3.4. Molecular Characterization of KSB Using ARDRA, BOX, ERIC, and REP PCR
Alu I,
Hae III,
Hinf I, and
Taq I provided distinctive and differentiating restriction patterns which were employed for the molecular characterization of all the isolates in the amplified ribosomal DNA restriction analysis (ARDRA; 16S rDNA PCR-RFLP technique). Using a 100 bp and 1 kb ladder as a molecular weight marker, the band patterns of the representative KSB strains are displayed in (
Figure 2). Cluster analysis was utilized to examine genetic relationships using Jaccard’s similarity coefficient-based banding pattern. The dendrogram obtained from KSB strains differentiated the 44 strains into two major clusters with a coefficient ranging from 0.54 to 1.00. At coefficient 0.54, all the isolates were divided into two clusters, A and B. Cluster A comprised of total 35 KSB strains out of which KSB 7, 35; KSB 6, 30; KSB 42, 43 were found genetically similar at similarity coefficient 1.00. Cluster B comprised of 9 strains in which KSB 34, 35 were also found similar at similarity coefficient 1.00.
A similarity matrix was generated by comparing combined REP, BOX, and ERIC PCR in a pair-wise comparison pattern. Jaccard similarity indices established on the basis of 292 bands of combined primer (BOX, ERIC, REP) PCR ranged from 0.46 to 0.90 (
Supplementary Figure S1). At coefficient 0.46, all the isolates were divided into two clusters, A and B. The first (A) cluster was divided into two sub clusters, A1 and A2. Sub-cluster A1 includes 27 strains KSB 1, KSB 24, KSB 8, KSB 9, KSB 13, KSB 25, KSB 2, KSB 4, KSB 16, KSB 14, KSB 17, KSB 15, KSB 3, KSB 11, KSB18, KSB 30, KSB 31, KSB 12, KSB 22, KSB 26, KSB 27, KSB 28, KSB 29, KSB 5, KSB 6, KSB 7, and KSB 10. Sub-cluster A2 comprises only four strains KSB 19, KSB 23, KSB 21, and KSB 30. Cluster 2nd (B) comprises a total of 13 strains and is further divided into two sub-clusters, B1 and B2. Sub-cluster B1 comprises eight strains KSB 32, KSB 33, KSB 34, KSB 35, KSB 36, KSB 37, KSB 39, and KSB 38. Sub-cluster B2 comprises four strains KSB 40. KSB 41, KSB 42, KSB 43, and KSB 44. The KSB isolates exhibited significant genetic diversity based on BOX, ERIC, and REP PCR profiles, and REP-PCR fingerprinting was found to be a highly reliable and reproducible approach in the current investigation to differentiate closely related bacterial strains. Recently, there have been significant advances in the characterization of microbial species brought about by the advent of more well-known molecular techniques, but there are still no simple ways to carry microbe identification all the way down to the strain level.
Similarly, Dong et al. [
38] evaluated the identification of KSB isolates by 16SrDNA sequencing and on the basis of the similarity pattern classified into fourteen distinct ARDRA types. The isolates were clustered in four genera as
Bacillus (A, B, C, E, F, G, and H);
Enterobacter (I, J, K, and L);
Pseudomonas (D and N); and
Achromobacter (M). According to Jain et al. [
32], the molecular diversity investigations using REP, ERIC, and BOX elements from repetitive DNA PCR revealed high genetic variation among the 30 zinc-tolerant bacteria. The study revealed that ERIC, REP, and BOX PCR profiles are highly reproducible and can easily distinguish between related bacterial species and ZTB isolates. It also revealed significant genetic divergence based on these profiles.
3.5. Determination of Physiological and PGPR Attributes of Potent KSB Strains
The major physiological attributes comprise temperature, salinity tolerance, pH, drought tolerance, and antibiotic sensitivity test. Potassium aids in activating various physiological and metabolic processes, thus enhancing the tissue water potentiality assisting in extreme temperature stress tolerance. The data indicated that the highest zone of solubilization was obtained at 30 °C, which was found to be the optimum temperature for growth of KSB isolates. It was further inferred that almost all the isolates showed significant solubilization in the temperature range of 30 °C to 50 °C. K solubilization increased at higher temperatures of incubation for all the bacterial strains as compared to that at low temperature range. A total of 41 strains were able to survive even at 50 °C with good KSI as compared to that at low temperatures, leading to an interpretation that the strains are more susceptible to high-temperature conditions (
Supplementary Sheet Table S3). Verma et al. [
17] conducted studies to ascertain the impact of the isolated bacteria grown on modified Aleksandrov broth at various temperatures ranging from 25 to 50 °C, demonstrating maximal solubilization (5.0 cm) at 25 °C and that it decreased with the rise in temperature. Other bacterial strains demonstrated substantial solubilization in the temperature range of 25 °C to 35 °C; however, bacterial strain UPS2C1 displayed maximum solubilization (4.13 cm) at 28 °C. All bacterial strains showed a decrease in K solubilization at a higher incubation temperature, i.e., 45 °C.
K plays an essential role in relation to the osmotic adjustment maintaining the turgor and regulating to all the metabolic activities under salinity stress. A range of varying concentrations (2–10%) of NaCl salt was added to the LB broth inoculated with the KSB isolates. Among the 44 isolates, 43 isolates were able to produce halo zones at 2%, 42 isolates at 4%, 39 isolates at 6%, and 12 isolates at 8%, whereas only 2 isolates were able to at 10% salt concentration. This clearly indicated that an increase in saline conditions inhibited the growth of the KSB strains (
Supplementary Sheet Table S4). The growth potential of seven isolates was evaluated by Bachani et al. (2016) [
41] in nutrient broth with varying NaCl concentrations from 0 to 20%, temperatures between 30 °C and 37 °C, and pH ranges from 3.0 to 8.0 and identified the halotolerant species as
A. soli MTCC 5918,
A. baumannii MTCC 5916, and
E. xiangfangensis MTCC 5917.
To determine the effect of pH stress on K solubilization, all the 44 KSB isolates were grown under different pH conditions. Changes of pH in the medium were determined with a change in the pH of the LB broth medium from a range of pH 4–10. All the 44 KSB isolates exhibited growth at all the pH conditions. When bacterial isolates were grown in a medium with a pH of 6.0, it was discovered that K solubilization was at its highest. K solubilization decreased as the pH of the medium increased. At pH 6.0, strain KSB 13 showed the highest levels of K solubilization, followed by strain KSB 38 (
Supplementary Sheet Table S5). Saha et al. [
18] investigated the impact of pH variation on a subset of seven KSB strains. With the exception of Bacillus axarquiensis BHU27, Bacillus safensis BHU12 showed the greatest bacterial cell development at pH 8.0. This was followed by pH 7.2, and pH 8.6 (p140.05). It was discovered that pH 9.2 resulted in noticeably lower bacterial cell proliferation.
K also regulates turgor maintenance, osmotic adjustment, and aquaporin function under drought conditions. To check this, artificial drought conditions for all the 44 KSB isolates were recorded for 48 h and growth was measured on a spectrophotometer at 660 nm in comparison without PEG in Aleksandrov broth. All the 44 KSB isolates exhibited growth at 5–25% PEG concentration whereas only 7 isolates showed growth at 30% PEG concentration (
Supplementary Sheet Table S6). Rodge et al. [
42] assessed the capacity of rhizobacteria to boost plant growth in drought conditions and confirmed it in a plate test by providing water stress conditions. According to the findings, under conditions of water stress, rhizobacteria from
Ficus religiosa dramatically increased root and shoot length compared to the controls.
An antibiotic sensitivity test was done by placing antibiotic discs of penicillin, ampicillin, neomycin, ceftizoxime, kanamycin, and tetracycline on the plates; the susceptibility or resistance of the bacterial strain towards a specific antibiotic was observed and the results are summarized in the table (
Supplementary Sheet Table S7). Results were in accordance with the findings described by Karthik et al. [
43], where the isolates were found to be sensitive to penicillin, gentamicin, neomycin, streptomycin, and vancomycin and resistant against ampicillin and methicillin. These findings led to the conclusion that antibiotic resistance helps the bacterial strain survive and adapt to harsh environmental circumstances during natural selection.
Plant growth-promoting rhizobacteria (PGPR) are a diverse group of soil bacteria that can solubilize potassium. They confer positive effects on the host plants in association, which stimulate the host plants’ growth through increased nutrient mobility, uptake, and enrichment through several mechanisms. As a result, they are vital for the preservation of soil ecology and fertility. Keeping all these benefits in consideration, all 44 KSB isolates were assessed for PGP attributes, i.e., phosphorus, zinc, silica, IAA, GA
3, siderophore, ammonia production, GNFM, ACC deaminase, biofilm, antagonistic activity, EPS, and HCN production and the results are summarized in the table (
Table 2 and
Table 3).
All the 44 KSB isolates were found to be positive for ammonia production, GNFM test, and ACC deaminase test, whereas none of the isolates were reported to be positive for the formation of biofilm. A total of 26 isolates were found to be positive for HCN production whereas only 9 isolates exhibited antagonistic activity. In order to evaluate the P solubilizing potential, the 44 KSB isolates were spot inoculated on Pikovskaya medium agar plates and their solubilization indices were recorded. All the isolates were found positive with KSB 13 (3.95 ± 0.07) and KSB 35 (3.95 ± 0.07) exhibiting the maximum SI followed by KSB34 (3.85 ± 0.14). The minimum value was exhibited by KSB 8 (2.02 ± 0.06). Similarly, Aliyat et al. [
44] demonstrated that the soluble phosphate in NBRIP medium ranged from 2.27 ≤ SPI ≤ 4.79, and the appearance of the halo zone in all strains was remarked after the third day of incubation.
Zinc in the plant depends on the total soluble Zn pool in the soil, with lower values resulting in reduced crop productivity and yield. Keeping this in view, KSB isolates were tested for their efficiency to solubilize zinc. KSB 38 (10.42 ± 0.13) showed maximum Zn solubilization followed by KSB 13 (10.40 ± 0.10) and KSB 39 (9.75 ± 0.13), whereas minimum by KSB 43 (3.77 ± 0.1). Similar studies were reported by Nagaraju et al. [
45], in which the isolates were evaluated for their efficiency of zinc solubilization on TRIS minimal medium. Results revealed that the isolate ZnSF-4 showed a maximum solubilization zone of 54 mm and least solubilization zone was observed in ZnSB-8 (7 mm). All the isolates were tested on Bunt and Rovira media supplemented with 0.25% magnesium trisilicate and the silica solubilization index (SSI) was calculated. Out of the total 44 isolates, 33 isolates showed positive results. SSI was observed in KSB 41 (3.73 ± 0.12), followed by KSB 13 (3.25 ± 0.15), and KSB 38 (3.02 ± 0.12), whereas the minimum was observed in KSB 19 (2.02 ± 0.14). The capacity of the isolates to solubilize silicate, produce indole acetic acid (IAA), stimulate plant development, and induce silicon (Si) uptake in plants was examined by Kang et al. [
46]. Based on its capacity to dissolve silica and the formation of IAA, a single bacterial isolate was selected.
Indole acetic acid (IAA) production is a major property of rhizospheric bacteria and stimulates and facilitates plant growth. The key precursor in the biosynthesis of IAA is tryptophan. All the KSB strains were tested for the qualitative and quantitative production of IAA. Maximum production was showed by KSB 41 (304.81 ± 0.79 µg/mL) followed by KSB 44 (260.77 ± 0.25 µg/mL) and KSB 13 (170.90 ± 0.15 µg/mL), whereas the minimum was showed by KSB 26 (11.12 ± 0.81 µg/mL). A total of 4 of the 63 KSB isolates tested by Shree et al. (2015) [
47] were able to produce IAA. Between 0.229 and 0.458 ng/mL, all were positive for IAA production. Gibberellins are tetracyclic diterpenoid acids involved in a number of developmental and physiological processes in plants. Among all the tested 44 KSB isolates maximum GA
3 was found in KSB 34 and minimum in KSB 37. Thirty fluorescent Pseudomonas sp. isolates were screened by Sharma et al. [
48] from the rhizosphere of apple and pears, and their production of gibberellins was determined to be between 116.1 and 485.8 g/mL.
On treatment with Nessler’s reagent, all the 44 KSB isolates developed a brown to yellow color, indicating that all the isolates exhibited positive results. Shree et al. [
47] concluded that all the isolates had the potential to produce ammonia, a PGPR trait that indirectly influences the growth of plants and microorganisms. The maximum ammonia production was observed in NKC-20 among all the isolates. The siderophore production efficacy of the 44 KSB isolates was observed using the chromo-azurol plate assay method. All the tested isolates were found to be positive, thus resulting in the formation of an orange halo zone around the colonies. KSB 41 (5.80 ± 0.10) followed by KSB 44 (5.55 ± 0.14) and KSB 34 (5.44 ± 0.13) showed the maximum zone of solubilization and KSB 42 (2.10 ± 0.20) exhibited the minimum value. Shree et al. [
47] identified four out of 63 isolates and only 2 were found to be positive for siderophore production. Plant growth-promoting rhizobacteria (PGPR) with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity are the immediate precursor to the plant hormone ethylene and have the potential to support plant growth and development in unfavorable environmental conditions. In the present study, all the 44 KSB isolates evinced the ACC deaminase activity by their growth on minimal media containing ACC as the sole nitrogen source. Pashapour et al. [
49] evaluated those 45 strains belonged to rhizobium and 2 bacterial strains belonged to Pseudomonas fluorescens. The results indicated that 15 strains produced ACC-deaminase.
The KSB isolates were grown on glucose nitrogen-free mineral agar (GNFM medium) and incubated for three to five days at 30 °C in order to assess their N
2 fixing capacity. After the incubation period, the color of the medium changed from green to yellow, which was indicative of the isolates’ capacity to fix N
2. All the isolates were found to be positive. As a potential alternative to reduce the effects of chemical fertilizer application, Akintokun et al. [
50] evaluated the N
2 fixing ability of the isolates as the first screening criterion. This trait is highly significant for plants. Total nitrogen fixation was present in 41 and 25% of the isolates from yellow and white maize, respectively. In order to study the antagonistic activity, inhibition tests with 44 KSB isolates against maize specific fungi were employed. A total of 9 KSB strains were found to exhibit antagonistic activity. According to Karthik et al. [
43], strain AR6 exhibited high antagonistic activity against the plant pathogenic fungus
A. niger. The ability of the rhizobacterial strain AR6 to produce protease, lipase, and amylase suggests that the most prominent characteristics of antagonism are the synthesis of hydrolytic enzymes.
The development of biofilms, in which bacterial cells adhere to abiotic and biotic surfaces, is a potential mechanism for mobilizing the K reserve in plants. In a biofilm, cells that are made up of polysaccharides, DNA, and junk proteins are immobilized inside a matrix of extracellular polymers that the cells themselves manufacture. In the current investigation, neither strain could cause the development of biofilms. In order to increase the amount of K released, Nagaraju et al. [
45] screened the isolates for biofilm development. When biofilm is used, four hypho-bacteria have shown a high capacity for solubilizing potassium (K), which is discharged at a significantly high rate. Production of HCN plays a role in suppressing soil-borne pathogens and is therefore crucial for maintaining pathogen control. The picric acid assay was used to evaluate the effectiveness of potent KSB isolates. Out of the total 44 KSB isolates, only 26 isolates were found to be positive. Similar work was demonstrated by Rodge et al. [
42] where only three isolates were found to be to be positive for HCN production, which acts as inducer of plant resistance; SMJ2II showed the fastest reaction in the picric acid assay. Exopolysaccharide (EPS) is a complex mixture of macro-molecular electrolytes found in the outer cells of bacteria that are discharged as mucus and that play a part in soil aggregation. It is characterized by the production of gummy secretions either around the colony periphery or by the entire colony in LB agar medium. All the 44 KSB isolates subjected to qualitative and quantitative estimation of exopolysaccharides were found to be positive. KSB 38 (2.16 ± 0.05 gm) exhibited the maximum value followed by KSB 44 (1.50 ± 0.09 gm) and KSB 43 (1.30 ± 0.01 gm), whereas the minimum value was showed by KSB 15 (0.05 ± 0.01 gm). Patel et al. [
51] reported the extraction of EPS from a selected four isolates of the total nine isolates and recorded its dry weight. KPEP3 and KPEP4 exhibited the highest EPS production with 0.259 g and 0.312 g, respectively, from 100 mL culture broth.
Further, the hydrolytic enzymes were determined in 44 KSB strains and all the 44 KSB isolates were found to be positive for phytase [
24], cellulase [
52], amylase [
53], and lipase [
53]. Except for KSB 1, KSB 32, KSB 33, and KSB 43, all the 40 isolates showed a significant solubilization index for protease production (
Table 4). Karthik et al. [
43] isolated 48 different cultures, and all these isolates exhibited extracellular protease activity on skim-milk agar plates. Only 16 KSB isolates were found evincing the chitinolytic activity, whereas the rest were found negative, as no zone was observed on the chitin agar plates. Similar studies were reported by Kuzu et al. [
54] including fifty-five strains from chitin wastes and which were tested for chitinolytic activity. After screening the strains for their ability to utilize chitin, 12 of them (2.18%) exhibited chitinolytic activity. Saiyad et al. [
24] screened five phytase-positive bacteria on phytase screening turbid agar media plates; on the basis of zone of hydrolysis, the highest SI 3.13 was recorded for isolate
B. coagulans II followed by
B. isolate E. asburiaecoagulans I,
Bu. Metallica, and
B. megaterium. Sawangjit [
52] evaluated eight cellulose-degrading bacteria from soil samples in the Samut Songkhram province, Thailand based on solubilization index. Geetha et al. [
53] reported 140 bacteria from rhizospheric soils and the bacterial cultures were cultivated on nutrient agar medium supplemented with egg yolk. Overnight incubation of the cultures produced clear zones around the colony, indicating lipase activity. The strains KG-50 and TG-60 showed significant lipase activity.