**Co-Inoculation of Rhizobacteria and Biochar Application Improves Growth and Nutrientsin Soybean and Enriches Soil Nutrients and Enzymes**

**Dilfuza Jabborova 1,2,3,\* , Stephan Wirth <sup>3</sup> , Annapurna Kannepalli <sup>2</sup> , Abdujalil Narimanov 1, Said Desouky <sup>4</sup> , Kakhramon Davranov <sup>5</sup> , Riyaz Z. Sayyed <sup>6</sup> , Hesham El Enshasy 7,8,9 , Roslinda Abd Malek <sup>7</sup> , Asad Syed <sup>10</sup> and Ali H. Bahkali <sup>10</sup>**


Received: 11 July 2020; Accepted: 27 July 2020; Published: 6 August 2020

**Abstract:** Gradual depletion in soil nutrients has affected soil fertility, soil nutrients, and the activities of soil enzymes. The applications of multifarious rhizobacteria can help to overcome these issues, however, the effect of co-inoculation of plant-growth promoting rhizobacteria (PGPR) and biochar on growth andnutrient levelsin soybean and on the level of soil nutrients and enzymes needs in-depth study. The present study aimed to evaluate the effect of co-inoculation of multifarious *Bradyrhizobium japonicum* USDA 110 and *Pseudomonas putida* TSAU1 and different levels (1 and 3%) of biochar on growth parameters and nutrient levelsin soybean and on the level of soil nutrients and enzymes. Effect of co-inoculation of rhizobacteria and biochar (1 and 3%) on the plant growth parameters and soil biochemicals were studied in pot assay experiments under greenhouse conditions. Both produced good amounts of indole-acetic acid; (22 and 16 μg mL<sup>−</sup>1), siderophores (79 and 87%SU), and phosphate solubilization (0.89 and 1.02 99 g mL<sup>−</sup>1). Co-inoculation of *B. japonicum* with *P. putida* and 3% biochar significantly improved the growth and nutrient content ofsoybean and the level of nutrients and enzymes in the soil, thus making the soil more fertile to support crop yield. The results of this research provide the basis of sustainable and chemical-free farming for improved yields and nutrients in soybean and improvement in soil biochemical properties.

**Keywords:** biochar; *Bradyrhizobium japonicum*; *Pseudomonas putida*; plant growth; plant nutrients; soil enzymes; soil nutrients; soybean

#### **1. Introduction**

The global climate scenario is experiencing a drastic depletion of soil nutrients due to various anthropogenic activities, burning of fossil fuel, and excess use of agrochemicals [1]. Applications of plant-growth promoting rhizobacteria (PGPR) and biochar have been advocated as an effective, cheap, and sustainable approach for the replenishment of crop health, crop nutrients, and soil nutrients and enzymes and for improving and sustaining soil fertility [2]. Furthermore, these amendments have a positive impact on the growth [3], development, and yield of several crops [4,5]. Various reports claimed that the application of plant growth-promoting rhizobacteria (PGPR) and biochar improves plant growth, plant nutrients, and physicochemical properties of soil [6–8]. Moreover, such applications of biochar also keep a check onatmospheric CO2 levels [9] and, thus, contribute todecrease global warming effects [10], while the use of PGPR to increase soil fertility and plant nutrients will help to reduce the doses of agrochemicals in the field [11].

A wide variety of symbiotic bacteria, such as *Rhizobium* sp. and *B. japonicum*, etc., have been reported to promote seed germination, the growth of root and shoot, andthe level of nutrients in soybean and also improve soil biochemical properties [4,5].Rhizobia-legumes symbiosis plays a vital role in increasing crop yields, reducing the use of inorganic nitrogen fertilizers and improving soil fertility [12]. Rhizobial species are commonly used as inoculants in various parts of the world for improving the yield of legumes. Co-inoculation with multifarious *Bradyrhizobium* sp. and *Pseudomonas* sp. improves plant growth, plant, and soil nutrients and enzymes through the production of siderophores [13], phytohormones [14], enzymes [15], exopolysaccharide [16], stress tolerance [17], and phosphate solubilization [18–23], etc. Thus, several studies reported increases in nodules number, nodule weight, nitrogen fixed, plant growth, and yield of legumes due to co-inoculation with plant growth promoting *Bradyrhizobium* sp. and *Pseudomonas* sp. [12–14], while the combination of biochar with PGPR further increases root length, shoot length, nodule per plant, seed number, and yield of crops [5].

The activity of PGPR bioinoculants helps in improving the level of extracellular soil enzymes that facilitates the decomposition of soil organic matter and ensures the availability of nutrients in the soil [15]. Among the soil enzymes, proteases and acid and alkaline phosphomonoesterase are the major enzymes that mediate the hydrolysis of the protein and phosphate (P) into bioavailable amino acids, organic nitrogen, and soluble P [16]. However, the activities of these enzymes are governed by many factors, such as soil properties, soil organic matter level, and the presence of organic compounds [24]. We hypothesized that co-inoculation with *B. japonicum*+*P. putida* and biochar would facilitate the beneficial effects on soybean plant growth, plant nutrients, and soil nutrients and enzymes.

The present study was aimed at evaluating the effects of co-inoculation of multiple plant growth-promoting traits positive in *Bradyrhizobium japonicum* USDA 110 and *Pseudomonas putida* TSAU1 and different levels (1 and 3%) of biochar on seed germination, growth parameters, and nutrient levels in soybean and the level of nutrients and enzymes in soil. The outcome of this study may provide a better way of increasing soil fertility and increasing the growth and yield of soybean. This approach has multiple dimensions; as utilization of biochar is not only a cheaper option but will also help in solving the management issues of biochar, it is expected to minimize the doses of agrochemicals and produce chemical-free food. The consortium effect of PGPR and application of biochar provide excellent benefits to the farmers as theyincur less investment and yield more crop productivity, and this organically grown crop has more demand with a good selling price.

#### **2. Materials and Methods**

#### *2.1. Bacterial Culture, Soybean, and Biochar*

*B. japonicum* USDA 110 and *P. putida* TSAU1 strains were collected from the culture collection of the Department of Microbiology and Biotechnology, National University of Uzbekistan, Tashkent, Uzbekistan. Soybean (*Glycine max* L. Merr.) seeds were obtained from Leibniz Centre for Agricultural Landscape Research (ZALF), Müncheberg, Germany.

The maize biochar (MBC) was collected from the Leibniz-Institute for Agriculture Engineering and Bioeconomy (ATB), Potsdam, Germany. Pyrolysis of MBC was carried out at 600 ◦C for 30 min and the chemical compositions of MBC were analyzed according to the method of Reibe et al. [25].

#### *2.2. Screening for the Production of PGP Metabolites*

*B. japonicum* USDA 110 and *P. putida* TSAU1 strains were screened for phosphate (P) solubilization on Pikovoskaya's agar and in Pikovoskaya's broth [26] for the production of indole-3-acetic acid (IAA)according to the method of Brick et al. [27], for production and estimation of siderophore according to the method of Patel et al. [28] and Payne [29], and the production and estimation of aminocyclopropane-1-carboxylate deaminase (ACCD) activity according to the method of Penrose and Glick [30]. The ACCD activity was measured as the amount of α-keto-butyrate produced per mg protein per h.

#### *2.3. Surface Sterilization, Germination, and Bacterization of Seeds*

Soybean seeds were sorted to eliminate broken, small, infected seeds and sterilized with 10% sodium hypochlorite solution for 5 min and washed three times with sterile, distilled water. Seeds were germinated in 85 mm × 15 mm tight-fitting plastic Petri dishes with 5 mL of water. *B. japonicum* USDA 110 and *P. putida* TSAU 1 broth rich in PGP metabolites were used for the inoculation of germinated seeds. Germinated seeds were first placed with sterile forceps into bacterial suspension (5 × 10<sup>6</sup> CFU g<sup>−</sup>1) for 10 min before planting, were air-dried, and then planted in plastic pots containing 400 g sandy loamy soil.

#### *2.4. Experimental Design*

The effect of rhizobacteria on the growth of soybean was studied in pot experiments in a greenhouse at ZALF, Müncheberg, Germany during July 2015. All the experiments were carried out in a randomized block design (RBD) with three replications. Experimental treatments included un-inoculated control (soil without biochar and soil with two levels of biochar (1 and 3%)), inoculation with *B. japonicum* USDA 110 (soil without biochar and soil with two levels of biochar (1 and 3%)), and co-inoculation with *B. japonicum* USDA 110 and *P. putida* TSAU 1 strains (soil without biochar and soil with two levels of biochar (1 and 3%)). The plants were grown in greenhouse conditions at 24 ◦C during the day and 16 ◦C at night for 30 days.

#### *2.5. Measurement of Plant Growth Parameters and Plant Nutrients*

Plants harvested after 30 days were subjected to the measurement of seed germination rate, root length, shoot length, root dry weight, shoot dry weight, and the number of nodules per plant of soybean. Plant nutrients, such as nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), sodium (Na), and calcium (Ca) were estimated from crushed plant tissue with an inductively coupled plasma optical emission spectrometer (ICP-OES; iCAP 6300 Duo, Thermo Fischer Scientific Inc., Waltham, MA, USA) via Mehlich-3 extraction [30]. The nitrogen and phosphorus contents of root and shoot were determined from dried powdered biomass. For nitrogen estimation, 1 g of plant biomass was digested with 10 mL concentrated H2SO4 and 5 g catalyst mixture in the digestion tube. The mixture was allowed to cool and then processed for distillation. The distillate was collected and titrated with H2SO4 blank (without leaf). Total nitrogen was calculated from the blank and sample titer reading [31]. For the estimation of P content, plant P was extracted with 0.5 N NaHCO3 (pH8.5)and treated with ascorbic acid in an acidic medium [32]. The intensity of blue color produced was measured and the amount of P was calculated from the standard curve of P. For the estimation of potassium content of plant biomass, 25 mL of ammonium acetate solution was added in 5 g of the biomass sample, the content was shaken for 5 min and filtered, and the amount of K from the filtrate was measured [33]. For the estimation of Na, Mg, and Ca, 1 g of plant extract was mixed with 80 mL of 0.5 N HC1 for 5 min at 25 ◦C followed by measurement of concentrations of these elements in the filtrate [34].

#### *2.6. Analysis of Soil Nutrient and Soil Enzymes*

The rootsoil (10 g) of experimental pots was air-dried soil, shaken with 100 mL ammonium acetate (0.5 M) for 30 min to effectively displace the available nutrients, and adhered to soil minerals. The soil organic carbon (SOC), nitrogen (N), phosphate (P), and potassium (K) content of soil were determined by the dry combustion method according to the method of Sims [35] and Nelson and Sommers [36] using a CNS analyzer (TruSpec, Leco Corp., St. Joseph, MI, USA). For this purpose, 10 mL of 1 N K2Cr2O7 and 20 mL of concentrated H2SO4was added in 1g soil, mixed thoroughly and diluted with 200 mL of distilled water followed by the addition of 10 mL each of H3PO4 and sodium fluoride. The resulting solution was used for the elemental analysis. Blank (without soil) served as control. Soil Organic Carbon (SOC) of soil sample was calculated with the help of blank and sample titer reading.

The acid and alkaline phosphomonoesterase activities were assayed according to the method of Tabatabai and Bremner [37].Moist soil (0.5 g) was placed in a 15 mL vial, and 2 mL of modified universal buffer (MUB) (pH 6.5 for the acid phosphatase assay or pH 11 for the alkaline phosphatase assay) and 0.5 mL of p-nitrophenyl phosphate substrate solution (0.05 M) were added to the vial, sequentially. The assay and control batches were replicated 3 times. The concentration of p-nitrophenol (p-NP) produced in the assays of acid and alkaline phosphomonoesterase activities were calculated from a p-NP calibration curve after subtracting the absorbance of the control at 400 nm. Protease activity was assayed according to the method of Ladd and Butler [38]. For this, 0.5 g of soil was weighed into a glass vial, and 2.5 mL of phosphate buffer (0.2 M, pH of 7.0) and 0.5 mL of N-benzoyl-L-arginine amide (BAA) substrate solution (0.03 M) were added. The ammonium released was calculated by relating the measured absorbance at 690 nm.

#### *2.7. Statistical Analyses*

All the experiments were performed in three replicates and the average of triplicate was considered. Experimental data were analyzed with the StatView Software (SAS Institute, Cary, NC, USA, 1998) using ANOVA. The significance of the effect of treatment was determined by the magnitude of the *p*-value (*p* < 0.05 < 0.001).

#### **3. Results**
