Journal of Genetic Engineering and Biotechnology (2017) 15, 379–391

H O S T E D BY
Academy of Scientific Research & Technology and
National Research Center, Egypt
Journal of Genetic Engineering and Biotechnology
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ORIGINAL ARTICLE

Phenotypic and genotypic characterization of

phosphate solubilizing bacteria and their efficiency
on the growth of maize
Amit Pande a,*, Prashant Pandey b, Simmi Mehra c, Mritunjay Singh d,
Suresh Kaushik e

a
Shri Venkateshwara University, Gajraula, Amroha 244236, India
b
Jaypee Hospital, Noida, Uttar Pradesh, 201304, India
c
Medanta The Medicity, Sec-38, Gurgaon, Haryana 122001, India
d
Allele Life Science, Sec-10, Noida 201301, India
e
Indian Agricultural Research Institute, New Delhi 110012, India

Received 6 March 2017; revised 10 April 2017; accepted 10 June 2017

Available online 27 June 2017

KEYWORDS Abstract Phosphate solubilizing bacteria (PSB) has ability to convert insoluble form of phospho-
Phosphorus; rous to an available form. Applications of PSB as inoculants increase the phosphorus uptake by
Phosphate solubilizing bac- plant in the agriculture field. In this study, isolation and identification of PSB were carried out in
teria (PSB); Indian agriculture field (Nainital region, Uttarakhand). A total of 8 phosphate solubilizing bacterial
Pikovskaya’s; colonies were isolated on the Pikovskaya’s (PKV) agar medium, containing insoluble tricalcium
Phosphate solubilization phosphate (TCP). The colonies showed clear halo zones around the bacterial growth were considered
index; as phosphate solubilizers. Out of 8 bacterial isolates, 3 isolates showed high phosphate solubilization
Tricalcium phosphate index (PSI) ranged from 4.88 ± 0.69 to 4.48 ± 0.30, lower pH ranging 3.08 ± 0.08 to 3.82 ± 0.12
and high phosphate solubilization varied from 305.49 ± 10 lg/ml to 277.72 ± 1.45 lg/ml, were
selected for further characterization. Based on the 16 S rRNA gene sequence analysis A4 isolate
and H6 isolate were closely related to Alcaligenes aquatilis (99%), and C1 isolate was closely related
to Burkholderia cepacia (99%). In addition, pot examination also showed the greatest efficiency in
promotion of maize growth compared to uninoculated plant. Isolated PSB were able to produce dif-
ferent organic acids (such as gluconic acids, formic acid, and citric acid) in the culture supernatant
and may consider as the principle mechanism for phosphate solubilization. This study clearly indi-
cates that A4, C1 and H6 isolates may use as a biofertilizers in ecological agricultural systems instead
of synthetic chemicals and may help to sustain environmental health and soil productivity.
Ó 2017 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research &
Technology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

* Corresponding author at: Shri Venkateshwara University, Department of Biotechnology, NH-24, Venkateshwara Nagar, Rajabpur, Gajraula,
Amroha, Uttar Pradesh 244236, India.
E-mail address: apamit@outlook.com (A. Pande).
Peer review under responsibility of National Research Center, Egypt.
http://dx.doi.org/10.1016/j.jgeb.2017.06.005
1687-157X Ó 2017 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
380 A. Pande et al.

1. Introduction and P. funicolosum). Since PSM are more suitable for high-
volume production of crop, the objective of this study was to
Phosphorus (P) is one of the major essential macronutrients of isolate the phosphate-solubilizing bacteria from rhizosphere
plants which regulates protein synthesis and plays an impor- of maize plants, to detect the phosphate-solubilizing ability,
tant role in biological development. Along with these essential to examine the production of organic acids and to characterize
functions, P is also associated with complex signal transduc- the microorganisms at the phenotypic and genotypic level. In
tion, macromolecular biosynthesis, energy transformations, addition, this study was to examine the effect of phosphate-
respiration and nitrogen fixation in legumes in the plant [1]. solubilizing bacteria as inoculants on plant growth.
Most of the P (95–99%) present in the soil is part of insoluble
compound and hence cannot be utilized by plants [2]. Since P is 2. Materials and methods
a stable element in soils, it does not form a gas (such as ammo-
nia), therefore cannot move far from where it is applied. The 2.1. Sample site and collection
reason for the stability of phosphate compounds in soils is that
they are highly reactive and reacts rapidly with other com- Soil samples were collected from eight different areas of Naini-
pounds (such as Al3+, Ca2+ and Fe3+), which become tal region (29°230 N 79°270 E/29.38°N 79.45°E/29.38; 79.45.) of
increasingly insoluble in the soil. Therefore, the release and Uttarakhand, India. The plants were dug out, the excess bulk
mobilization of insoluble and fixed forms of phosphorus is soil was removed by gently shaking, and the soil adhering the
an important aspect of increasing soil phosphorus availability. root was considered rhizosphere soil [12] and collected in ster-
To overcome this problem, most of the farmers regularly use ilized plastic bags.
chemical phosphate fertilizers which get incorporated into
the soil. This applied phosphorus easily transforms into an 2.2. Isolation of phosphate solubilizing bacteria
insoluble and stable form with limited availability to plants
and only 5% or less of the total amount of P in soil is available
for plant nutrition [3,4]. Due to the negative environmental One gram (1 g) of each soil sample was dispersed in 9 ml of
impacts of chemical fertilizers and their increasing costs, the autoclaved distilled water, filtered by 125 mm WhatmanÒ
use of PSB is advantageous in the sustainable agricultural qualitative filter paper, Grade 1. One ml of the above filtered
practices. The use of microbial inoculants possessing P- solution was again transferred to 9 ml of sterile distilled water
solubilizing activities in soils is considered as an to form 102 dilution. Similarly 103, 104, 105, 106, 107
environmental-friendly alternative to further applications of and 108 serials were made for each soil sample [13]. The seri-
chemical based P fertilizers [5]. Microbial intervention of ally diluted (104, 105 and 106) soil samples were plated
PSB seems to be an effective way to enhance the phosphorus (0.1 ml) on the Pikovskaya’s agar containing tricalcium phos-
availability in soil. phate (TCP) as the phosphate source [14]. The Pikovskaya’s
The main mechanism of phosphate solubilization is the pro- medium consisted of yeast extract 0.50 (g/l), dextrose 10.00
duction of some organic acids. Among the organic acids pro- (g/l), calcium phosphate 5.00 (g/l), ammonium sulfate 0.50
duced, gluconic, formic acid, 2-ketogluconic, citric, oxalic, (g/l), potassium chloride 0.20 (g/l), magnesium sulfate 0.10
lactic, isovaleric, succinic, glycolic and acetic acids produced (g/l), manganese sulfate 0.0001 (g/l), ferrous sulfate 0.0001
from P- solubilizing bacteria. Production of these organic acids (g/l), agar 15 (g/l) and dissolved in 1000 mL distilled water.
results in the lowering of pH in the surroundings. The lowering The pH of the media was adjusted to 7.0 before autoclaving
in pH of the medium suggests the release of organic acids by the at 15 lbs pressure (121 °C) for 15 min. Mix well and pour into
P-solubilizing microorganisms via the direct oxidation pathway sterile Petri plates (25 ml/plate) under laminar flow hood and
that occurs on the outer face of the cytoplasmic membrane allowed to solidify. Plates were incubated in inverted position
[6,7]. These acids are the product of the microbial metabolism in incubator for up to 7 days at 27–30 °C and colonies with a
[8]. Many reports suggest a positive correlation between lower- clear halo were marked positive for phosphates solubilization
ing of pH and soluble P concentration in the medium. Some of and were considered as PSB. These selected colonies were
the alternate mechanisms suggested are production of chelating again subcultured (2–3 times) by striking method till the pure
compounds, inorganic acids sulfuric, nitric and carbonic acids. cultures were obtained on the same PVK media when grown at
There are several reports of phytase producing organism. 30 °C. Isolated bacteria were kept on PVK agar slant at 4 °C
Richardson AE (1997) reported insoluble P solubilization by for further study.
secreting phytase enzyme in Pseudomonas sp. [9]. Several spe-
cies of microorganisms are able to secrete phytohormones such 2.3. Morphological characterization
as auxins, gibberellins, cytokinins, and nitric oxide which
directly involved in plant growth and productivity [10,11]. The bacterial species form characteristic colonies on Pikovs-
Microorganisms such as Alcaligenes sp., Aerobactor aerogenes, kaya’s Agar Media. Morphology of the isolates was studied
Achromobacter sp., Actinomadura oligospora, Burkholderia sp., by Gram staining using kit (K001-1KT, Hi Media) by the stan-
Pseudomonas sp., Bacillus sp. and Rhizobium sp. are the most dard procedure [15]. The stained cells were observed under
important phosphate solubilizers in soils. Apart from bacterial compound microscope. The Gram reaction and cell morphol-
sp. some fungal stains are also reported as phosphate solubiliz- ogy for efficient PSB strains were recorded. Motility of each
ers such as Aspergillus sp. (A. spergillus awamori, A. niger, A. bacterial strain was checked using HiMedia MIU medium
tereus, A. flavus, A. nidulans, A. foetidus, A. wentii.) and (SL042). Bacterial strain with positive growth away from stab-
Penicillium sp. (Penicillium digitatum, P lilacinium, P balaji, line causing turbidity was considered as motile.
Phenotypic and genotypic characterization of phosphate solubilizing bacteria 381

2.4. Phenotypic identification of bacteria as working solution. Aliquots of 0.2, 0.3, 0.5, 0.8, 1.0, 1.5, 2.0,
2.5, 3.0 ml of working solution and 0.25 ml of Barton’s reagent
Isolates were also tested for Indole test, Urease test, Catalase were added to each flask and the volume was made up to 5 ml
test, Voges-Proskauer (VP) test, Methyl Red (MR) test and with distilled water. After 10 min, the intensity of yellow color
Citrate test [16]. The phenotypic properties of isolates were developed was read at 430 nm spectrophotometrically. Stan-
determined as described in Bergey’s manual of systematic bac- dard curve was prepared by plotting absorbance at 430 nm
teriology [17]. vs concentration of P.

2.5. Analysis of phosphate solubilizing activity 2.5.3. Quantitative measurement of pH of the media
Initial pH and change in pH were also recorded on same inter-
The qualitative as well as quantitative analysis of phosphate val (2nd, 4th, 6th and 8th day) by digital pH meter (OAK-
solubilizing activity of the selected isolates was conducted by TON, pH 700). The experiments were conducted in
plate screening method and broth culture method, respectively. triplicates and values were expressed as their mean.

2.5.1. Qualitative measurement of phosphate solubilization 2.6. Pot experiment

Bacterial isolates were screened for their TCP solubilizing
activity on PKV plates. Isolates were spot inoculated on the The experiment was carried out in a greenhouse during May–
center of agar plate aseptically. All the plates were incubated June 2016 with four replications. A pot experiment consisted
at 28 °C ± 2 °C for 7 days. A clear zone around a growing col- of one control (non-inoculated with PSB) and 3 PSB species.
ony indicated phosphate solubilization and was measured as Test was conducted in pots (5 cm in diameter) containing
phosphate solubilization index (SI). SI was calculated as the 500 gm of soil. A 15 cm depth of soil was used from the agri-
ratio of the total diameter (colony + halo zone) to the colony culture field. Unsterile soil (silt loam, pH 7.5–8.0 mg/100 g;
diameter [18]. total organic C, 0.10%; available N, 36.8 mg/100 g; available
P, 5 mg/100 g; available K, 12 mg/100 g; total Ca,
Colony diameter þ Halo zone diameter 39.816 mg/100 g; total Na 22 mg/100 g, total Mg,
PSI ¼
Colony diameter 40.1 mg/100 g) was thoroughly mixed, passed through a
All the observations were recorded in triplicate. Strains 2 mm sieve and dried in sunlight for 7 days, and then sterilized
developing clear zones around their colonies could easily be three times by dry heat treatment for 2 h. at 121 °C. Tricalcium
identified as PSBs. phosphate (TCP) was supplied as soil P fertilizer at the rate of
160 mg kg1 based on the nutrient requirements of maize.
2.5.2. Quantitative measurement of phosphate solubilization For testing the efficiency of isolated phosphate solubilizing
bacteria, its effect was observed on growth of maize plant
Quantitative estimation of solubilized P by bacterial isolate
in vitro. Seeds were surface sterilized with sodium hypochlorite
was done by the vanadomolybdophosphoric yellow color
(2%) and alcohol (70%) for 1 min. Then seeds were washed
method [19] in pikovskaya’s broth containing 5000 mg/ml tri-
with autoclaved distilled H2O to remove small amount of
calcium phosphate.
chemicals. The broth culture of each 3 isolates (single colonies)
The phosphate solubilizing ability of each isolate was tested
was made in Pikovskaya’s medium. For this purpose, PSB sus-
using insoluble tricalcium phosphate [Ca3(PO4)2] as sole P
pensions were prepared by culturing on Pikovskaya’s media
source in Pikovskaya’s medium. 10 ml of pikovskaya’s broth
and incubating on an orbital shaker at 160 rpm, 30 °C, for
containing 5000 mg/ml P in the form of tricalcium phosphate
48 h. Then bacterial cells were harvested by centrifugation
was inoculated with 0.1 ml of bacterial culture (inoculum
for 5 min at 13,081g (10,000 rpm), 21 °C, in RAMI centrifuge,
adjusted to 2  108 CFU/ml) at 28 °C ± 2.0 °C up to 8 days.
and washed with sterile distilled water. The cell pellet was
After incubation, 1 ml of the supernatant was taken out on
resuspended with sterile distilled water, and then cells were fur-
2nd, 4th, 6th and 8th day.
ther adjusted to 2.0  108 colony forming units (CFU)/ml (at
The supernatant was obtained by centrifugation at
O.D 430). After making the broth culture, the equal numbers
10,000 rpm for 20 min and was passed through a 0.45 mM mil-
of seeds of maize were soaked in each broth culture separately
lipore filter and then 0.1 ml of the supernatant (filtered) was
for 12 hrs at 28 °C. The equal number (4 seeds in each pot) of
mixed with 0.25 ml of Barton’s reagent and volume was made
seed was potted in the sterile soil. Previously prepared 2 mL of
up to 5 ml with double distilled water (ddw). After 10 min, the
broth culture was uniformly applied on seeds and then again
intensity of yellow color was read on spectrophotometer (UV–
covered with uniform 3 cm layer of soil. One pot was kept as
VIS Spectrophotometer-SL-159, Elico, India) at 430 nm and
control in which the seeds were potted in sterile soil without
the amount of P-solubilized was extrapolated from the stan-
soaking in any culture of phosphate solubilizing bacteria. Pots
dard curve. The experiments were conducted in triplicates
were watered daily to maintain soil field capacity during the
and values were expressed as their mean.
study period. Agronomic variables (shoot and root length
A standard curve was prepared by dissolving 0.02195 gm of
and dry weigh) were evaluated 20 days after inoculation. At
potassium dihydrogen orthophosphate/Monopotassium phos-
the end of the study, plant growth parameters – such as% of
phate (dried at 60 °C for 1 h and then cooled in desiccators) in
Seed germination, shoot length, root length, shoot dry matter,
100 ml of double distilled water (ddw) and labeled as stock P
root dry matter and number of leaves were calculated.
solution. A further dilution of 15 ml of stock solution was
taken and volume was made up to 25 ml with ddw and labeled % of Seed germination was calculated by following formula-
382 A. Pande et al.

Number of seeds germinated  100 taxonomical classification of individual organisms [20]. For
% of Seed germination ¼
Number of seeds planted the determination of closest type strains NCBI Blast was used
[21].

2.7. Analysis of organic acids 2.9. Phylogenetic analysis

HPLC reverse-phase chromatography was used for the analy- Sequence data were compared visually and sequences were
sis of organic acids produced by PSB strains in broth medium. aligned using the Clustal W software and distances were calcu-
On 8th day, supernatant was taken from bacterial cultures that lated according to Kimura’s two-parameter method [22]. Phy-
had been centrifuged at 13,000 rpm for 15 min. Samples were logenetic trees were produced using the neighbor-joining
filtered through a 0.45 mM Millipore filter. 20 ll of filtrates method. Bootstrap analysis was based on 1000 resamplings.
was then injected into an HPLC column (Symmetry, C-18, The MEGA (Molecular Evolutionary Genetics analysis) 6.05
250 mm  4.6 mm, 5 lm) using a glass syringe. The operating package was used for all phylogenetic analysis [23]. The final
conditions consisted of 20 mM KH2PO4 and methanol (60:40v/ sequence was submitted to GenBank [24].
v) were used as mobile phase at a constant flow rate of 1 ml/
min and the column was operated at 30 °C. Retention time 2.10. Statistical analysis
(RT) of each signal was recorded at a wavelength of 256 nm.
The software used for HPLC analyses was the chemstation.
Three/four replicates were used for each experiment. The data
HPLC profiles of the culture filtrates were analyzed by com-
were analyzed by analysis of variance (ANOVA) and the
parison with the elution profiles of pure organic acids (Bio-
means were compared with Tukey’s test at P < 0.05. In the
Rad Standard containing citric acid, gluconic acid, formic
field experiment the plots with different treatments were
acid, pyruvic acid and oxalic acid) and the peak areas of their
arranged in a randomized complete block design with three
standards.
replicates per treatment.
2.8. Genomic DNA isolation, PCR amplification and sequencing
3. Results
of 16S rDNA gene
3.1. Isolation and identification of phosphate solubilizing
Total genomic DNA was extracted by CTAB method. After bacteria (PSB)
DNA extraction, PCR reaction was performed in a veriti ther-
mal cycler (Thermo Fisher Scientific). The universal primers
(27F Forward primer 50 -AGAGTTTGATCCTGGCTCAG-30 In the present study, the collected soil samples were evaluated
and 939r reverse primer 50 -CTTGTGCGGGCCCCCGTCAA in vitro for P solubilizing bacteria in Pikovskaya’s (PKV) plates
TTC-30 ) were used for the amplification of the 16S rDNA gene supplemented with 1.5% (w/v) agar. A total of 8 phosphate
fragment. PCR amplifications were carried out in 50 ll PCR solubilizing bacterial colonies were isolated on PVK agar med-
reaction mixture consisted of 3 ll template DNA (100 ng – ium, containing tricalcium phosphate (TCP). Out of 8 bacterial
1.0 lg), 25 ll 2X KAPA Taq ready mix [containing dNTPs isolates, 3 isolates (A4, C1 and H6) were found to be potent
(0.2 mM of each dNTP at 1X), MgCl2 (1.5 mM at 1X)], and phosphate solubilizers showing clear halo zone around its col-
1 ll of both forward and reverse primers. The amplification ony. The halo zone formation around the bacterial colonies
cycle consisted of an initial denaturation step of 3 min at could be due to the production of organic acids or due to
93 °C, followed by 35 cycles of 30 sec at 95 °C (denaturation), the production of polysaccharides or due to the activity of
30 sec at 60 °C (annealing) and 30 sec at 72 °C (extension), phosphatase enzymes of phosphate solubilizing bacterial
with a final extension step for 3 min at 72 °C. PCR products strains [25–28].
were electrophoresed using 1.5% agarose stained with ethid-
ium bromide (0.5 lg/ml), and visualized using a Gel luminax 3.2. Analysis of phosphate solubilizing activity
312. PCR products of isolates were purified using PCR purifi-
cation kit (Qiagen, Germany) according to the manufacturer’s 3.2.1. Qualitative measurement of phosphate solubilization
instructions and the amplified product was sequenced on San- Qualitative P-solubilization potential was anticipated by
ger sequencing platform at Pragati Biomedical, New Delhi for observing the large clear/halo zones around the bacterial colo-
sequencing. All the bacterial isolates were classified by BLAST nies on Pikovskaya agar media and PSI was calculated. 3 iso-
analysis of their respective 16S rRNA gene partial sequences. lates were found to have 4.0 phosphate solubilizing index
Various databases including eztaxon, NCBI and Ribosomal (PSI). Maximum PSI was observed by C1 (4.88 ± 0.69) fol-
Database Classifier (RDC) were used to determine the exact lowed by H6 (4.64 ± 1.12) and A4 (4.48 ± 0.30) (Table 1).

Table 1 Qualitative estimation of phosphate solubilization efficiency of A4, C1 and H6.

Bacterial isolates Colony diameter (cm) Halo zone diameter Phosphate solubilization
(zone of solubilization in cm) index (PSI)
A4 1.53 ± 0.11 5.33 ± 0.25 4.48 ± 0.30
C1 1.7 ± 0.26 6.46 ± 0.49 4.88 ± 0.69
H6 1.73 ± 0.37 5.96 ± 0.72 4.64 ± 1.12
Phenotypic and genotypic characterization of phosphate solubilizing bacteria 383

Figure 1 Phosphate solubilizing activity of A4, C1 and H6 isolates during 8 days of incubation.

Figure 2 Standard curve.

3.2.2. Quantitative measurement of phosphate solubilization lization was observed by C1 (305.49 ± 10.72 lg/ml) followed
As shown in Fig. 1, Ca3(PO4)2 containing medium solubiliza- by H6 (282.38 ± 11.81 lg/ml) and A4 (277.72 ± 1.45 lg/ml).
tion is slow in the first two days and then becomes fast, reach- It was also found that the growth of the isolate caused a signif-
ing the highest (305.49 ± 10.7281 lg/ml) in 8th day and after icant increase of acidity in Ca3(PO4)2 containing medium. In
8th day no further concentration of soluble P change was seen 8th days, pH in the culture medium declined to 3.08 ± 0.08
(data not presented). The amount of P-solubilized was extrap- from an initial pH of 7.11 (Fig. 3) and after 8th day, no further
olated from the standard curve (Fig. 2). Maximum P solubi- pH change was seen (data not presented). Minimum pH was
384 A. Pande et al.

Figure 3 Change in pH by 3 efficient phosphate solubilizing bacterial stain in liquid broth media during 8 days of incubation.

observed by C1 (3.08 ± 0.08) followed by A4 (3.36 ± 0.11) leaves and leaves length were recorded. The shoots were sepa-
and H6 (3.82 ± 0.12). rated from the roots at 0.5 cm above the surface of soil. The
roots were washed out with tap water to remove the soil par-
3.3. Morphological characterization of PSB ticles. The dry weight of roots and shoots was measured after
drying in an oven for 48 h at 60 °C and compared with control
Bacterial species were further examined for their Gram’s reac- plant. A4 isolates showed shoots height 5.75 ± 0.46 cm,
tion and shape. Characteristically, all the isolates were Gram shoots fresh weight 1.07 ± 0.23 gm, shoots dry weight 0.46
negative and of rod shaped with flagellum, motile and most ± 0.01 gm, roots height 12.9 ± 1.8 cm, roots fresh weight
of were shining. The results are summarized in Table 2. 0.52 ± 0.17 cm and roots dry weight 0.25 ± 0.02 cm and
leaves height 11.15 ± 0.94 cm. C1 isolates showed shoots
3.4. Biochemical characterization of PSB height 6.5 ± 0.58 cm, shoots fresh weight 1.09 ± 0.41 cm,
shoots dry weight 0.48 ± 0.01 cm, roots height 13.875
± 1.6 cm, roots fresh weight 0.61 ± 0.04 gm and roots dry
These isolates were further characterized by a series of bio-
weight 0.52 ± 0.12 gm and leaves height 13.075 ± 0.62 cm.
chemical reactions and identified as genus Burkholderia and
H6 isolates showed shoots height 6.15 ± 0.5 cm, shoots fresh
Alcaligenes. These bacteria were well known identified as phos-
weight 1.04 ± 0.1 gm, shoots dry weight 0.43 ± 0.17 gm, roots
phate solubilizers in many studies. [29–32]. All 3 bacteria have
height 13.27 ± 1.34 cm, roots fresh weight 0.54 ± 0.05 gm
shown positivity for urease, catalase test, Voges-Proskauer
and roots dry weight 0.26 ± 0.04 gm and leaves height
(VP) test and citrate test. Bacteria isolate C1 was negative
12.35 ± 0.51 gm. After comparing, we found that there was
for indole test and Methyl Red (MR) test. However, bacteria
significant difference (p < 0.05) between treated seed and
isolate A4 and H6 were negative for Methyl Red (MR) test.
without treated seed (control). The results are summarized in
The results are summarized in Table 2.
Table 3.
3.5. Pot examination
3.6. Production of organic acid

For determination of plant parameters, all maize plants were

Analyses of organic acids production by HPLC are presented
harvested after 20 days and the growth parameters, such as
in the chromatogram in Fig. 4 and were compared with their
shoots and roots height, fresh and dry weight and number of

Table 2 Morphological characterization and biochemical characterization of A4, C1 and H6 isolates.

Bacterial Cell shape Gram Motility Indole Urease Catalase Voges-proskauer Methyl Citrate
isolates staining test test test test (VP) test red (MR) test test
A4 Rod shaped, Larger, Single flagellum,  Motile + + + +  +
Shining, Yellowish colored, Smooth
C1 Rod shaped, Small, Single flagellum,  Motile  + + +  +
Shining, Cream colored, Smooth
H6 Rod shaped, Larger, Single flagellum,  Motile + + + +  +
Shining, Yellowish colored, Smooth
Phenotypic and genotypic characterization of phosphate solubilizing bacteria 385

standard. The results revealed that three different kinds of

13.075 ± 0.62 (b)

Maximum leaves

11.15 ± 0.94 (b)

12.35 ± 0.51 (b)

organic acids - gluconic acid, formic acid and citric acid along

5.85 ± 0.53 (a)

with unknown acid were produced from PSB isolates. C1 iso-

length (cm)
lates showed the presence of two organic acids (gluconic acid
and formic acid) and one unknown acid. Two isolates (A4
and H6) produce only one organic acid (citric acid). The

Values are expressed as means ± standard deviation of four independent data. Data were analyzed by ANOVA and the means were compared using Tukey’s test at P < 0.05.
results are summarized in Table 4.
Number of
3.7. Genomic DNA isolation, PCR amplification and sequencing
leaves

3 of 16S rDNA gene

3
3
3
0.25 ± 0.02 (b)

0.26 ± 0.04 (b)

The extracted DNA was analyzed on agarose gel electrophore-
0.13 ± 0.01 (a)
Dry weight (g)

sis. A good quality of intact DNA having high molecular

0.52 ± 0.12

weight was obtained. The genomic DNA of 3 bacterial isolates

was amplified by 16S rRNA and was run on agarose gel
(1.5%). The isolates indicated the positive signal in the form
of discrete and distinct 1000 bp bands on gel. Results are
Fresh weight (g)

shown in Fig. 5.
0.52 ± 0.17 (b)

0.54 ± 0.05 (b)

0.31 ± 0.01 (a)

0.61 ± 0.04

3.8. Genotypic identification and phylogenetic analysis

The Blast search performed against GenBank revealed a large

number of similar 16S rRNA gene sequences. The blast results
Maximum length (cm)

of most promising bacterial isolates showed >99% similarities

between available GenBank entries in which C1 isolate was
13.875 ± 1.6 (b)
13.27 ± 1.34 (b)

identified as Burkholderia cepacia and A4 and H6 were identi-

12.9 ± 1.8 (b)
7.4 ± 0.14 (a)

fied as Alcaligenes aquatilis. Results are shown in Table 5.

These sequences were submitted to the NCBI database and
Root

the accession numbers were obtained. Fig. 6 showed a phylo-

genetic tree including the isolates (C1, A4 and H6) from this
study and some closely related sequences obtained from NCBI.
(b)
(b)
(b)
(a)
Dry weight (g)

Two distant phylogenetic groups corresponding to the genera

0.20 ± 0.07
0.46 ± 0.01
0.48 ± 0.01
0.43 ± 0.17
Effect of phosphate solubilizing bacteria (A4, C1 and H6) on plant growth.

Burkholderia sp. and Alcaligenes sp. were obtained. In the phy-

Mean values (mean ± S.D) sharing the same letter do not differ significantly at P  0.05.

logenetic group of genus Burkholderia, isolate C1 was closely

related to Burkholderia cepacia. The isolates A4 and H6
formed a cluster together and were closely related to Alcalige-
nes aquatilis. The number beside the node is the statistical
Fresh weight (g)

1.07 ± 0.23 (b)

1.09 ± 0.41 (b)
0.58 ± 0.02 (a)

1.04 ± 0.1 (b)

bootstrap value. In brackets are the GenBank accession num-

bers of the 16S rRNA genes. The tree was rooted using Enter-
obacter cloacae ATCC13047 (AJ251469) as outgroup.

4. Discussion
5.75 ± 0.46 (b)
6.5 ± 0.58 (b)
6.15 ± 0.5 (b)
3.4 ± 0.18 (a)
Height (cm)

PSB is a phosphate-solubilizing microorganism which can be

routinely screened by a plate assay method using Pikovskaya
Shoot

medium. The bacteria will grow on this medium and form a

clear zone around the colony [33,34]. These bacteria can con-
vert tricalcium phosphate in the medium from insoluble to sol-
germination
% Of seed

uble forms [35]. Previous reports described some Burkholderia

strains as being efficient phosphate solubilizers [36,37]. Phos-
100%
100%
100%
100%

phate solubilization potential has been attributed to the strains

ability to reduce pH of the surroundings, either by releasing
organic acids or protons [38]. Organic acids, such as gluconic
(a)
(a)
(a)
(a)

acid, formic acid, oxalic acid, and citric acid, secreted by

0.36 ± 0.02
0.33 ± 0.01
0.35 ± 0.02
0.34 ± 0.03
weight (g)

PSB can directly solubilize mineral phosphate as a result of

anion exchange or indirectly chelate both Fe and Al ions asso-
Seed

ciated with phosphate. This leads to increased P availability,

which ultimately increases plant P uptake.
Table 3

Control

In the present study, maize rhizosphere was selected for iso-

lation of phosphate solubilizing bacteria. This habitat was cho-
H6
A4
C1

sen due to greater possibility of occurrence of phosphate

386 A. Pande et al.

Figure 4 (a) HPLC chromatogram of organic acids produced from A4 isolates. The corresponding peak detected in culture medium was
of citric acid. (b) HPLC chromatogram of organic acids produced from C1 isolate. The corresponding peaks detected in culture medium
were of gluconic acid and formic acid including unknown peak. (c) HPLC chromatogram of organic acids produced from H6 isolates. The
corresponding peak detected in culture medium was of citric acid.

Table 4 Variety and quantity of organic acid produced by A4, C1 and H6 isolates.
Sample Name of organic acid Retention time (Min) Amount of organic acid in pH of the
culture filtrate (lg/ml) liquid media
Standard Culture filtrate
A4 Citric acid 2.39 2.38 1.68 3.36
C1 Gluconic acid 1.89 1.90 0.803167052 3.1
Formic acid 1.49 1.42 7.48114555
H6 Citric acid 2.39 2.40 0.682604544 3.45
Phenotypic and genotypic characterization of phosphate solubilizing bacteria 387

culture medium and the P mineralization by the organic phos-

phorus mineralizing bacteria, suggesting that mineralization of
organic phosphorus may have different mechanisms of phos-
phorus solubilizing [43].
In addition, biochemical tests performed for the PSB iso-
lates led to their probable identification up to genus level.
Results for some of the common tests performed are listed in
Table 2. As shown in Table 2, Isolate C1, A4 and H6 were
gram negative, rod-shaped and motile. Isolate C1 was negative
for indole test and methyl red (MR) test, and positive for
urease test, catalase test, Voges-Proskauer (VP) test and citrate
test. Isolates A4 and H6 were negative for methyl red (MR)
test and positive for other tests.
Increased growth and P uptake of several crop plants due
to PSB inoculation have been reported in a number of studies
conducted under both growth chamber and greenhouse condi-
tions [44–46,38,47,48].
The effects of PSB on the growth of maize in a pot trial
found that all 3 isolates showed significant stimulating effect
on maize growth (P  0.05) in root and shoot height, and root
and shoot fresh and dry weight and leaves length with respect
to control (Table 3). Generally, available phosphorus in soil
can be increased by low-molecular-mass organic acids pro-
duced from PSB [49]. The results of this study demonstrated
that three types of organic acids were produced along with
one unknown acid by PSB (on day 8). This was a significant
characteristic of these bacteria, including a drop in pH and sol-
uble phosphorus which indicates that organic acid production
may have a central role in the solubilization of an insoluble
Figure 5 Electrophoresis amplification result of three selected phosphate source. Fig. 4b demonstrates that C1 isolates pro-
isolates based on 16 S rRNA genes. M: Marker 1 KB, Well 1–3: duced two organic acids such as gluconic acid (0.80 lg/ml)
Isolate A4, Isolate C1 and Isolate H6. and formic acid (7.48 lg/ml) and one unknown acid, while
A4 isolate (Fig. 4a) and H6 isolate (Fig. 4c) produced only
solubilizing bacteria. Panhwar et al. found considerably higher citric acid along with two different concentrations 1.68 lg/ml
number of PSB population in the rhizosphere in comparison and 0.68 lg/ml, respectively and the results are summarized
with non-rhizospheric or bulk soil [39]. Barea et al. also in Table 4.
screened several rhizospheric bacteria for phosphate solubiliz- In this study, multiple organic acid isolate of C1 showed
ing potential [40]. In this study, 8 bacterial isolates produced a phosphate solubilizing (305.49 ± 10 lg/ml) activity and a pH
halo zone around colonies, of these the 3 best isolates were (3.08 ± 0.08) drop greater than single organic acid isolates
selected based on the basis of phosphate solubilizing index of A4 (277.72 ± 1.45 lg/ml, pH 3.36 ± 0.11) and H6
(>4), lower pH (<4) and phosphate solubilizing ability (282.38 ± 11.81 lg/ml, pH 3.82 ± 0.12). This result similar
(>250 lg/ml). We have found the soluble P concentration with the findings of Chen et al. [41,50], who reported that a
was inversely correlated with pH in culture medium during combination of multiple organic acids could accomplish min-
the growth period of bacteria. This was reported earlier by eral solubilization and pH drop better than a single organic
Chen et al. and Xiang et al. [41,42]. The data showed negative acid. However, the phosphate solubilization ability may also
correlation (r = 0.68) between pH and soluble P content of depend on other factors such as substrate, medium, tempera-
the medium and the positive correlation (r = 0.96) between ture, time, and other mechanisms in addition to organic acid
soluble P content and PSI, suggested that acidification of the production.
medium can facilitate phosphate solubilization. However, Valverde et al. [51] reported that Burkholderia sp. produced
our results contrasted with the report of Tao et al., which gluconic acid, acetic acid and citric acid, and showed that these
documented that there was no correlation between the pH of bacterial genera had high efficiency in solubilizing insoluble

Table 5 Molecular characterization of A4, C1 and H6 isolates.

Isolate Most closely related organism 16 S rRNA accession no. Strain
Species % Similarity Sequence query coverage (%)
A4 Alcaligenes aquatilis 99% 97% KX345319 LMG22996
C1 Burkholderia cepacia 99% 97% KY435830 ATCC 25416
H6 Alcaligenes aquatilis 99% 96% KX345927 LMG 22996
388 A. Pande et al.

Figure 6 Phylogenetic tree based on 16S rDNA gene sequences showing the position of Burkholderia cepacia (C1), Alcaligenes aquatilis
(A4) and Alcaligenes aquatilis (H6) strains with regard to related species, which was generated based on pairwise nucleotide distance of the
Kimura 2-parameter using the neighbor-joining method included in the MEGA 6.05 software package. The scale bar indicates 0.02
substitutions per nucleotide position. The number beside the node is the statistical bootstrap value. In brackets are the GenBank accession
numbers of the 16S rRNA genes.

phosphate. Other organic acids could be produced by them as (higher than uninoculated plants); including shoot height (6.5
well, such as butyric acid, lactic acid, succinic acid, malic acid, ± 0.58 cm), shoot fresh weight (1.09 ± 0.41 gm), and shoot
glycolic acid, fumaric acid [52], propionic acid, formic acid [53] dry weight (0.48 ± 0.01 gm), root length (13.875 ± 1.6 cm),
and unknown organic acids [54]. Furthermore, C1 was the best root fresh weight (0.61 ± 0.04 gm) root dry weight (0.52
isolate for significantly enhancing all plant growth parameters ± 0.12 gm) and leaves height (13.075 ± 0.62 cm). Regarding
Phenotypic and genotypic characterization of phosphate solubilizing bacteria 389

the ability of mineral phosphate solubilization and types of tial. Previous studies have also reported that some Burkholderia
organic acids of C1, we found that this isolate had high ability species are efficient phosphate solubilizers [68].
for solubilization of tricalcium phosphate, and produced glu- Based on the results found in present study, we concluded
conic acid and formic acid. This indicated that gluconic acid that Alcaligenes aquatilis may consider as novel phosphate sol-
and formic acid may be related to the mechanism of PSB in ubilizer. In addition, Burkholderia cepacia strain had great
promoting maize growth. Our findings correspond to results potential for use as soil inoculants when compared to others
reported by Afzal and Bano [55] who reported that wheat (Tri- isolates. These PSB isolates hold good prospects in future for
ticum aestivum) inoculated with PSB (Pseudomonas sp. strain sustainable agricultural practice with minimal chemical inputs
54RB) significantly increased root and shoot weight, plant and enhances organic farming. Production and utilization of
height, spike length, grain yield and P uptake higher than biofertilizer formulation using these rhizobacterial isolates in
the control. Similar results were also found in cowpea (Vigna agricultural fields can increase soil fertility and can increase
unguiculata) which revealed the enhancement of nodulation, the crop yield. However, before using these bacterial isolates
root and shoot biomass, straw and grain yield and P and N as a biofertilizer, it should be investigated for crop
uptake of plants inoculated with Gluconacetobacter sp. and productivity.
Burkholderia sp. [56].
Moreover, the cultures were also identified using the mod- 5. Conclusion
ern 16S rRNA technique. According to the sequence of the 16S
rRNA gene, 1 isolates belong to Burkholderia sp. and other In conclusion, 3 bacterial strains from maize rhizosphere soil
two belongs to the Alcaligenes sp. Sequences from 3 isolates in Nainital region were isolated, purified, characterized and
were almost 99% similar to other 16S rRNA sequences from identified by 16S rRNA gene sequencing. These bacterial
the NCBI database. As shown in Table 5, bacterial isolates strains were identified as belonging to the genera Burkholderia
of C1 were identified as Burkholderia cepacia (99% sequence and Alcaligenes. From this study, we have isolated efficient
homology) and were belong to B. cepacia complex (Bcc) and phosphate solubilizing bacteria, which released high amounts
bacterial isolates of A4 and H6 were identified as Alcaligenes of P in broth media and released 3 different kinds of organic
aquatilis (99% sequence homology). In the rhizosphere of acid, were detected from the culture medium by HPLC analy-
maize, Burkholderia cepacia represents probably one of the sis. The results of our isolates highlight their importance as
predominant bacterial species [57]. Some studies also revealed phosphate solubilizers and recommend these strains as biofer-
that B. cepacia is present in large numbers associated with the tilizers. Further studies could be performed to evaluate their
roots and the rhizosphere of maize, [58,59]. Furthermore, B. effect on plant growth promoting properties under greenhouse
cepacia has been reported to compete, survive, and colonize conditions as well as the field ones.
roots of various maize cultivars [60], to enhance the productiv-
ity of several crop plants [61], and to antagonize and suppress
all the major soilborne fungal pathogens of maize, such as Acknowledgments
those belonging to the genus Fusarium [62,63]. In addition to
phosphate solubilization, Burkholderia sp., especially The authors gratefully acknowledge Ms. Supriya Pandey and
Burkholderia cepacia has potential for antimicrobial activity Mr. Avinash Negi for their support during this work.
and promoting plant growth of maize [64].
Phylogenetic tree based on 16S rRNA sequences of C1, A4 References
and H6 was constructed with other closely related species of
Burkholderia and Alcaligenes, obtained from NCBI (Fig. 6). [1] M.S. Khan, A. Zaidi, M. Ahemad, M. Oves, P.A. Wani, Plant
This phylogenetic analysis revealed that C1 isolates closely growth promotion by phosphate solubilizing fungi current
matched with Burkholderia cepacia, B. seminalis and B. viet- perspective, Arch. Agron. Soil Sci. 56 (2010) 73–98.
namiensis and both A4 and H6 isolates closely matched with [2] M.E.P. Corona, I.V.D. Klundert, J.T.A. Verhoeven,
Alcaligenes aquatilis which are completely separated from Availability of organic and inorganic phosphorus compounds
Enterobacter sp. by 100 bootstrap values. However, 16S as phosphorus sources for carex species, New Phytol. 133 (1996)
225–231.
rRNA based identification has limited utility in the genus
[3] R. Pundarikakshudu, Studies of the phosphate dynamics in a
Burkholderia, especially within the B. cepacia complex, where vertisol in relation to the yield and nutrient uptake of rainfed
it cannot be used as a mean to accurately distinguish this to cotton, Exp. Agric. 25 (1989) 39–45.
the species level [65,66]. In order to identify C1 to the species [4] A.M. Boronin, Rhizosphere bacteria of the genus Pseudomonas
level, molecular analysis of phylogenetic marker, recA, should enabling plant growth and development, Sorovsky Educ. Mag.
be used for further study. The recA gene has been widely 10 (1998) 25–31.
applied in bacterial systematics [67] and has proven very useful [5] A. Zaidi, M.S. Khan, M. Ahemad, M. Oves, P.A. Wani, Recent
for the identification of B. cepacia complex species, with phy- advances in plant growth promotion by phosphate-solubilizing
logenetic analysis of sequence variation within the gene microbes, in: M.S. Khan et al. (Eds.), Microbial Strategies for
enabling discrimination of all nine current species within the Crop Improvement, Springer-Verlag, Berlin Heidelberg, 2009,
pp. 23–50.
B. cepacia complex [65].
[6] R. Maliha, K. Samina, A. Najma, A. Sadia, L. Farooq, Organic
Moreover, decreasing pH in media and production of acids production and phosphate solubilization by phosphate
organic acid (Citric acid) also conform the phosphate solubilizing microorganisms under in vitro conditions, Pak. J.
solubilizing activity of A4 and H6 isolates and may consider Biol. Sci. 7 (2004) 187–196.
first time as novel phosphate solubilizing bacteria. In summary, [7] S. Babu-Khan, T.C. Yeo, W.L. Martin, M.R. Duron, R.D.
the rhizosphere soil of maize in uttarakhand region was a good Rogers, A.H. Goldstein, Appl. Environ. Microbiol. 61 (1995)
source of Burkholderia sp. with phosphate solubilizing poten- 972–978.
390 A. Pande et al.

[8] S.N. Trolove, M.J. Hedley, G.J.D. Kirk, N.S. Bolan, P. [29] A. Peix, P.F. Mateos, C. Rodriguez-Barrueco, E. Martinez-
Loganathan, Progress in selected areas of rhizosphere research Molina, Velazquez and E. Growth promotion of common bean
on P acquisition, Aust. J. Soil Res. 41 (2003) 471–499. (Phaseolus vulgaris L.) by a strain of Burkholderia cepacia
[9] A.E. Richardson, P.A. Hadobas, Soil isolates of Pseudomonas under growth chamber conditions, Soil Biol. Biochem. 33 (2001)
spp. that utilize inositol phosphates, Can. J. Microbiol. 43 (1997) 1927–1935.
509–516. [30] M.M. Collavino, P.A. Sansberro, L.A. Mroginski, O.M.
[10] S. Fibach-Paldi, S. Burdman, Y. Okon, Key physiological Aguilar, Comparison of in vitro solubilization activity of
properties contributing to rhizosphere adaptation and plant diverse phosphate-solubilizing bacteria native to acid soil and
growth promotion abilities of Azospirillum brasilense, FEMS their ability to promote Phaseolus vulgaris growth, Biol. Fertil.
Microbiol. Lett. 326 (2012) 99–108. Soils 46 (2010) 727–738.
[11] A. Rojas, G. Holguin, B.R. Glick, Y. Bashan, Synergism [31] D. Kothamasi, S. Kothamasi, A. Bhattacharyya, R.C. Kuhad,
between Phyllobacterium sp. (N2-fixer) and Bacillus C.R. Babu, Arbuscular mycorrhizae and phosphate solubilising
licheniformis (P-solubilizer), both from a semiarid mangrove bacteria of the rhizosphere of the mangrove ecosystem of Great
rhizosphere, FEMS Microbiol. Ecol. 35 (2001) 181–187. Nicobar island, India, Biol. Fertil. Soils 42 (2006) 358–361.
[12] I. Von der Weid, E. Paiva, A. Nobrega, J.D. van Elsas, L. [32] V. Kumar, R.K. Behl, N. Narula, Establishment of phosphate-
Seldin, Diversity of Paenibacillus polymyxa strains isolated from solubilizing strains of Azotobacter sp. chroococcum in the
the rhizosphere of maize planted in Cerrado soil, Res. rhizosphere and their effect on wheat cultivars under greenhouse
Microbiol. 151 (2000) 369–381. conditions, Microbiol. Res. 156 (2001) 87–93.
[13] Y.H. Hou, Q.F. Wang, J.H. Shen, J.L. Miao, G.Y. Li, [33] R.I. Pikovskaya, Mobilization of phosphorus in soil in
Molecular identification of a halotolerant bacterium NJ82 connection with the vital activity of some microbial species,
from Antarctic Sea ice and preliminary study on its salt Microbiologia 17 (1948) 362–370.
tolerance, Microbiology 35 (2008) 486–490. [34] W.B. Nilsson, R.N. Paranjype, A. DePaola, M.S. Strom,
[14] R.I. Pikovskaya, Mobilization of phosphorous in soil in the Sequence polymorphism of the 16S rRNA gene of Vibrio
connection with vital activity of some microbial species, vulnificus is a possible indicator of strain virulence, J. Clin.
Mikorobiologiya 17 (1948) 362–370. Microbiol. 41 (2003) 442–446.
[15] H.D. Isenberg, Essential Procedures for Clinical Microbiology, [35] S.S. Pal, Interactions of an acid tolerant strain of phosphate
American Society for Microbiology, Washington D.C., 1995. solubilizing bacteria with a few acid tolerant crops, Plant Soil
[16] Fadden. Mc, Biochemical Tests for Identification of Medical 198 (1998) 167–177.
Bacteria, Williams and Wilkins, Baltimore, USA, 1980, pp. 51–54. [36] A.R. Torres, W.L. Araújo, L. Cursino, M. Hungria, F.
[17] N.R. Krieg, G. Holt, Bergey’s Manual of Determinative Plotegher, F.L. Mostasso, J.L. Azevedo, Diversity of
Bacteriology, 9th ed., Williams and Wilkins, Baltimore, 1994. endophytic enterobacteria associated with different host
[18] Edi-Premono, M.A. Moawad, L.G. Vleck, Effect of phosphate plants, J. Microbiol. 46 (2008) 373–379.
solubilizing Pseudomonas putida on the growth of maize and its [37] K. Khalimi, D.N. Suprapta, Y. Nitta, Effect of Pantoea
survival in the rhizosphere, Indones. J. Crop Sci. 11 (1996) 13–23. agglomerans on growth promotion and yield of rice, Agric.
[19] N.S. Subba Rao, Phosphate solubilization by soil Sci. Res. J. 2 (2012) 240–249.
microorganisms, Advances in Agricultural Microbiology, [38] P. Hariprasad, S.R. Niranjana, Isolation and characterization of
Oxford & IBH Publishing Co, 1982, pp. 1–149. phosphate solubilizing rhizobacteria to improve plant health of
[20] A.A.A. Hamid, S. Hamdan, S.H.Z. Ariffin, F. Huyop, tomato, Plant Soil 316 (2009) 13–24.
Molecular prediction of dehalogenase producing [39] Q.A. Panhwar, R. Othman, Rahman Z. Abdul, S. Meon, M.
microorganism using 16S rDNA analysis of 2,2- Ismail, Isolation and characterization of phosphate solubilizing
dichloropropionate (dalapon) degrading bacterium isolated bacteria from aerobic rice, Afr. J. Biotechnol. 11 (2011) 2711–
from volcanic soil, J. Biol. Sci. 10 (2010) 190–199. 2719.
[21] Z. Zhang, S. Schwartz, L. Wagner, W. Miller, A greedy [40] J.M. Barea, E. Navarro, E. Montoya, Production of plant
algorithm for aligning DNA sequences, J. Comput. Biol. 7 growth regulators by rhizosphere phosphate solubilizing
(2000) 203–214. bacteria, J. Appl. Microbiol. 40 (1976) 129–134.
[22] M. Kimura, A simple method for estimating evolutionary rates [41] Y.P. Chen, P.D. Rekha, A.B. Arun, F.T. Shen, W.A. Lai, C.C.
of base substitutions through comparative studies of nucleotide Young, Phosphate solubilizing bacteria from subtropical soil
sequences, Mol. Evol. 16 (1980) 111–120. and their tricalcium phosphate solubilizing abilities, Appl. Soil
[23] K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, Ecol. 34 (2006) 33–41.
Molecular evolutionary genetics analysis version 6.0, Mol. Biol. [42] W.L. Xiang, H.Z. Liang, S. Liu, F. Luo, J. Tang, M.Y. Li, Z.M.
Evol. 30 (2013) 2725–2729. Che, Isolation and performance evaluation of halotolerant
[24] J.D. Thompson, T.J. Gibson, F. Plewniak, F. Jeanmougin, D.G. phosphate solubilizing bacteria from the rhizospheric soils of
Higgins, The CLUSTAL_X windows interface: flexible historic Dagong Brine Well in China, World J. Microbiol.
strategies for multiple sequence alignment aided by quality Biotechnol. 27 (2011) 2629–2637.
analysis tools, Nucleic Acids Res. 25 (1997) 4876–4882. [43] G.C. Tao, S.J. Tian, M.Y. Cai, G.H. Xie, Phosphate-
[25] A.K. Halder, P.K. Chakrabartty, Solubilization of inorganic solubilizing and -mineralizing abilities of bacteria isolated
phosphate by Rhizobium, Folia Microbiol. 38 (1993) 325–330. from soils, Pedosphere 18 (2008) 515–523.
[26] D.H. Goenadi, I. Sisweto, Y. Sugiarto, Bioactivation of poorly [44] R. Dey, K.K. Pal, D.M. Bhatt, S.M. Chauhan, Growth
soluble phosphate rocks with a phosphorus-solubilizing fungus, promotion and yield enhancement of peanut (Arachis
Soil Sci. Soc. Am. J. 64 (2000) 927–932. hypogaea L.) by application of plant growth-promoting
[27] D. Paul, S.N. Sinha, Isolation of phosphate solubilizing bacteria rhizobacteria, Microbiol. Res. 159 (2004) 371–394.
and total heterotrophic bacteria from river water and study of [45] L.A. Fernández, P. Zalba, M.A. Gómez, M.A. Sagardoy,
phosphatase activity of phosphate solubilizing bacteria, Adv. Phosphate-solubilization activity of bacterial strains in soil and
Appl. Sci. Res. 4 (2013) 409–412. their effect on soybean growth under greenhouse conditions,
[28] D. Paul, S.N. Sinha, Phosphate solubilization potential and Biol. Fertil. Soils 43 (2007) 805–809.
phosphatase activity of some bacterial strains isolated from [46] A. Vikram, H. Hamzehzarghani, Effect of phosphate solubilizing
thermal power plant effluent exposed water of river Ganga bacteria on nodulation and growth parameters of greengram
CIBTech, J. Microbiol. 2 (2013) 1–7. (Vigna radiata L. Wilczek), Res. J. Microbiol. 3 (2008) 62–72.
Phenotypic and genotypic characterization of phosphate solubilizing bacteria 391

[47] U. Bharucha, K. Patel, U.B. Trivedi, Optimization of indole Productivity with Rhizosphere Bacteria), CSIRO Division of
acetic acid production by Pseudomonas putida UB1 and its Soils, Adelaide, 1994, pp. 201–203.
effect as plant growth-promoting rhizobacteria on mustard [59] F. Di Cello, A. Bevivino, L. Chiarini, R. Fani, D. Paffetti, S.
(Brassica nigra), Agric. Res. 2 (2013) 215–221. Tabacchioni, C. Dalmastri, Biodiversity of a Burkholderia
[48] T. Sivakumar, T. Shankar, P. Vijayabaskar, V. cepacia population isolated from the maize rhizosphere at
Ramasubramanian, Plant growth promoting activity of nickel different plant growth stages, Appl. Environ. Microbiol. 63
tolerant Bacillus cereus TS1, J. Agric. Technol. 8 (2012) 2101– (1997) 4485–4493.
2113. [60] K.P. Hebbar, A.G. Davey, J. Merrin, P.J. Dart, Rhizobacteria
[49] M.A.B. Mia, Z.H. Shamsuddin, M. Mahmood, Effects of of maize antagonistic to Fusarium moniliforme, a soil-borne
rhizobia and plant growth promoting bacteria inoculation on fungal pathogen: colonization of rhizosphere and roots, Soil
germination and seedling vigor of lowland rice, Afric. J. Biol. Biochem. 24 (1992) 989–997.
Biotechnol. 11 (2012) 3758–3765. [61] S. Tabacchioni, A. Bevivino, L. Chiarini, P. Visca, M. Del
[50] O.K. Song, S.J. Lee, Y.S. Lee, S.C. Lee, K.K. Kim, Y.L. Choi, Gallo, Characteristics of two rhizosphere isolates of
Solubilization of insoluble inorganic phosphate by Burkholderia Pseudomonas cepacia and their potential plant-growth-
cepacia DA23 isolated from cultivated soil, Brazilian J. promoting activity, Microb. Rel. 2 (1993) 161–168.
Microbiol. 39 (2008) 151–156. [62] K.P. Hebbar, M.H. Martel, T. Heulin, Suppression of pre- and
[51] A. Valverde, P. Delvasto, A. Peix, E. Vel zquez, I. Santa-Regina, postemergence damping-off in corn by Burkholderia cepacia,
A. Ballester, C. Rodr guez-Barrueco, C. Garc a-Balboa, J.M. Eur. J. Plant Pathol. 104 (1998) 29–36.
Igual, Burkholderia ferrariae sp. nov., isolated from an iron ore [63] J.M. Klinger, R.P. Stowe, D.C. Obenhuber, T.O. Groves, S.K.
in Brazil, Int. J. Syst. Evol. Microbiol. 56 (2006) 2421–2425. Mishra, D.L. Pierson, Evaluation of the Biolog automated
[52] E. Frossard, L.M. Condron, A. Oberson, S. Sinaj, J.C. Fardeau, microbial identification system, Appl. Environ. Microbiol. 58
Processes governing phosphorus availability in temperate soils, (1992) 2089–2092.
J. Environ. Qual. 29, 15–23. [64] P.N. Bhattacharyya, D.K. Jha, Plant growth-promoting
[53] H. Rodriguez, R. Fraga, Phosphate solubilizing bacteria and rhizobacteria (PGPR): emergence in agriculture, World J.
their role in plant growth promotion, Biotechnol. Adv. 17 (2000) Microbiol. Biotechnol. 28 (2012) 1327–1350.
(1999) 319–339. [65] J.J. LiPuma, B.J. Dulaney, J.D. McMenamin, P.W. Whitby, T.
[54] E. Perez, M. Sulbaran, M.M. Ball, L.A. Yarzábal, Isolation and L. Stull, T. Coenye, P. Vandamme, Development of rRNA-
characterization of mineral phosphatesolubilizing bacteria based PCR assays for identification of Burkholderia cepacia
naturally colonizing a limonitic crust in the south-eastern complex isolates recovered from cystic fibrosis patients, J. Clin.
Venezuelan region, Soil Biol. Biochem. 39 (2007) 2905–2914. Microbiol. 37 (1999) 3167–3170.
[55] A. Afzal, A. Bano, Rhizobium and phosphate solubilizing [66] E. Mahenthiralingam, J. Bischof, S.K. Byrne, C. Radomski, J.E.
bacteria improve the yield and phosphorus uptake in wheat Davies, Y. Av-Gay, P. Vandamme, DNA-Based diagnostic
(Triticum aestivum), Int. J. Agri. Biol. 10 (2008) 85–88. approaches for identification of Burkholderia cepacia complex,
[56] M.S. Linu, J. Stephen, M.S. Jisha, Phosphate solubilizing Burkholderia vietnamiensis, Burkholderia multivorans,
Gluconacetobacter sp., Burkholderia sp. and their potential Burkholderia stabilis, and Burkholderia cepacia genomovars I
interaction with cowpea (Vigna unguiculata (L.) Walp.), Int. J. and III, J. Clin. Microbiol. 38 (2000) 3165–3173.
Agric. Res. 4 (2009) 79–87. [67] S. Karlin, G.M. Weinstock, V. Brendel, Bacterial classifications
[57] C. Nacamulli, A. Bevivino, C. Dalmastri, S. Tabacchioni, L. derived from recA protein sequence comparisons, J. Bacteriol.
Chiarini, Perturbation of maize rhizosphere microflora 177 (1995) 6881–6893.
following seed bacterization with Burkholderia cepacia MCI 7, [68] J. Caballero-Mellado, J. Onofre-Lemus, P. Estrada-de Los
FEMS Microbiol. Ecol. 23 (1997) 183–193. Santos, L. Martı́nez-Aguilar, The tomato rhizosphere, an
[58] K.P. Hebbar, M.H. Martel, T. Heulin, Burkholderia cepacia, a environment rich in nitrogen-fixing Burkholderia species with
plant growth promoting rhizobacterial associate of maize, in: M. capabilities of interest for agriculture and bioremediation, Appl.
H. Ryder, P.M. Stephens, G.D. Bowen (Eds.), Improving Plant Environ. Microbiol. 73 (2007) 5308–5319.