factor for obtaining the benefits from inoculation. Bacillus sp., promotes growth and yield of different crops, in addition to improving the nutrient absorption capacity, resulting in more vigorous plants (Shafi et al., 2017). Dotto et al. (2010); Rodrigues et al. (2006), reported contrasting productivity results of different maize hybrids with bacterial application. Lima et al. (2011), reported positive results in the development of plant shoot and root, improving its establishment and consequently the grain yield in his research of corn seeds with Bacillus spp., sown in soil that received NPK fertilization.
The success of B. Subtilis in promoting plant growth is related to the biological characteristics of these microorganisms, which are easy to maintain (Lanna-Filho et al., 2010). The growth promotion caused by Bacillus spp., is the result of increased nitrogen fixation, solubilization of nutrients, synthesis of phytohormones and improved soil conditions (Lanna-Filho et al., 2010).

Conclusions
The inoculation of Bacillus sp., demonstrated different results in soybean and corn crops. In soybeans, the application of Bacillus sp., showed better results in microbiological attributes in the highest dose, (twice the commercial dose). In the other attributes there were no major changes. It is important to note that bacteria inoculation does not cause damage in the culture production. This result will be further investigated in the future, with the objective of developing a commercial product based on Bacillus sp., exposing a positive evolution in plant development and yield.
In the studied corn crops, the inoculation of Bacillus sp. resulted in an increase in plant shoot, root and yield, in the commercial dose, with great importance in the increase of microbial population of the soil, and consequently higher corn yield. Further studies with Bacillus sp., are still needed to determined its role as cell protector that keep viable during the seed germination period and others variables that can interfere with their survival. Nonetheless, there is a great potential for the development of commercial inoculants.