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378
AJCS 4(6):378-383 (2010) ISSN:1835-2707
Stress induced phosphate solubilization by Arthrobacter sp. and Bacillus sp. isolated from tomato
rhizosphere
Samiran Banerjee1*, Rakhi Palit2, Chandan Sengupta3, and Dominic Standing4
1Department of Soil Science, University of Saskatchewan, Saskatoon, Canada
2Department of Plant Sciences, University of Saskatchewan, Saskatoon, Canada
3Department of Botany, University of Kalyani, West Bengal, India
4School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom
*Corresponding author: s.banerjee@usask.ca
Abstract
The importance of rhizospheric microbial phosphate solubilization has now been well documented. However, the performance of these
microbes is greatly affected by various environmental stresses such as salt stress, pH stress, temperature stress etc. In this study, two
stress tolerant phosphate solubilizing rhizobacteria Arthrobacter sp. and Bacillus sp. have been isolated from tomato rhizosphere and
characterized with various morphological and biochemical tests. Phosphate solubilizing bacteria were screened on the basis of their
phosphate solubilization and strains with high phosphate solubilizing ability were then tested against wide range of temperature, pH, and
salt stresses. Their ability to solubilize other insoluble phosphates, such as ferric phosphate (FePO4) and aluminum phosphate (AlPO4)
was also studied. In addition to phosphate solubilizing ability these strains also demonstrated various plant growth promoting and
biocontrol activities including indole acetic acid (IAA) production. These two strains have the potential to be used as plant growth
promoting rhizobacteria (PGPR).
Keywords: Rhizobacteria, phosphate solubilization, environmental stress, plant growth promoting rhizobacteria.
Introduction
Phosphorus (P) is an essential nutrient for plant growth and
development constituting up to 0.2% plant dry weight (Harrison
et al., 2002). Phosphorus is typically insoluble or poorly soluble
in soils. Although the average P content of soils is about 0.05%
(W/W), only 0.1 % of the total phosphorus exists in plant
accessible form (Illmer and Schimmer, 1995). As a result large
amounts of soluble forms of P fertilizers are applied to attain
maximum crop production. However, the applied soluble forms
of P fertilizers are easily precipitated into insoluble forms such
as tricalcium phosphate [Ca3(PO4)2], FePO4, and AlPO4 (Achal
et al., 2007). It has been found that approximately 75–90% of
applied P fertilizer is precipitated by Ca, Fe and Al metal
cations and these insoluble forms are not efficiently taken up by
the plants. This again leads to an excess application of P
fertilizer to crop fields (Khan et al., 2007). The unavailable
phosphates built up in soils are enough to sustain maximum
crop yields globally for about 100 years (Goldstein et al., 1993;
Khan et al., 2007). Additionally, excess P application also
enhances the potential for P loss to surface waters through
overland or subsurface flow, which accelerates freshwater
eutrophication. Plants take up inorganic phosphate in two
soluble forms: the monobasic (H2PO4
−) and the dibasic
(HPO4
2−) ions (Vessey, 2003). Some soil microorganisms are
able to solubilize these insoluble P forms through the process of
organic acid production, chelation, and ion exchange reactions
and make them available to plants (Vessey, 2003). Seed or soil
inoculations with phosphate solubilizing microbes (PSM) have
largely been used to improve crop growth and production by
solubilizing of fixed and applied phosphates (Nauyital et al.,
2000). The existence of microorganisms able to solubilize
various forms of calcium phosphate has been reported
frequently but relatively few studies investigated the
solubilization of other phosphates such as AlPO4 and FePO4.
Microbes in alkaline soils in India are confronted with high salt,
high pH, and high temperature and microbial phosphate
solubilization is highly sensitive to these environmental stresses
(Johri et al., 1999). The production of food and forage in
semiarid and arid regions of the world can be increased by the
application of PSMs capable of withstanding such abiotic
stresses. Moreover, PSMs may also show plant growth
promoting activities such as indole acetic (IAA), gibberellic
acid, cytokinins, ethylene production, hydrogen cyanide (HCN)
production, asymbiotic nitrogen fixation and resistance to soil
borne pathogens etc (Cattelan et al., 1999). The aforementioned
characteristics are necessary for an efficient biofertilizer
(Ahmad et al., 2008). The objective of our study was to isolate
and characterize phosphate solubilizing rhizobacteria that are
able to solubilize various insoluble phosphates efficiently under
environmental stresses.
379
Materials and methods
Isolation of phosphate solubilizing rhizobacteria
Phosphate solubilizing rhizobacteria were isolated from the
rhizosphere of tomato grown in a tropical agricultural field at
Kalyani, West Bengal India (22°59′N, 88°28′E). The soil in
Kalyani is typically mild alkaline alluvial soil. The soils
adhered to tomato roots were collected in sterile distilled water
prior to serial dilution. Serially diluted (up to 10-5) sample
aliquots were spread onto Petri plates containing Pikovskaya
(PKV) agar (Pikovskaya, 1948). Appearance of halo zones
around some of the colonies suggested their phosphate
solubilizing ability (Vyas et al., 2007). Eleven bacterial
colonies were isolated from 10-3 dilution and were inoculated
separately into conical flasks containing Pikovskayas broth and
incubated at room temperature (25±2 ºC) on an orbital shaker
for 2 days. Three replicated cultures were centrifuged at 8000g
for 20 minutes at room temperature (25±2 ºC) and 2 ml aliquots
of the supernatant were taken and soluble phosphorous
estimated colorimetrically following the chloromolybdic acid -
stannous chloride method (Jackson, 1967) at 600 nm. The
corresponding amount of soluble phosphate was calculated
from a standard curve of KH2PO4 (9 points; r2 = 0.99). Two
strains (labeled TRSB10 and TRSB 16) with highest phosphate
solubilizing efficiency were used for further characterization
(efficiency at solubilizing Ca3(PO4)2, AlPO4 and FePO4 for 6
days). The Ca3(PO4)2 solubilization assay was performed in
similar way as described above. For AlPO4 and FePO4
solubilization a modified Pikovskaya’s broth was used. The
broth contained 4.0 g/l AlPO4 or 6.0g/l FePO4.2H2O, yielding
an equivalent amount of phosphorus as in the standard PVK
medium (5.0g/l Ca3(PO4)2) , together with 0.5 g CaCO3 per
liter to avoid lowering of pH in the broth. Estimation of the
number of colony forming units (CFU) in Pikovskaya’s broth
was done for 6 days. Phosphate solubilization is primarily
contributed by the production of organic acids which reduces
the pH of the medium (Vessey, 2003). Therefore, the pH of
Pikovskaya’s broth (control and inoculated) was measured for 6
days.
Estimation of stress induced phosphate solubilizing capacity
For determination of phosphate solubilization under salt, pH,
and temperature stressed conditions, Pikovskaya’s broth with
Ca3(PO4)2 was used. Pikovskaya’s broth (100 ml) with different
concentrations of NaCl (0%, 2.5%, 5%, 10% and 20% w/v) was
prepared for salt induced phosphate solubilization. To induce
pH stress the pH of Pikovskayas broth was adjusted to 5
different levels (pH 8, 9, 10, 11) by 1N HCl or 1M NaOH.
Flasks with salt and pH stress induced Pikovskaya’s broth were
inoculated with the two bacterial strains and incubated at room
temperature (25±2 ºC) for 3 days. For estimation of high
temperature induced phosphate solubilization, Pikovskaya’s
broth (100 ml) was inoculated with the strains were incubated
for 3 days at three different temperatures (370C, 450C or 500C).
In all cases, the quantity of solubilized Ca3(PO4)2 was measured
colorimetrically as described above.
Fig 1. Tricalcium phosphate solubilization by various strains
isolated from tomato rhizosphere. Each value is the mean of
three replicates. Error bars show one standard error of the
mean.
Table 1. F values showing the effect of time, cfu, and pH on
Ca3(PO4)2 solubilization.
TRSB 10 TRSB 16
Time 131.9*** 79.79***
Cfu 93.14*** 129.1***
pH 1.517 131.1***
Time:cfu 14.72** 59.23***
Time:pH 2.920 71.64***
Cfu:pH 4.658 21.20**
Time:cfu:pH 7.2119* 9.396*
*** P<0.001 ** P<0.01 * P<0.05
Morphological and biochemical characterization
Morphological and biochemical characteristics were studied
according to a microbiology manual (Cappucino and Sherman,
1982).
Study of PGPR characteristics
Production of IAA was estimated according to a modified Brick
et al. (1991) method (Ahmad et al., 2008). Two bacterial strains
were inoculated in 100 ml Pikovskaya broth and incubated for
24 hrs at room temperature (25±2 ºC). One ml of the inoculated
broth for each strain was recultured in freshly prepared 100 ml
Pikovskaya broth with 1 ml of 0.2 % L-tryptophan. These
cultures were incubated at room temperature (25±2 ºC) for a
further 24 hrs and 5ml aliquots were centrifuged at 8000 g for
10 min. Two ml of supernatant were mixed with 4 ml of freshly
prepared Salkawaski reagent (100 ml of 35 % perchloric acid
plus 2 ml 0.5 M FeCl3 solution). The intensity of the resultant
pink color was measured at 530 nm after 30 min of dark
incubation.
380
Fig 2. Solubilization various phosphates by Arthrobacter sp.
(TRSB10) and Bacillus sp. (TRSB16) Each value is the mean
of three replicates. Error bars show one standard error of the
mean.
The corresponding amount of IAA was calculated from a
standard curve (7 points; r2 = 0.98). Production of HCN was
detected according to Bakker and Schipper (1987) method.
Single isolates were streaked onto petri plates of solidified
King’s B medium and a single disc of filter paper was placed in
the lid of each petri plate. The petri plates were then sealed with
parafilm® and incubated at 250C for 4 days, whereas one
uninoculated set was kept as control. Color change in filter
paper from deep yellow to dark brown was visually assessed for
production of HCN. Ammonia production was detected
according to Cappuccino and Sherman (1992) and Ahmad et
al., (2008). In brief, all the isolates were grown separately in
different culture tubes containing 10 ml of peptone water. After
4 days of incubation at 300C, 1 ml Nessler’s reagent was added
in each tube to determine the presence ammonia. Brown to
yellow colour indicated the ammonia production.
Statistical analysis
The results were graphically presented by using Sigmaplot 11.0
(Systat Software Inc., Chicago, USA). The effect of CFU, pH,
and time (individual and combined) on Ca3(PO4)2 solubilization
was analyzed by analysis of variance test with R software
(www.r-project.org).
Results
The phosphate solubilizing bacteria isolated from tomato
rhizosphere strains demonstrated a wide range (17-189 μg/ml)
of Ca3(PO4)2 solubilization after 2 days (Fig.1). Two strains
(TRSB10 and TRSB16) with highest phosphate solubilizing
efficiency were selected for further characterization. Both
bacterial strains showed high efficiency to solubilize Ca3(PO4)2
, AlPO4 and FePO4 (Fig 2). In the time-course of phosphate
solubilization assay, TRSB16 consistently showed high rates of
solubilization of Ca3(PO4)2 (239 μg/ml), AlPO4 (144 μg/ml),
and FePO4 (92 μg/ml). Relatively low solubilization of
Ca3(PO4)2 (180 μg/ml), AlPO4 (34 μg/ml), and FePO4 (71
μg/ml) was observed in case TRSB10. The level of Ca3(PO4)2
solubilization by TRSB16 was highest on the fourth day
whereas TRSB10 showed maximum solubilization on the third
day. For AlPO4 both strains showed maximum solubility on the
fourth day. In case of FePO4, phosphate solubilization by
TRSB16 was highest on the fifth day but TRSB10 solubilized
highest amount of FePO4 on fourth day. The solubilization of
insoluble phosphate was found to be proportional to the number
of CFU (Fig. 3). A steady increase in CFU number was
observed up to Day 3 after which the microbial population size
decreased gradually. The production of organic acid is thought
to be responsible for phosphate solubilization which again
reduces the pH of the medium. The pH of Pokivskaya’s broth
decreases sharply after Day 1 which is followed by a gradual
decline. This supports the production of organic acids and
explains the aforementioned phosphate solubilization patterns.
The F values obtained from analysis of variance show that the
effect of three factors viz. time, cfu, and pH is different in
TRSB 10 and TRSB 16 (Table 1). Time and cfu were found to
have high statistically significant impact on Ca3(PO4)2
solubilization by both the strains. However, pH has significant
effect on TRSB 16 only. These strains showed different levels
of phosphate solubilization under various stresses (Fig 4). The
production of soluble phosphate by TRSB16 was maximum at
pH 10 and in case of TRSB10 it was pH 9. Both strains showed
lowest phosphate solubilization at pH 8. At 2.5% salt
concentration both TRSB10 and TRSB 16 showed the highest
solubilization followed by a sharp decrease at subsequent salt
concentrations. Phosphate solubilization was highest by
TRSB10 at 450 C (165.37 μg/ml), however, for TRSB16 it
decreased steadily with the increasing temperature.
Figure 3. Change in pH and CFU during 6-days of incubation.
Each value is the mean of three replicates. Error bars show one
standard error of the mean.
381
Table 2. Morphological, biochemical, and plant growth
promoting characteristics of TRSB 10 and TRSB 16.
Characteristics TRSB 10 TRSB 16
Morphological-
Gram staining + +
Motility - +
Pigmentation - -
Biochemical-
Catalase + +
Amylase + +
Urease - +
Oxidase - +
Methyl red - +
H2S production - -
Gelatin hydrolysis - +
Citrate utilization - +
Casein hydrolysis + +
Esculin hydrolysis + -
Starch hydrolysis + +
Voges Proskauer test - +
Plant growth promoting-
IAA production 3 μg/ml 20.3 μg/ml
Ammonia production + +
Hydrogen cyanide production - +
Antibacterial activity-
Xanthomonus + -
Pseudomonas - -
However, both strains showed efficient phosphate solubilization
at 500 C with the amount of solubilized phosphate exceeding
100 μg/ml. With regard to biochemical characteristics, TRSB16
was more consistent than TRSB 10 (Table 2). TRSB16 is
highly efficient IAA producer (20.3 μg/ml), however, TRSB10
also showed significant IAA production (3 μg/ml). Although
both TRSB10 and TRSB16 showed ammonia production but
only TRSB16 was HCN producer. Interestingly, TRSB10 was
found to possess antimicrobial activity. Based on their
morphological and biochemical characteristics TRSB10 and
TRSB16 were identified as Arthrobacter sp and Bacillus sp
respectively (Institute of Microbial Technology, Chandigarh,
India).
Discussion
Phosphate-solubilizing bacteria are known to improve
solubilization of fixed soil phosphorus and applied phosphates,
resulting in higher crop yields. Phosphate solubilizing microbes
have been routinely isolated from rhizospheric soil of various
plants such as rice (Chaiharn and Lumyong, 2009), wheat
(Ahmad et al., 2008), soybean (Son et al., 2006), mustard
(Chandra et al., 2007), aubergine (Ponmurugan and Gopi,
2006), and chili (Ponmurugan and Gopi, 2006). However, the
prevalence of PSB in tomato rhizosphere is not well-
investigated in alluvial soil. Alluvial soils occupy approxi-
mately 75 million ha in India constituting the largest soil group
in India. Our study shows that tomato rhizosphere comprises of
abundant PSB with a range of P solubilizing efficiency. Ability
to solubilize various insoluble phosphates is always desirable to
be a competent PGPR. These two isolated strains were found
highly efficient solubilizers of three common insoluble
phosphates. Solubilization of Ca3(PO4)2 was found in similar
range as reported by Chen et al. (2007), Pandey et al. (2006),
Johri et al. (1999). However, AlPO4 and FePO4 solubilization
Fig 4. Stress induced solubilization of tricalcium phosphate in
Pikovskya’s broth. Each value is the mean of three replicates.
Error bars show one standard error of the mean.
rate was found to be higher than the reported values by Henri et
al. (2008) and Sulbaran et al. (2009). Phosphate solubilizing
rhizobacteria are always confronted with various environmental
stresses. The ability to withstand the adverse environmental
conditions such as high salinity, high/low pH, and high
temperature is significant not only for rhizobacterial survival in
tropical agricultural soils but also to be used as biofertilizer.
Stress induced phosphate solubilization has been studied by
several researchers (Gaind and Gaur, 1991; Johri et al., 1999;
Nautiyal et al., 2000; Son et al., 2006). Phosphate solubili-
zation rate of these two strains under stressed condition was
significantly higher than the reported values by Gaind and Gaur
(1991), Johri et al. (1999), Nautiyal et al. (2000) and was in the
similar range as reported by Son et al. (2006) for Pantoea
agglomerans R-42. Plant growth promoting rhizobacteria can
enhance plant growth directly by secreting plant growth
promoting substances and making available some nutrients
present in the environment and indirectly by anticipating or
minimizing the influence of soil-borne phytopathogens (Ahmad
et al., 2008). Auxin is the most effective plant growth hormone
and among different auxins IAA is the commonest one, which
is mainly produced by tryptophan dependent pathway.
Rhizobacterial IAA production plays a significant role in the
host plant’s growth. Indole acetic acid production in microbial
has been investigated by several researchers (Ahmad et al.,
2008; Ghosh et al., 2008; Gulati et al., 2009). Both the strains
especially TRSB16 are highly efficient IAA producer.
382
Production of ammonia is an important attribute of PGPR that
influences plant growth indirectly (Wani et al., 2007).
Production of this secondary metabolite was found in both
bacterial strains. Hydrogen cyanide is a secondary metabolite
implicated in plant protection. Thus the ability to produce HCN
is a desired quality of plant growth promoting rhizobacteria. By
synthesizing HCN some rhizobacteria inhibit plant disease
development thus strengthening the host’s disease resistance
mechanism (Schippers et al., 1990). The presence of HCN in
the soil can also act as an efficient biological weed control
measure by inhibiting seed germination and seedling vigor. The
strain TRSB16 was found to be a strong HCN producer.
Tomato (Lycopersicon esculentum) is the second most
important vegetable crop after potato. World’s total production
was approximately 126.2 million tons in 2007 (FAOSTAT,
2007) and India is the fourth largest producer. Therefore,
tomato production plays a key role in Indian agriculture.
Application of P fertilizer is important for tomato as P
limitation can result in stunted growth. To achieve maximum
yield, every year large quantity of P fertilizer is applied,
majority of which becomes immobilized and unavailable to
plants. Phosphate solubilizing bacteria isolated from tomato
rhizosphere can play a critical role by making P available to
tomato plants. Furthermore, the PSB with PGPR functions such
as IAA, HCN, ammonia production and biocontrol activities
may have profound impact on tomato plants growth. In this
study, we have shown that TRSB10 and TRSB16, isolated from
tomato rhizosphere are efficient phosphate solubilizers, which
have various PGPR activities and are also able to perform
significantly under unfavorable environmental conditions.
These two strains show potential as plant growth beneficial
inoculants in alkaline soil regions. Further studies on the
rhizocompetence of these two strains are recommended.
Acknowledgements
Many thanks to Dr. Moumita Datta for her assistance in
laboratory. We would also like extend our sincere thanks to the
editor and two anonymous reviewers for their insightful
suggestions.
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