**1. Introduction**

Rhizobium bacteria contribute combined nitrogen to many leguminous plants while occurring inside specialized root structures called nodules [1]. The productivity of soybeans (*Glycine max*) is enhanced significantly through nodule occupancy by prolific fixers such as *B. diazoefficiens* USDA 110 [2]. Early during plant growth, root hairs signal soil-borne rhizobia by releasing isoflavonoids [3]. Rhizobia respond to isoflavonoids such as genistein by synthesizing Nod Factors, which in turn initiate curling of soy root hairs, followed by infection thread and nodule formation [4,5]. The nitrogen contributing efficacy of rhizobia varies widely, necessitating screening and evaluation of candidate bacterial strains for application in fields. Successful colonization of the infection thread and nodules require temporal adhesion and colonization of rhizobia at the root hair surface. Adhesion to the root surface is dependent on physical proximity, facilitated by bacterial motility in the soil. Adherence and colonization by proximal bacteria depend on the physiochemical compatibility of both the root and rhizobial surfaces.

**Citation:** Sandhu, A.K.; Subramanian, S.; Brözel, V.S. Surface Properties and Adherence of *Bradyrhizobium diazoefficiens* to *Glycine max* Roots Are Altered When Grown in Soil Extracted Nutrients. *Nitrogen* **2021**, *2*, 461–473. https://doi.org/ 10.3390/nitrogen2040031

Academic Editor: Jacynthe Dessureault-Rompré

Received: 14 October 2021 Accepted: 11 November 2021 Published: 15 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Surface properties of bacterial cells have a considerable influence on attachment to the root surface, so compatible surface properties contribute to increased adhesion. More hydrophobic mutants of *Bradyrhizobium japonicum* have been associated with more competitive nodule formation [6]. Bacterial lipopolysaccharides (LPS) have direct relation to hydrophobicity and more hydrophobic LPS is positively correlated to surface hydrophobicity [7,8]. *Bradyrhizobium* producing altered or limited extracellular polysaccharides (EPS) were defective in biofilm formation and binding to soy lectin and resulted in pseudo nodules [9,10]. Along with surface properties, there is evidence that motility can provide the cell with some significant advantages to adhere to the root surface [11,12]. On the other hand, not much is known about soy root surface properties. Soy roots produce surfaceexposed lectins which sequester cognate sugar moieties at the surface of bacteria [13,14], especially in acidic soil. Lectins are proteinaceous, specific carbohydrate-binding proteins. They interact with carbohydrates in a highly specific but non-covalent manner [15]. In more alkaline soil, root hairs produce the protein rhicadhesin which promotes the bacterial attachment [16]. The surface properties of rhizobia therefore play an important role in the initial adhesion to soy root surface.

Bacteria respond to specific chemical and physical cues by condition-specific gene expression and altered phenotype. This is also the case with *Bradyrhizobium*, as phenotypic variations have been reported for populations cultured in different sugar sources. While *B. diazoefficiens* swims by sub-polar flagella, L-arabinose induces the production of lateral flagella, which lead to swarming on moist surfaces [17]. The EPS composition of *B. japonicum* varies by the available sugar sources [9]. *Bradyrhizobium* are generally cultured using a mineral salt medium with peptone, yeast extract and either arabinose or mannitol as a carbon source. We asked whether *Bradyrhizobium* adapts its adhesion-specific phenotype when growing in soil. Arguing that the primary drivers of phenotypic change would be water-diffusible substances able to enter the cell, we cultured *Bradyrhizobium* in soybean field aqueous soil extract and characterized adhesion-pertaining phenotypes.

## **2. Materials and Methods**

## *2.1. Bacterial Strains and Culture Media*

*B. diazoefficiens* USDA 110, 126, 3384 and *B. elkanii* USDA 26 were obtained from the NRRL Culture Collection of the Agricultural Research Service, United State Department of Agriculture. Cultures were grown in liquid or on solid PSY and SESOM, a filter-sterilized liquid soil extract [18]. PSY was prepared as mentioned by Mesa et al. [19], containing per liter: KH2PO4, 300 mg; Na2HPO4, 300 mg; CaCl2·2H2O, 5 mg; MgSO4·7H2O, 100 mg; peptone, 3 g; yeast extract, 1 g; H3BO3, 10 mg; ZnSO4·7H2O, 1 mg; CuSO4·5H2O, 0.5 mg; Na2MoO4·2H2O, 0.1 mg; MnCl2·4H2O, 0.1 mg; FeCl3, 0.19 mg; thiamine-HCl, 1 mg; biotin, 1 mg; Na-panthothenate, 1 mg; L-arabinose.

Soil for SESOM was obtained from a field under soybean cultivation directly after harvest, was dried at 55 ◦C and stored at 4 ◦C. SESOM was prepared as described previously [18] by adding 200 g of dry soil to 1 l of prewarmed (60 ◦C) sterile MOPS buffer (10 mM, pH 7.0) ina2L Erlenmeyer and shaken for 4 h at 150 rpm. To remove soil particles, the suspension was filtered sequentially through filter paper and cellulose acetate filters of pore sizes 8, 4, 1.2, 0.8, 0.4 and 0.2 μM. The clear extract was filtered to sterility through a 0.2 μM bottle top filter. Each extract was checked for sterility by plating 20 μL on R2A agar and incubating at 30 ◦C for 72 h. The sterile SESOM was supplemented with 0.1 g/L Bacto peptone and 0.1 g/L L(+)-Arabinose.

### *2.2. Motility*

The effect of culture medium on swimming motility was determined by inoculating the center of low percentage agar plates (0.35%). Precultures were prepared by inoculating grown in PSY and SESOM and incubating at 30 ◦C for 24 h while shaking at 250 rpm. Cultures were diluted to A600 of 0.100 and 20 μL was drop inoculated at the center of PSY

or SESOM low agar plates. Plates were incubated at 30 ◦C for 10 d when the colony radius was measured.

#### *2.3. Microbial Adhesion to Hydrocarbons*

Microbial adhesion to hydrocarbons (MATH) was assessed to analyze surface hydrophobicity of cultures. Exponential phase cultures (50 mL) prepared as outlined above were harvested by centrifugation for 10 min at 10,000× *g* and resuspended in 15 mL sterile Phosphate Urea Magnesium Sulfate Buffer (PUM) containing 22.2 g/L K2HPO4·3H2O, 7.26 g/L KH2PO4, 1.8g/L Urea and 0.02 g/L MgSO4·7H2O with pH adjusted to 7.1 [20,21]. Following a second round of harvesting, cells were suspended in 15 mL PUM by vortexing. To three acid washed glass tubes, we added 4 mL cell suspension, and supplemented with 1 mL n-hexadecane, retaining the final 3 mL suspension as blank. Cell suspended in PUM were exposed to hexadecane by vortexing for 1 min and allowing the mixture to separate at room temperature for 1 h. After separation, 1 mL of the aqueous phase was withdrawn very carefully using acid washed, sterile glass pipettes, and transferred to a quartz cuvette. The absorbance was measured at 600 nm, using hexadecane-supplemented PUM without bacteria added as blank, termed Absorbance Math (AM). The absorbance of untreated cell suspensions was measured at 600 nm, using PUM buffer as blank, and termed Absorbance original (AO). The fraction of cells that partitioned to the hydrocarbon phase was calculated as:

$$\mathsf{F}\mathsf{p} = 1 - \left(\mathsf{A}\_{\mathsf{M}} / \mathsf{A}\_{\mathsf{O}}\right)$$

#### *2.4. Lectin Binding Assay*

Surface-exposed sugar moieties were characterized using a collection of 24 lectins (Vector Laboratories, Burlingame, CA, USA) (Table 1). Nine of these were fluoresceinconjugated, and the remaining fifteen were biotinylated. Exponential phase 24 h old cultures were prepared as described above, harvested, and washed with HSB buffer (2.383 g/L HEPES, 8.766 g/L NaCl, 0.011 g/L CaCl2 and 0.8 g/L sodium azide) (Vector Laboratories), and resuspended to absorbance of 0.100 at 600 nm. Lectins were diluted to 20 μg/ mL in HSB, and 100 μL added to 50 μL of cell suspension. After mixing by brief vortexing, lectins were allowed to bind for 10 min at room temperature, followed by vortexing and a further 10 min rest period. The cells were harvested by centrifugation at 16,200× *g* for 7 min. Unbound lectin was removed by three wash cycles using HSB buffer. For fluorescein-conjugated lectins, the resulting pellet was resuspended in 20 μL of HSB and the entire sample transferred to a clean microscopic slide. The drop of cell suspension was covered with a glass coverslip, pressed slightly to reduce cell movement, and the sides sealed using clear nail varnish to prevent evaporation. For biotinylated lectins, lectinexposed washed cells were supplemented with 50 μL of 20 μg/mL streptavidin-FITC and left to bind for 30 min. Unbound streptavidin-FITC was removed by washing cells twice. The pellet was resuspended in 20 μL HSB and transferred to a microscope slide.

Samples were viewed by fluorescence microscopy using an Olympus BX53 Upright Compound Microscope with 466/40 nm excitation and a 525/50 nm emission filter, captured using an Olympus DP70 digital camera. Sample images were captured using 100, 200, 400, 600, 800, 1000 and 1200 ms exposure time. Binding intensity was scored by the shortest exposure time yielding visible fluorescence, with 100 ms scoring level 4, and <1200 ms scoring level 0. The proportion of cells displaying fluorescence was determined by Image J by preparing the binary image under Process. The resulting binary image was further processed for analysis by using the following programs sequentially > Mask > Watershed > Fill holes. For the analyses, the analyze particles program was selected under the Analyze icon.


**Table 1.** List of lectins used.

\* Vector Laboratories.
