The synergistic action of Azospirillum brasilense and effective microorganisms promotes growth and increases the productivity of green corn

In view of the current dissemination of even more sustainable techniques, which allow the producer to explore the microbial biodiversity native to the property's soil and obtain effective microorganisms (EM), the hypothesis was tested that the biopriming of corn seeds using the co-inoculation of A. brasilense + EM could promote growth and increase the productivity of green corn. Additionally, the effect of treating seeds with humic acids was evaluated. For this, a field experiment was conducted using the following treatments: A. brasilense; EM; A. brasilense + EM; humic acids (Solo Humics) and Control. Co-inoculation of A. brasilense + EM potentiated the growth-promoting effects of diazotrophic (height and stem diameter increased 30 days after sowing DAS, fresh and dry mass of aerial part at 90 DAS and dry mass of root at 60 and 90 DAS) and increased productivity of green corn (presented the highest averages for ear weight with and without straw). The treatment of seeds with humic acids showed promising effects only in the initial stages of crop development – when the plants evaluated were at 30 DAS. However, in the final evaluation phase (90 DAS) the effect of these acids became less satisfactory than microbial inoculation. The enrichment of inoculants of A. brasilense with EM is therefore a viable alternative for promoting growth and increasing productivity in the cultivation of green maize in addition to being a low complexity technology and even cheaper than the simple use of commercial inoculants.

In view of the current dissemination of even more sustainable techniques, which allow the producer to explore the microbial biodiversity native to the property's soil and obtain effective microorganisms (EM), the hypothesis was tested that the biopriming of corn seeds using the co-inoculation of A. brasilense + EM could promote growth and increase the productivity of green corn. Additionally, the effect of treating seeds with humic acids was evaluated. For this, a field experiment was conducted using the following treatments: A. brasilense; EM; A. brasilense + EM; humic acids (Solo Humics) and Control. Co-inoculation of A. brasilense + EM potentiated the growth-promoting effects of diazotrophic (height and stem diameter increased 30 days after sowing -DAS, fresh and dry mass of aerial part at 90 DAS and dry mass of root at 60 and 90 DAS) and increased productivity of green corn (presented the highest averages for ear weight with and without straw). The treatment of seeds with humic acids showed promising effects only in the initial stages of crop developmentwhen the plants evaluated were at 30 DAS. However, in the final evaluation phase (90 DAS) the effect of these acids became less satisfactory than microbial inoculation. The enrichment of inoculants of A. brasilense with EM is therefore a viable alternative for promoting growth and increasing productivity in the cultivation of green maize in addition to being a low complexity technology and even cheaper than the simple use of commercial inoculants. Keywords: rhizobacteria, commercial inoculants, Zea mays L.

INTRODUCTION
One of the biggest challenges of modern agriculture is balancing the growing demand for food with the rational use and occupation of the environment. In this context, the development and application of technologies that guarantee productivity and that do not generate negative environmental effects have been stimulated. Researchers that prospect new microbial strains that use functional traits of microorganisms to control phytopathogens or promote plant growth have become very common [1,2,3], aiming at alternatives to pesticide and fertilizer use in crops [4].
The first microbial inoculant for plants was developed about 100 years ago. However, in recent decades, farmer receptivity of these products has considerably improved due to the expressive improvements in crop productivity [5]. The main inoculants currently found on the market consist of unique microbial strains, with proven efficiency. Despite this, in the last decade, mixed inoculants have conquered the market. Co-inoculations are intended to associate different functional traits, often expressed by different microorganisms [6,7,8]. In this way, biological nitrogen fixation (BNF), phytohormone synthesis, the ability of antibiosis to pathogens, as well as solubilization of different nutrients can be achieved with the use of the same inoculant, formulated from the mixture of two or more strains.
The use of commercial inoculants contributes considerably to cost reduction of chemical fertilization in different cultures, inexpensive and low complexity techniques, which allows for obtaining efficient microbial formulations from the native microbial biodiversity of the property's soil, and decreasing the general expenses with planting. These techniques use effective microorganisms (EM). EM are consortia of microorganisms that, when used as inoculants, increase the microbial diversity of the soil [9], absorption of P and Zn [10], favors photosynthetic processes [11], assist in the decomposition of organic matter [12], have an antagonistic effect to soil pathogens [13], anticipate seed germination [14], and contribute to increased grain productivity [8,15,16]. These microorganisms are easy to obtain and are mainly recommended for use in family farming.
Therefore, taking into account the positive effects of the use of inoculants based on Azospirillum brasilense, in a single lineage, for the corn culture [17,18,19] and taking into account the lack of tests that associate this diazotrophic to EM in this culture, we decided to test the hypothesis that the biopriming of corn seeds using the co-inoculation of A. brasilense + EM could promote growth and increase the productivity of green corn. Additionally, we also tested the treatment of seeds with humic acids. Humic acids stimulate the production of plant hormones, especially auxin, resulting in the growth and development of roots [20,21,22,23]. These acids also altered soil chemistry and microbial dynamics in the rhizosphere [24], influencing soil-microorganism-plant interactions, mainly on nutrient availability and assimilation [22]. Given the above, the objective of this work was to evaluate the effect of biopriming of corn seeds with EM and A. brasilense isolated and in co-inoculation as well as the effect of the treatment of seeds with humic acids on the growth and productivity of green maize.

EM preparation
EM were attracted to "baits", using rice as organic matter. These baits were prepared by cooking approximately 700 grams of unsalted rice, which was placed on 15 x 20 cm wooden boards. Five boards were installed on exposed soil, after the total removal of the litter, according to the methodology of Porto et al. (2020) [15]. After installation, a layer of litter was added as a cover ( Figure 1b) and the apparatus was covered with a fine TNT canvas that were placed on wooden supports in order to protect against raindrops. The installation took place in a legal reserve Semidecidual Seasonal Forests, also located in the Fazendinha farm area (18°24'22" S, 50º23'34" W at 544 m elevation) (Figure 1a). The colonization of the baits by the local microbiota was monitored daily, from the 11th day of incubation, and total colonization was observed on the 15th day when the boards were removed and taken to the Microbiology Laboratory of the Goiás State University, Sudoeste campus, for isolation of EM.
The colonies were selected from the baits following the methodology proposed by Bonfim et al. (2011) [26], with only those containing pink, blue, yellow, and orange colors maintained.
To activate the EM, the selected colonies were added to five liters of cane molasses diluted in 50 liters of clean water (without chlorine). Thus, the EM production process was carried out in nonsterile conditions. The mixture was placed in a 70-liter plastic drum, the container being hermetically closed and left to rest in the shade for 25 days. To reduce the pressure of the fermentation process (anaerobic), the drum was opened every two days. At 25 days, the fermentation process ceased and the inoculant showed the expected characteristics, which were orange in color and a pleasant sweet odor. The number of colony forming unit (CFUs) per mL of the inoculant was quantified using the serial dilution technique [27].

Seed treatment and sowing
Seeds of simple hybrid corn MG 711 from Morgan Sementes® were subjected to biopriming using the inocula of interest (EM and Azospirillum brasilense) and treated with humic acids (Solo Humics). The EM were applied at a concentration of 3.4 x 10 5 CFU mL -1 , under the dose of 20 mL 5 kg -1 of seeds. The inoculation of Azospirillum brasilense was performed using commercial inoculant of the strains AbV5 and AbV6, brand AzoTotal® of the company Total Biotecnologia Indústria e Comércio Ltda, registered with MAPA under the number PR-93923 10074-1. The application was done at 2 x 10 8 CFU mL -1 (100 mL 25kg -1 seed). In the treatment of co-inoculation, the seeds received AzoTotal® and EM in equal proportions (20 mL 5 kg -1 of seeds). The soil conditioner used was the humic acid of the brand SoloHumics® (mixture of humic and fulvic acids in H2O, peat and KOH) registered in MAPA number-BA-32601-1, being applied in the dose of 50 mL 5kg -1 of seeds. Seed treatment was carried out with the aid of transparent plastic bags and a 50-mL syringe. The treated seeds were mixed and dried in the shade for 20 minutes before sowing.
The experiment was designed in randomized blocks, with five treatments: biopriming with A. brasilense; biopriming with EM; biopriming with A. brasilense + EM; treatment with humic acids (Solo Humics) and without biopriming or untreated seeds (Control). The experimental plots consisted of seven six-meter lines, with 0.50-meter spacing between lines, with four repetitions. Sowing was carried out aiming to reach a population of 65,000 plants ha -1 considering the variety of corn used.
The seeds were planted on Nov. 26, 2019, with the aid of a seven-row mechanized planter, with three cm deep grooves. At the time of sowing, chemical fertilization was carried out with 400 kg ha -1 in formulation 8-20-20, being the sources (N, P2O5 and K2O), respectively nitrogen, triple superphosphate and potassium chloride. At 60 days after sowing (DAS), foliar fertilization was applied using the commercial product Nitamin® (33 percent N) 4 L ha -1 .
Data were collected at 30, 60, and 90 (DAS) (December 2019 to February 2020). At 30 DAS, the following response variables were evaluated: plant height (m) and stem diameter (cm); at 60 and 90 DAS: plant height (m), stem diameter (cm), fresh and dry mass of the aerial part (g), and fresh and dry mass of the roots (g). At 90 DAS, the productivity characteristics of green corn were also evaluated. For this, the ears were harvested and evaluated for the average weight of commercial ears with and without straw (g) as well as average length and diameter of commercial ears without straw. Commercial ears were considered to be longer than 15 cm, as Moreira et al. (2010) [28] and with an average diameter greater than four cm, grenades and free from injuries caused by insect pests and diseases.
The data was obtained with the aid of a tape measure, manual caliper, and precision scale, being collected from 15 plants, obtained from the four central lines of the useful area of the 15m² plot. To determine the dry mass of the aerial part, the plants were cut close to the soil. The roots were extracted from the soil with the aid of a hoe, and a radius of 30 cm of soil was removed around the roots. These were later washed to remove the adhered soil. The plant material was dried in an oven with forced aeration at 55ºC until it reached constant mass.
The data was submitted to analysis of variance (ANOVA) and when they were proven significant by the F test, the averages were compared by the Tukey test at five percent probability. Statistical analyses were conducted using RStudio 1.2.1335 [29].

RESULTS
The biopriming of corn seeds with A. brasilense + EM positively affected the plant height only at 30 DAS, so that the plants submitted to this treatment had the highest average for this variable (0.64 m). This average was significantly higher than that observed for the Control (0.57 m) (difference between means = 7.00, p = 0.005) and also for plants submitted to EM only (0.58 m) (difference between means = 5.55, p = 0.010) (Figure 2a, c and e). At 30 DAS, the treatment of seeds with Solo Humics also positively affected growth, with the average height (0.62 m) being significantly different from that observed for the Control (difference between means = 5.27, p = 0.018).  at 30 (a and b) (Figure 2b).
At 60 and 90 DAS, the different treatments did not affect the height or the diameter of the plants (Figure 2d and f), however, when evaluating the total growth through fresh and dry mass, different responses were observed. At 60 DAS, the biopriming of corn seeds with A. brasilense + EM promoted the worst result in terms of fresh mass of the aerial part (654.03 g) (Figure 3a). However, at 90 DAS, this treatment, as well as the treatment using only A. brasilense as inoculum, led to a greater accumulation of fresh matter in the aerial part, at 584.53 grams and 589.98 grams, respectively (Figure 3c).  at 60 (a and b) and 90 days (c and d)  At 60 DAS, the fresh root mass was positively affected by all inoculation treatments, with the worst averages observed in the plants of the Control treatment (82.94 g) (Figure 3b), but at 90 DAS, the plants subjected to EM, A. brasilense + EM and A. brasilense, had the highest averages observed for this variable (118.44 grams, 109.43 grams, and 107.72 grams, respectively) (Figure 3d). In this period, the plants of the Control treatment showed the worst averages observed for fresh root weight (78.41 grams).
In relation to the dry mass of the aerial part, at 60 DAS, the inoculation treatments were not more efficient than the Control in promoting accumulation, with the highest averages observed in the plants treated with A. brasilense (238.87 grams), similar to those observed in the Control plants (218.98 grams) (Figure 4a). However, at 90 DAS, the plants submitted to A. brasilense + EM were significantly superior to the others in accumulating dry mass in the aerial part (123.4 grams) ( Figure  4c). brasilense + EM, humic acids (Solo Humics) and Control. In the boxplot, the black line represents the median, the blue line the average and points observed outside are outliers. Above the boxplots, means followed by the same letter do not differ significantly by Tukey's test at 5% probability.
The highest averages for dry root weight were observed in plants submitted to A. brasilense + EM (28.70 grams) and in those treated with Solo humics (28.48 grams), at 60 DAS (Figure 4b). However, at 90 DAS, the plants treated with Solo humics dropped their accumulation (20.19 grams) and the plants subjected to A. brasilense + EM were superior to the others in accumulating dry mass at the root (39.46 grams) (Figure 4d).
At 90 DAS, when productivity was assessed, a tendency was observed for seed inoculation with A. brasilense + EM to positively affect the weight of the ears (304 grams) and the weight of the ears with straw (371.71 grams) (Figure 5a and b). This co-inoculation provided percentage gains of 25.73 percent for ear weight and 21.3 percent for ear weight with straw, compared to Control plants. However, the diameter of the ears was not significantly affected by this treatment (Figure  5c).
The inoculation treatments positively affected the length of the ears, with the worst averages observed in those from Control plants (15.91 cm) and the highest observed in ears from plants treated with EM (18 cm) (Figure 5d and e), with an average of 13.13 percent higher.

The biopriming of corn seeds with A. brasilense + EM positively affected plant growth
As expected, the co-inoculation of corn seeds with A. brasilense + EM positively affected plant height and stem diameter. This is because mixed inoculations are made up of a range of microorganisms with different functional traits, which can have multiple effects on plant growth and productivity [30]. The synergistic action of microorganisms has been explored in several studies that have evaluated since the synergistic action of mycorrhizal fungi with bacteria that promote plant growth [31], to the joint action of specific microbial strains associated with effective microorganisms [9]. Therefore, the enrichment of the inoculant of A. brasilense with EM potentiated the effect of diazotrophic in promoting plant growth.
Similarly, the treatment of seeds with Solo humics also positively affected plant height and diameter at 30 DAS. In general, humic acids can modify the dynamics of ammoniacal N, promoting an increase in the availability of NH4 + and reduction of NH3and, consequently, reduce the processes of volatilization and leaching of N, favoring the use by cultures [32]. Therefore, at this early stage of development, a better absorption of N induced by these acids culminated in plant growth promotion. Similar results were observed by Baldotto and Baldotto (2016) [33] in corn plants at the early stage of development. However, in the later stages of development (60 and 90 DAS), these acids lost their effectiveness in relation to inoculation treatments, with regard to accumulation of fresh mass, dry mass, and productivity. Batista et al. (2018) [34] also found that, at 137 DAS, the use of humic/fulvic acids does not affect morphological characteristics or components of corn yield and productivity.
At 90 DAS, the co-inoculation A. brasilense + EM positively affected the accumulation of fresh and dry mass in the aerial part and root. The effects of A. brasilense on growth may be associated with its diazotrophic character for corn [35,36] and also due to its ability to synthesize phytohormones as indole-3-acetic acid (IAA) [37,38], gibberellins [39] and cytokinins [40], which likely also contributed to the higher average height and stem diameter observed at 30 DAS. These effects were intensified by the synergistic action of the microorganisms that make up EM.
EM can contain anaerobic, aerobic microorganisms, as well as dozens of microorganisms with different ecological functions (lactic acid-producing bacteria, yeasts, photosynthetic bacteria, fungi and actinomycetes) [26,41]. This diversity of functions can be added to the effect of rhizobacteria, such as A. brasilense and enhance plant growth. Sharma and Kumawat (2012) [42] demonstrated that the inoculation of EM preparations can culminate in greater BNF in the soybean culture. These mixed cultures of natural organisms can be used as inoculants in a safe manner without risk of environmental impacts [9].

The biopriming of corn seeds with A. brasilense + EM positively affected the weight of the ears
The positive effects of A. brasilense + EM co-inoculation on growth also culminated in a greater weight of corn ears. Megali et al. (2015) [43] demonstrated that the administration of effective microorganisms can not only increase corn productivity, but also reduce the diversity of insect pests that attack plants. Currently, most research evaluates the individual effect of using A. brasilense [44,45,46] or EM on maize crops. In research carried out by Galindo et al. (2019) [47], inoculation with A. brasilense increased the leaf chlorophyll index, stem diameter, ear length, and the efficiency of N use, with a positive effect on the yield and operating profit of the corn grain. Alternatively, Teixeira et al. (2017) [16] found that high doses of EM provided up to a 38 percent increase in plant height, 144 percent in fresh mass of aerial part, 71 percent in root length, 199 percent in fresh mass of root, 23 percent in ear mass with seeds, 20 percent in weight of grains, and 16 grain in weight of 1,000 grains.
Research involving the evaluation of the joint action of A. brasilense + EM in the green corn crop has not yet been published. Ribeiro et al. (2020) [8] tested the triple co-inoculation of B. japonicum + A. brasilense + EM in soybeans and found promising effects on the growth and productivity of the Brasmax Desafio RR cultivar. This work confirmed the hypothesis that the coinoculation of A. brasilense with EM can positively affect the growth and productivity of green corn, demonstrating that the enrichment of inoculants based on this diazotrophic, which aims to increase production essentially through BNF, while EM can make the treatment more effective, constituting an alternative not only to guarantee higher yields, but also to reduce the expenses with biological inputs applied to the corn crop as small producers could easily produce part of their own inoculant.

CONCLUSIONS
The co-inoculation of A. brasilense + EM is efficient in promoting the growth and productivity of green corn. Despite the treatment of seeds with humic acids having promising effects in the early stages of corn development, the effect of these acids became less satisfactory than microbial inoculation in the final evaluation phase. The biopriming of corn seeds with A. brasilense + EM is a viable technique to ensure greater productivity and sustainability of the corn crop as it is a low cost and low complexity technology.