In vitro evaluation of plant growth-promoting mechanisms

The ability to convert inorganic tri-calcium (Ca) phosphate–Ca₃(PO₄)₂ –into more soluble mono- and di-calcium phosphates was evaluated using qualitative plate-based and quantitative broth-based analyses. For the qualitative assay, each bacterial strain was spot-inoculated on Pikovskaya’s (PVK) agar medium plates containing 0.5% tri-Ca phosphate [36]. After incubating the plates for 14 days at 30°C, phosphate-solubilization was determined by the occurrence of a clear halo around bacterial growth. The phosphate-solubilization ability on plates was expressed using the solubilization index [26], where solubilization index (SI) = (halo + colony diameter) / colony diameter. To quantify the amount of phosphate solubilized, each bacterial strain was inoculated in PVK liquid broth to a concentration of 10⁶ cfu/mL and incubated for 72 hrs at 30°C in a shaking incubator (180 rpm). Subsequently, the culture supernatant was obtained via centrifugation for 10 min at 8000x g, and 1 mL of this supernatant was mixed with 500 μL of 10% (w/v) trichloroacetic acid and 4 mL of the colour reagent (1: 1: 1: 2 ratio of 3M H₂SO₄: 2.5% (w/v) ammonium molybdate: 10% (w/v) ascorbic acid: distilled water). After incubation for 15 min at room temperature, the absorbance of the resulting solution was measured at 820 nm. The amount of soluble phosphates produced by each strain per mL of the PVK medium (μg/mL) was estimated using a standard KH₂PO₄ curve [37].

The ability of each bacterial strain to hydrolyze phytate (an organic form of plant-unavailable phosphate) using the phytase enzyme was evaluated in both qualitative and quantitative assays. For qualitative evaluation, each strain was spot-inoculated onto phytase screening medium (PSM) agar plates containing sodium phytate [38]. After incubating the plates for 14 days at 30°C, phytate hydrolyzation was determined by the development of a clear halo around bacterial growth and was expressed as SI. For quantitative evaluation, each bacterial strain was inoculated to a concentration of 10⁶ cfu/mL in PSM broth and incubated at 30°C for 72 hrs in a shaking incubator (180 rpm). The culture supernatant was then extracted via centrifugation for 15 min at 8000x g and 150 μL of the supernatant was mixed with 600 μL of a solution containing 0.1M Tris-HCl, 2mM sodium phytate and 2mM CaCl₂. Following the incubation period of 30 min at 37°C, 750 μL of 5% (w/v) trichloroacetic acid and 750 μL of the colour reagent (4: 1 ratio of 1.5% (w/v) ammonium molybdate in 5.5% (v/v) H₂SO₄: 2.7% (w/v) ferrous sulfate solution) were added. After 5 min of incubation, the absorbance of the resulting solution was measured at 700 nm. The standard curve of KH₂PO₄ was used to estimate the amount of soluble phosphorus released by each bacterial strain by hydrolyzing phytate. One unit (U) of phytase activity was defined as the amount of phytase enzyme required to liberate 1 nmol of soluble phosphorus per minute under the given assay conditions and is expressed per mL of PSM culture [39].

To evaluate siderophore production by the six bacterial strains, each strain was spot-inoculated on chrome azurol S (CAS) agar plates and incubated for 7 days at 30°C [40]. The colour change from blue to orange/deep yellow around the bacterial growth on the CAS agar plates indicated the production of siderophores by bacteria. This area of the orange halo was measured and expressed as cm² [28].

The ability of the six bacterial strains to modulate vital plant hormones (IAA and ethylene) in order to enhance the growth and development of the host plant was analyzed. To evaluate the in vitro production of IAA, each bacterial strain (ca. 10⁶ cfu/mL) was inoculated into Luria Bertani broth amended with 5 mM L-tryptophan and incubated for 72 hrs at 28°C in a shaking incubator (150 rpm) [41]. After centrifugation (8000x g; 15 min), 1 mL of culture supernatant was mixed with 100 μL of orthophosphoric acid (10mM) and then 2 mL of the Salkowski’s reagent (1: 30: 50 ratio of 0.5M FeCl₃: 95% (w/w) sulfuric acid: distilled water) was added. The resulting solution was incubated for 15 min at room temperature and the absorbance of the colour that developed was measured at 530 nm [42]. A standard curve of pure IAA was used to estimate the amount of IAA produced by each strain per mL of the growth medium [43].

The ACC deaminase activity of the six bacterial strains was examined using in vitro and in vivo techniques described by Penrose and Glick [44]. Each strain was grown to stationary phase in tryptic soy broth (nutrient-rich medium) at 30 ºC. Bacterial cells of each strain were harvested via centrifugation at 8000x g. To induce the ACC deaminase activity, bacterial cells were suspended in DF salts minimal medium (nutrient-poor medium) amended with 3mM ACC as the sole source of nitrogen and grown for 24 hrs at 30 ºC in a shaking incubator (200 rpm). The bacterial cells were harvested via centrifugation (8000x g), washed and suspended either in 0.1M Tris-HCl for in vitro analysis or in 0.03M MgSO₄ for in vivo analysis. For the in vitro assay, bacterial cells suspended in 0.1M Tris-HCl were mixed with toluene, and a portion of the toluene-treated cells was mixed with 0.5M ACC and incubated for 15 min at 30 ºC. After adding 0.56M HCl, the solution was mixed, and the supernatant was collected by centrifugation (16000x g). The supernatant was then mixed with 0.56M HCl and 2,4-dinitrophenylhydrazine reagent (0.2% 2,4-dinitrophenylhydrazine in 2M HCl) and incubated for 30 min at 30 ºC. After adding 2M NaOH, the absorbance of the resulting solution was measured at 540 nm. ACC deaminase activity was quantified using pure α-ketobutyrate as the standard and expressed as the amount of α-ketobutyrate produced per mg protein per hour. To analyze ACC deaminase activity in vivo, a gnotobiotic root elongation assay was used involving ethylene-sensitive plants–canola (Brassica napus) and tomato (Solanum lycopersicum). Canola seeds (var. Rugby Roundup ready) were obtained from the SeCan Association’s Alberta branch (Lamont, AB, Canada). Tomato seeds (var. Celebrity) were obtained from the West Coast Seed Company, Delta, BC, Canada. For surface-sterilization, seeds were immersed in 30% hydrogen peroxide for 90 s and washed thrice in sterile distilled water. Effectiveness of the surface sterilization was confirmed by imprinting ten randomly selected canola and tomato seeds on tryptic soy agar (TSA) plates which were incubated for 48 hrs at 30°C to check for surface contamination. The ACC-induced bacterial cells of each strain suspended in 0.03M MgSO₄ (OD₆₀₀ = 0.15) were used in this assay. Surface-sterilized canola and tomato seeds were incubated in petri dishes for 1 hr with one of the following treatments: sterile 0.03M MgSO₄ (control) or bacterial suspensions of each of the six strains. Following the incubation period, 7 seeds per plant species from each treatment were aseptically placed in sterile CYG™ germination pouches (Mega International, Newport, MN, USA) containing 15 mL sterile distilled water. Subsequently, the pouches were incubated in a growth chamber (Conviron CMP3244, Conviron Products Company, Winnipeg, MB, Canada) maintained at 20 ºC with a day/night cycle beginning with 12 hrs of dark followed by 12 hrs of light, with light intensity set to 18 μmol/m²/s. The primary root lengths of canola and tomato seedlings from each treatment were measured five days after germination.

The ability of the six bacterial strains to secrete key cell wall degrading enzymes (chitinase, β-1,3-glucanase, protease and cellulase) was evaluated in vitro. Qualitative evaluation of chitinase activity included spot-inoculating each strain on chitin agar plates containing 1.62 g nutrient broth (Sigma-Aldrich, USA), 0.5 g NaCl, 6 g M 9 salts (Difco, USA), 8 g colloidal chitin and 15 g agar per litre [45]. After incubation for 7 days at 30°C, a clear halo surrounding the bacterial growth indicated positive chitinase activity and the width of the clearance zone was calculated as = (halo + colony diameter)–(colony diameter). The amount of colloidal chitin converted to simple sugars due to the chitinase activity was quantified by inoculating each bacterial strain to a concentration of 10⁶ cfu/mL in liquid chitin medium. After incubating for 5 days at 30°C in a shaking incubator (150 rpm), the culture supernatant was separated by centrifugation for 15 min at 8000x g and 500 μL of the supernatant was mixed with 500 μL of 1 M phosphate buffer and 500 μL of colloidal chitin solution containing 10 mg chitin. The resulting solution was incubated for 30 min at 37°C and centrifuged for 3 min at 8000x g to collect the supernatant. The supernatant (1 mL) was mixed with dinitrosalicylic acid (2 mL) and heated for 5 min in a boiling water bath. The absorbance of the final solution was measured at 575 nm [37]. Using glucose as the standard, the chitinase enzyme activity was estimated by measuring the release of reducing sugars from chitin. One unit (U) of chitinase activity was defined as the amount of chitinase enzyme that resulted in the release of 1 μmol of glucose from colloidal chitin per minute.

The β-1,3-glucanase activity was evaluated qualitatively by spot-inoculating each bacterial strain on plates containing β-1,3-glucan (laminarin) as the sole carbon source (5 g/L) along with other essential nutrients outlined by Renwick et al. [46]. Following incubation for 3 days at 30°C, plates were stained with Congo Re d (0.6 g/L) and left at room temperature for 90 min. The hydrolysis of glucan (i.e. glucanase activity) was indicated by the development of a yellow/orange zone around the bacterial growth on plates and the width of this yellow/orange zone was measured. For the quantitative determination of β-1,3-glucanase activity, the aforementioned laminarin medium (without agar) was inoculated with each bacterial strain (ca. 10⁶ cfu/mL) and incubated for 5 days at 30°C in a shaking incubator (150 rpm). The culture supernatant was extracted by centrifugation for 15 min at 8000x g and 500 μL of the supernatant was mixed with 500 μL of 1 M citrate buffer (pH 5.0) and 500 μL of 4% laminarin. After an incubation period of 30 min at 37°C, 2 mL of dinitrosalicylic acid was added and the solution was heated for 5 min in a boiling water bath. The absorbance of the resulting solution was measured at 500 nm [37]. The β-1,3-glucanase activity was estimated by measuring the release of reducing sugars from laminarin using glucose as the standard. One unit (U) of β-1,3-glucanase activity was defined as the amount of β-1,3-glucanase enzyme that resulted in the release of 1 μmol of glucose from laminarin per minute.

To evaluate the protease enzyme activity qualitatively, each bacterial strain was spot-inoculated on casein–yeast extract (CYE) agar plates amended with 7% skimmed milk powder [24]. The plates were incubated for 7 days at 30°C, and the development of a clear zone surrounding the bacterial growth indicated protease activity. The width of the clear zone was measured. To quantify the protease enzyme activity, each bacterial strain was inoculated to a concentration of 10⁶ cfu/mL in CYE liquid medium amended with 7% skimmed milk powder and incubated for 5 days at 30°C in a shaking incubator (150 rpm). The culture supernatant was extracted by centrifugation for 15 min at 8000x g and 500 μL of the supernatant was mixed with 500 μL of 0.2 M phosphate buffer and 500 μL of 1% azocasein. After incubation for 30 min at 37°C, 2 mL of 10% (w/v) trichloroacetic acid was added and the solution was further incubated for 5 min at room temperature. The absorbance was measured at 440 nm after the addition of 1M NaOH (1mL). Protease activity was determined by measuring the release of reducing amino acids from azocasein using tyrosine as the standard. One unit of protease enzyme activity was defined as the amount of the protease enzyme that resulted in the release of 1 μmol of tyrosine from azocasein per minute [37].

The cellulase enzyme activity for each bacterial strain was assessed qualitatively by spot-inoculating on CYE agar plates amended with 1% sodium carboxymethylcellulose [47]. After incubating for 48 hrs at 30°C, the plates were flooded with Congo red solution (0.5% w/v) and left at room temperature for 30 min. Subsequently, plates were drained and rinsed with 1 mol/L NaCl and the development of a clear zone around the bacterial growth indicated cellulase activity. The width of the clearance zone was measured. For quantitative evaluation of cellulase activity, each strain was inoculated to a concentration of 10⁶ cfu/mL in CYE liquid medium amended with 1% sodium carboxymethylcellulose and incubated for 5 days at 30°C in a shaking incubator (150 rpm). The culture supernatant was extracted via centrifugation for 15 min at 8000x g and 500 μL of the supernatant was mixed with 500 μL of 1M citrate buffer and 500 μL of 1% carboxymethylcellulose. After an incubation period of 30 min at 37°C, dinitrosalicylic acid (2 mL) was added and the solution was heated for 5 min in a boiling water bath. The absorbance of the resulting mixture was measured at 500 nm [45]. Using glucose as the standard, cellulase activity was estimated by measuring the release of reducing sugars from carboxymethylcellulose. One unit (U) of cellulase enzyme activity was defined as the amount of cellulase enzyme that resulted in the release of 1 μmol of glucose from carboxymethylcellulose per minute [37].

The six bacterial strains were analyzed for in vitro production of ammonia by inoculating each strain (ca. 10⁶ cfu/mL) into 10 mL of peptone water and incubating for 72 hrs at 30°C. After that, 500 μL of Nessler’s reagent was added and the development of a brown-yellow colour indicated ammonia production [48].

Catalase activity was evaluated for each bacterial strain by mixing a loopful of fresh bacterial culture with 50 μL of 3% (v/v) hydrogen peroxide on a sterile glass slide and incubating at room temperature for 1 min. The evolution of oxygen, i.e. development of gas bubbles, indicated a positive catalase reaction [24].