Bacterial strains
Four bacterial strains were selected from the Multifunctional Microorganisms Collection from Embrapa Milho e Sorgo. The strains B116 and B2084 can solubilize P and were identified as B. thuringiensis and B. subtillis, respectively (Oliveira et al. 2009; Abreu et al. 2017; Velloso et al. 2020). The strains A1626 and A2142, two nitrogen-fixing bacteria from the genus Azospirillum, were isolated from sorghum stalk and maize rhizosphere soil, respectively. All strains promoted maize growth in hydroponic or field conditions (Sousa et al. 2018, 2021; Velloso et al. 2020).
Identification of Azospirillum species
Bacterial genomic DNA was extracted with the Wizard Genomic DNA Purification Kit (Promega, USA) and amplified with the 16S rDNA primers 8F and 1492R (Turner et al. 1999). PCR reactions were performed with 30 ng of bacterial genomic DNA, 2.5 µL 10X PCR buffer (20 mM Tris–HCl pH 8.4, 50 mM KCl), 0.4 µM of each primer, 100 µM dNTP, 2.5 mM MgCl2, and 1 U Taq DNA polymerase (Invitrogen, USA) in a total volume of 25 µL. PCR was performed with the following conditions: 95°C for 2 min, 30 cycles of 30 s at 94°C, 30 s at 55°C and 2 min at 72°C. Finally, reactions were incubated for 10 min at 72°C. The amplification products were purified with the ExoSAP-IT Kit (USB, USA), and sequenced with the primers 8F, 1492R, 515F (Turner et al. 1999) and 902R (Hodkinson and Lutzoni 2009) using Big Dye Terminator v3.1 kit, as recommended by the manufacturer (Applied Biosystems, USA). The samples were analyzed in the automated sequencer ABI PRISM 3500 XL Genetic Analyzer (Applied Biosystems, USA) and DNA sequences were compared using the BlastN program (Altschul et al. 1997). The 16S rRNA gene sequences were deposited in GenBank under accession numbers MW646094 (A1626) and MW646095 (A2142).
Compatibility test
The bacterial strains were confronted on the same Petri dish to evaluate their compatibility as reported by Cuesta et al. (2012), with modifications. Individual strains were grown in TSB (Trypticase Soy Broth) medium at 28º C overnight. Then, 100 µL of the liquid culture of one strain was spread with a Drigalski loop on the surface of Petri dish containing PDA medium (200 g L− 1 potato, 20 g L− 1 dextrose and 15 g L− 1 agar). After drying, 25 µL of the culture of another strain were inoculated adding four drops at equidistant points on the PDA surface. The plates were incubated at 28°C ± 2 for 5 days in triplicate and the strain’s compatibility was determined by the absence of an inhibition zone.
P solubilization and mineralization
One isolated colony of each strain grown on BDA plates (200 g L− 1 potato, 20 g L− 1 dextrose and 15 g L− 1 agar) was transferred to TSB medium (Trypticase Soy Broth) and incubated overnight at 28º C. After this period, a bacterial suspension in the concentration of 5 × 107 CFU (colony-forming unit) mL− 1 was transferred, in triplicate, to 100 mL of the NBRIP culture medium (Nautiyal 1999), either individually or co-inoculated with another strain at the same concentration. The NBRIP medium was supplemented separately with 25 g L− 1 Ca3(PO4)2 (Ca-P), 5 g L− 1 FePO4 (Fe-P) and sodium phytate at 1 g L− 1. The samples were incubated at 28°C for nine days at 120 rpm and centrifuged at 5.000 × g for 10 min. The supernatant was filtered on Whatman filter paper No. 42 and the concentration of soluble P was determined by the colorimetric method (Murphy and Riley 1962). Additionally, the pH of the filtrate from all samples, including the controls, was determined.
In vitro indole acetic acid (IAA) production
The production of tryptophan-dependent IAA molecules was measured by the colorimetric method according to Patten and Glick (1996). Each strain was grown in 50 mL of liquid TSB culture medium supplemented with 1.0 mg mL− 1 of DL-tryptophan and incubated at 30°C for five days at 100 rpm in the dark. After centrifugation for 10 min at 5,500 rpm, 0.1 mL of the supernatant was mixed with 0.1 mL of the Salkowski reagent (Loper and Schroth 1986) and incubated for 20 min in the dark. The concentration of IAA molecules in the supernatant was determined by the colorimetric measurement at 540 nm (Labsystems Multiskan, USA) in triplicate and compared to a standard curve.
Maize plant growth under hydroponic conditions
The hydroponic experiment was conducted with 11 treatments (four individual strains, six co-inoculations and one negative control (non-inoculated) (Table 1), arranged in a completely randomized design with three replicates with five maize seedlings each.
Table 1
Identification of the treatments with Azospirillum brasilense and Bacillus spp. strains.
Strains
|
Identification
|
Non-inoculated
|
NaCl 0.85% (w/v)
|
B116
|
Bacillus thuringiensis
|
B2084
|
B. subtilis
|
A1626
|
Azospirillum brasilense
|
A2142
|
A. brasilense
|
B116 x B2084
|
B. thuringiensis + B. subtilis
|
B116 x A1626
|
B. thuringiensis + A. brasilense
|
B116 x A2142
|
B. thuringiensis + A. brasilense
|
B2084 x A1626
|
B. subtilis + A. brasilense
|
B2084 x A2142
|
B. subtilis + A. brasilense
|
A1626 x A2142
|
A. brasilense + A. brasilense
|
For the preparation of microbial inoculants, the strains were grown individually in liquid TSB culture medium at 28°C and 150 rpm for three days. After the incubation period, cultures were centrifuged at 6,000 rpm for 10 min. Bacterial suspensions were adjusted to absorbances equal to or higher than 1, at wavelength of 550 nm, to obtain a concentration of 107 CFU mL− 1 after resuspension in 2.0 L of 0.85% (w/v) NaCl.
The maize seedlings were evaluated as described by Sousa et al. (2021). Maize seeds were surface disinfested with 0.5% (v/v) sodium hypochlorite for five minutes, washed and soaked for four hours in deionized water and transferred to germination paper rolls. After seed germination for three days, uniform seedlings were transferred to trays containing eight liters of half strength Hoagland´s nutrient solution pH 5.65 (Liu et al. 1998) and acclimatized for seven days. After acclimatization, the roots were incubated for six hours at room temperature with the bacterial suspension, prepared as described previously. In the control, the plants were incubated in 0.85% (w/v) saline solution. The trays with maize seedlings were manually agitated at frequent intervals to facilitate contact of the bacteria with the roots. After the incubation period, the inoculum excess was removed by gentle shaking and the seedlings were incubated in nutrient solution. The nutrient solution was changed every three days and the plants were kept for another ten days in a growth chamber with constant aeration at controlled day/night temperatures (27/20°C) with a 12 h photoperiod and light intensity of 330 µmol photons m− 2 s− 1. In all experiments, roots were separated from the shoot and photographed with a digital camera (Nikon D300S SLR). The obtained images were analyzed with the softwares RootReader2D and WinRhizo v. 4.0 (Regent Systems, Canada) to measure traits related to root morphology, such as total root length (L), total root surface area (SA), and surface area of roots with diameters between 0–1 mm (SA1), 1–2 mm (SA2) and larger than 2 mm (SA3) (cm2) (Sousa et al. 2012). Dry weight measurements were carried out for roots and shoots, which were placed separately in paper bags, dried in a forced circulation oven at 65°C and weighed on a precision scale until constant weight.
Greenhouse experiment
For the greenhouse experiment, 11 treatments described for hydroponics (Table 1) were arranged in a completely randomized design, with four replicates (Table 1). Pots containing 5 kg of a Latossolo Vermelho, very clayey texture (Typic Haplustox, Brazilian savanna) were used, with the following chemical and physical characteristics in the top soil (0–20 cm): pH-water = 5,24; Al = 0,4; Ca = 2,5; Mg = 0,2 (cmolc dm− 3); CEC (cation exchange capacity) = 11,8 cmolc dm− 3; P = 2,2; K = 30,3 (mg dm− 3); V (base saturation) = 23,2 % and clay content = 740 g kg− 1.
Twenty days before sowing, soil acidity correction was carried out based on chemical analysis. The liming requirement was calculated to reach a base saturation of 70% with the application of 6.0 Mg ha− 1 of dolomitic limestone (43% CaO, 14% MgO, 80% PRNT) and 1.0 Mg ha− 1 of phosphogypsum (17% Ca, 14% S). Irrigation was performed to maintain soil humidity at 80% of the field capacity. For soil fertilization, urea (90 kg ha− 1 of N), triple superphosphate (TSP) (450 kg ha− 1 of P2O5), 500 kg ha− 1 of KCl and 50 kg ha− 1 of commercial formulation of micronutrient FTE – fritted trace elements (9.0% Zn, 1.6% B, 0.8% Cu, 3.0% Fe, 2.6% Mn and 0.1% Mo) were applied.
The bacterial inoculation was prepared as follows: cells from 50 mL cultures incubated for 72 h in LB medium were harvested by centrifugation at 10,000 × g for 10 min, resuspended in a 0.85% (w/v) NaCl solution and the optical densities were adjusted to 1.0 absorbance at 540 nm, corresponding to 108 cells mL− 1. Subsequently, the bacterial suspension was added in a sterilized mineral coal in the proportion of 30% (w/v) of liquid inoculant (10 mL pot− 1 for individual strain or 5 mL pot− 1 of each strain for co-inoculation). The inoculant (bacteria + mineral coal) was pelletized as a seed coat onto maize seeds, using 4% (w/w) cassava starch gum as adhesive. Strains were inoculated on five maize seeds (cultivar AG 7098), leaving three plants per pot eight days after sowing. The side-dress fertilization was performed with urea (90 kg N ha− 1) at 20 days after sowing.
The plants were harvested at 45 days after sowing, and roots and shoots were separated and dried in a forced air circulation oven at the temperature of 65o C until constant weight to obtain dry matter. Then, the plant material was ground in a Wiley mill, and chemical analyses were conducted for determining the N, P and K concentration in shoots and roots in the Laboratory of Plant Chemical Analysis at Embrapa Milho e Sorgo using ICP-OES (Nogueira and Souza 2005). N, P and K content were calculated by multiplying the N, P and K concentrations with the dry weight, which was performed separately for roots and shoots. The rhizosphere soil was collected for determination of available phosphorus content, extractor Melich-1 (Silva 2009) and acid and alkaline phosphatase activity.
Phosphatase activity in soil
The determination of phosphatase activity was performed according to the methodology described by Tabatabai and Bremner (1970). For acid phosphatase analysis, the sample’s pH was adjusted to 6.5 with 1 M HCl, and for alkaline phosphatase activity, the pH was corrected to 11 with 1 M NaOH. The p-nitrophenol (PNP) concentration was determined in triplicate by a colorimetric measurement at 540 nm (Labsystems Multiskan, USA) and compared to a standard curve.
Statistical analysis
The data was submitted to variance analysis using the software SISVAR 5.6 (Ferreira 2011) and the means were compared by the LSD test at 5% level of probability.