Preliminary study on phosphate solubilizing Bacillus subtilis strain Q3 and Paenibacillus sp. strain Q6 for improving cotton growth under alkaline conditions

Isolation of rhizobacteria from cotton rhizosphere

Soil samples were collected from the rhizosphere of cotton plants cultivated on farmer fields at two sites (Bahawalpur and Haroonabad). Isolation of bacterial colonies was performed using the dilution plate technique described by Dworkin & Foster (1958). For purification of bacterial colonies, samples were streaked onto general-purpose agar plates prepared from glucose (10 g), K₂HPO₄ (2.5 g), KH₂PO₄ (2.5 g), (NH₄)₂HPO₄ (1.0 g), MgSO₄.7H₂O (0.2 g), FeSO₄.7H₂O (0.01 g), MnSO₄.7H₂O (0.007 g) and agar powder (15 g) per liter of deionized water and final pH adjusted to 7.2. These plates were incubated at 30 °C for 2 days. Isolated single colonies were removed and re-streaked to fresh general purpose agar plate and incubated as described above. The technique was repeated and the single colony cultures were preserved for further experimentation.

Characterization

The bacterial isolates were characterized for catalase activity as described by MacFaddin (1980). A urease test was carried out using sterilized urea broth (Shruti, Arun & Yuvneet, 2013). The ability of rhizobacterial strains to solubilize inorganic phosphate was investigated using Pikovskaya’s agar medium (Pikovskaya, 1948). The ammonia production test was performed using Nessler’s reagent, following methods by Cappuccino & Sherman (1992). The Gram staining and cell shape of bacteria was studied after 48 h of growth on agar plates (Vincent, 1970).

Osmoadaptation assay

Osmoadaptation assays of bacterial isolates were performed as described by Zahir et al. (2010). Salinity tolerance of bacterial isolates was assessed at 1.42 (control), 4, 8 and 12 dS m⁻¹ salinity levels developed by adding calculated amounts of mixed salts (NaCl, MgSO₄, CaCl₂ and Na₂SO₄). Salts were calculated by using quadratic equation as described by Haider & Ghafoor (1992) for the development of salinity levels in broth. The broth was autoclaved at 121 °C for 30 min and then 25 mL of broth was taken in 50 mL flasks and inoculated with rhizobacterial isolates. Flasks were incubated at 30 °C and after 3 days of incubation, absorbance (optical density) was measured by using spectrophotometer (Model Carry 60; Agilent Tech., Santa Clara, CA, USA) at 600 nm wavelength.

Growth of PGPR at different pH levels

The growth of bacterial isolates was determined at three pH levels using methods by Shruti, Arun & Yuvneet (2013). The purpose of the study was to assess the ability of isolates to grow under alkaline conditions. Nutrient broth was poured into test tubes and pH was adjusted to 4.0, 7.0 and 10.0. The tubes were autoclaved, cooled and inoculated with the respective strain and incubated at 30 °C for 24 h. The optical density (OD) was measured by using a spectrophotometer (Model Carry 60; Agilent Tech., Santa Clara, CA, USA) at 600 nm wavelength.

Root colonization assay

Root colonization ability of phosphate solubilizing isolates in cotton was studied under axenic conditions, following methods by Simons et al. (1996). Glass jars were sterilized and filled with sterilized sand. Half strength Hoagland solution was used to moisten the sand. Surface-sterilized cotton seeds were submerged for ten minutes in the broth of the respective strains and were then sown. Jars were placed in growth chambers at 28 ± 1 °C. After 1 week, root tips (0.2 g) were removed and added to 5 mL sterilized distilled water and shaken vigorously for 30 min using an orbital shaker (Memmert, Schwabach, Germany) at 100 rpm. Bacterial suspension solutions were made from 10⁻¹ to 10⁻⁶. One mL of each dilution was poured onto petri plates filled with sterilized general purpose media that were then incubated at 28 ± 1 °C. The CFU mL⁻¹ was calculated and colonies were counted by using a digital colony counter (J.P Selecta, Barcelona, Spain).

Quantitative estimation of phosphate solubilization

Cultures showing positive results in the agar medium examinations were further assessed for P solubilization quantitatively using methods described by Clescerie, Greenberg & Eaton (1998). For this purpose, cultures were inoculated in 50 mL of Pikovskaya’s broth and incubated at 30 °C for 48 h. The broth was centrifuged and 5 mL of the supernatant was taken following by addition of 5 mL of Vanadomolybdate solution. The volume was made up to 25 mL and incubated overnight for development of yellow color. The absorbance was measured by using spectrophotometer (Model Carry 60, Agilent Tech., Santa Clara, CA, USA) at 420 nm wavelength.

Evaluation of phosphate solubilizing PGPR under axenic conditions

Based on in vitro characterization of PGPR strains, seven phosphate solubilizing strains were selected for their plant growth promoting abilities under axenic conditions. The experiment was conducted at College of Agriculture and Environmental Sciences, the Islamia University of Bahawalpur. Cotton seeds were surface sterilized by submersion in 95% ethanol for 30 s, followed by submersion in 0.2% HgCl₂ solution for 3 min. Seeds were then washed thoroughly with sterilized water. Three surface-sterilized seeds were inoculated in each of the seven selected isolates by submerging them in the respective broth for ten minutes. In case of the control, surface-sterilized seeds were treated with sterilized broth without inoculation. Inoculated seeds were transplanted to autoclaved glass jars filled with sand. Sterilized Hoagland solution (Hoagland & Arnon, 1950) (modified by adding 1.5 g L⁻¹ phosphate rock (Sigma-Aldrich, St. Louis, MO, USA) as source of phosphorus instead of KH₂PO₄) was added in the jars to provide nutrients to the seedlings. Jars were arranged in a completely randomized design (CRD) with three replications in a growth room with controlled temperature (30 °C), humidity (55–67%) and light intensity (1,300–1,400 µmoles/m²/s) with a 16 h light and 8 h dark cycle. Growth parameters, i.e., shoot length, root length, shoot fresh weight, root fresh weight, shoot dry weight, root dry weight and root/shoot ratio, were recorded after 20 days of germination.

16S rDNA sequencing of selected strains

Two strains (Q3 and Q6) were selected and identified through amplification, sequencing and bioinformatics analysis of their 16S rDNA gene sequences. For this purpose, crude DNA of the selected isolates Q3 and Q6 was extracted from the cell culture using proteinase K treatment (Cheneby et al., 2004). The 16S rDNA sequence was amplified in a thermocycler (Eppendorf, Hauppauge, NY, USA) using the universal primers for forward 785F (5′-GGATTAGATACCCTGGTA-3′) and reverse 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) reactions. The PCR reaction was carried out using 2.5 µL crude DNA as a template following the program as described by Hussain et al. (2011). The size of the amplified 16S rDNA was confirmed by separation on 1% agarose gel along with GeneRuler 1kb DNA (Fermentas, Burlington, Canada). The 16S rDNA PCR product was purified using a PCR Purification Kit (Favorgen, Changzhi Township, Taiwan) and amplified PCR products were sequenced using a commercial service offered by Macrogen Seoul, Korea (http://macrogen.com/eng/).

Strains were identified using a partial sequence of the 16S rDNA gene on MEGA 7.0.14 software (Pennsylvania State University, USA) and BLASTn search on NCBI servers. Sequences of closely related and validly published type strains (n = 15) used for constructing the phylogenetic tree were selected and retrieved from the MEGA database. The phylogenetic and molecular analyses were performed with selected closely related taxa according to procedure using MEGA version 7.0.14 (Kumar, Stecher & Tamura, 2016). The evolutionary history was inferred using the Neighbor-Joining method (Saitou & Nei, 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura, Nei & Kumar, 2004) and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated. The stability of the relationship was assessed by bootstrap analysis by performing 500 re-samplings for the tree topology of the neighbour-joining method L 195. We replaced “but” with “with”.

Statistical analysis

The data obtained from above tests were subjected to statistical analysis using MS Excel (Microsoft Office 10) and analysis of variance techniques (ANOVA) in Statistix 8.1. The means were compared using LSD at 5% probability level (p ≤ 0.05) (Steel, Torrie & Dicky, 1997).

Characterization

All 20 strains we isolated were positive for catalase. One strain (Q19) was positive for urease. Seven strains, i.e., Q2, Q3, Q6, Q7, Q8, Q13 and Q14, formed a clear zone around the colony in Pikovskaya agar medium and were found to show phosphorus solubilization capacities. Nine strains, i.e., Q2, Q3, Q6, Q8, Q13, Q14, Q15, Q18 and Q19, possessed the ability to produce ammonia. Eleven strains including Q3 and Q6 showed positive results for Gram staining while all the tested strains were rod shaped bacteria (Table 1).

Salinity tolerance of rhizobacterial strains varied among tested strains (Table 2). As the salinity level increased, most of the rhizobacterial growth decreased. At the highest NaCl salinity level (12 dS m⁻¹), the maximum optical density was given by strain Q6 followed by Q3, Q12 and Q11. The data reveals that maximum growth (OD at 620 nm) of bacterial isolates occurred at neutral and alkaline pH (Table 3). All rhizobacterial isolates showed poor growth under acidic pH. At neutral pH, the bacterial isolate Q13 showed maximum growth followed by Q6, Q7, Q2 and Q12. At alkaline pH (10.0), Q13 showed maximum growth followed by Q14 and Q1.

All selected strains efficiently colonized cotton roots with the maximum root colonization (4.34 × 10⁶ cfu g⁻¹) was observed in the case of strain Q3 followed by Q6 (Table 4). All strains were solubilizing inorganic phosphate in liquid broth (Fig. 1) but maximum optical density was given by Q3 (2.605 ± 0.06) followed by Q6 (2.44  ± 0.003) and Q7 (2.38 ± 0.12).

Effect of phosphate solubilizing PGPR on the growth of cotton seedlings

The seven isolates with phosphate solubilizing capacities that showed potential growth at the higher salinity levels were selected to investigate their plant growth promoting activities for cotton seedlings under axenic conditions. Results indicated that inoculation with these strains increased the growth of cotton seedlings as compared to the un-inoculated control.

Inoculation of phosphate solubilizing rhizobacteria significantly enhanced the shoot length (Fig. 2). Increase in shoot length was 71%, 59%, 50%, 46%, 24%, and 22% for Q3, Q6, Q13, Q7, Q14 and Q2, respectively, as compared to the un-inoculated control. Increases in the root length of the cotton seedlings were promising for all strains (Fig. 3). The maximum increase in the root length was observed with strain Q3 (162%), followed by Q6, Q13, Q8 and Q7. Phosphate solubilizing rhizobacteria significantly increased the shoot fresh weight of cotton seedlings compared to the control (Fig. 4). The maximum increase in the shoot fresh weight was observed with strain Q6 (87%), followed by Q3 (75%), Q7 (63%) and Q13 (60%) as compared to the un-inoculated control. The results of three isolates, i.e., Q2, Q8 and Q14, were non-significant when compared with control.

Inoculation of phosphate solubilizing rhizobacteria promoted a significant enhancement in the root fresh weight as compared to the un-inoculated control (Fig. 5). Maximum increase in the root fresh weight was observed with strain Q3 (3 fold) followed by Q6 (277%), Q7 (176%) and Q2 (150%), which were significantly higher than un-inoculated control. The two strains Q8 and Q14 were found to be non-significant compared with the control. The results regarding the root/shoot ratio revealed that phosphate solubilizing rhizobacterial strains contributed to enhancement of the root shoot ratio. The strain Q3 showed maximum increase in root/shoot ratio (50%) followed by Q6 and Q8 compared to the un-inoculated control (Fig. 6).

Shoot dry weight was significantly improved by rhizobacterial strains except Q2 where the increase was non-significant when compared with control. Maximum shoot dry weight of cotton seedlings was improved by Q6 followed by Q3 (Fig. 7). The PSB strains also improved the root dry weight of cotton seedlings however this increase was non-significant in case of Q2, Q8, Q13 and Q14 when compared with control. Maximum root dry weight was observed due to inoculation with Q3 followed by Q6 and Q7 where the results were significantly better than in-inoculated control (Fig. 8).

Identification of selected strains through 16S rDNA sequencing

The 16S rDNA genes 1000 bp and 1203 bp amplified from the strains Q3 and Q6 were sequenced and the sequence results were deposited in the GenBank database under the accession numbers KX788864 and KX788865 for Q3 and Q6, respectively.

The BlastN analysis of the 16S rDNA amplicon indicated their maximum similarity with the bacterial strains belonging to genus Bacillus and Paenibacillus, respectively. The analysis of the 16S rDNA of the bacterial strains Q3 and Q6 was carried out by constructing the phylogenetic tree following the neighbor joining method (Figs. 9 and 10). The bacterial strains Q3 and Q6 were observed to be phylogenetically positioned in the cluster comprising the bacterial strains belonging to the genus Bacillus and Paenibacillus, respectively. Following the phylogenetic relationship of the strain Q3 with B. subtilis (Fig. 9) and Q6 with several Paenibacillus sp. (Fig. 10), these bacterial isolates were named Bacillus subtilis strain Q3 and Paenibacillus sp. strain Q6, respectively.