Introduction
Phosphorus (P) is the second important key plant nutrient which affects the
overall growth of plants by influencing the key metabolic processes such as
cell division and development, energy transport (ATP, ADP), signal
transduction, macromolecular biosynthesis, photosynthesis and respiration
(Shenoy and Kalagudi, 2005; Khan et al., 2009, 2014). Soils contain very
little total P (0.02–0.5 % (w/w); Fernandez et al., 2007), of which
only 0.1 % is available to plants (Zou et al., 1992). Thus, P needs to be
applied to soils as soluble P fertilizers; a small part (1 %) is utilized
by plants and the remainder (∼ 99 %) is rapidly converted into
insoluble complexes (Mehta et al., 2014) due to precipitation reactions with
Al3+ and Fe3+ in acidic and Ca2+ in calcareous soils (Khan et
al., 2009). These metal ion complexes precipitate about 80 % of added P
fertilizer. Hence, the recovery efficiency of P is not more than 20 % of
applied P in the world soils (Qureshi et al., 2012). Considering the low
recovery of applied and native P and the high cost of chemical phosphatic
fertilizers in addition to an increasing concern about environmental
degradation (Aziz et al., 2006; Khan et al., 2014), it is important to find
viable solutions to increase P fertilizer use efficiency. Two management
options can be effective: (i) increasing the recovery and solubility of
applied P fertilizers and (ii) replacing the expensive chemical P fertilizers
with novel, cheaper, more ecologically friendly but nevertheless efficient P
sources, such as indigenous rock phosphates (RPs).
Interest in the use of RPs as alternative P sources has increased in recent
times due to their relatively low cost and utilization potential (Zapata and
Zaharah, 2002; Akande et al., 2010). It has been suggested that the
production of P fertilizer from RP is estimated to peak within the next 30
years because of the rising costs of synthetic fertilizers presently
available on the market (Cordell et al., 2009; Beardsley, 2011; Ekelöf et
al., 2014). The application of RPs directly to the soils has yielded some
positive results in acidic soils, but the efficacy of such material is almost
negligible in neutral and alkaline soils (Begum et al., 2004). However, there
are reports that Syrian RP was an effective P fertilizer for rape plants
(Brassica napus L.) in alkaline soil (pH = 7.72; Habib et al.,
1999) and for maize grown in an acidic Lily soil (pH = 3.95; Alloush and
Clark, 2001). Therefore, efforts have been made to find suitable ways to
improve the solubility and efficiency of indigenous RPs.
Numerous studies have been conducted to evaluate the efficiency of different
amendments to increase the availability and solubility of P from native and
applied sources including RP. Among these, organic amendments, including
animal manure, plant residues and green manure (Alloush, 2003; Toor, 2009;
Aria et al., 2010; Adesanwo et al., 2012), composts (Nishanth and Biswas,
2008; Wickramatilake et al., 2010; Saleem et al., 2013), and bacterial
inoculation (Panhwar et al., 2011; Gupta et al., 2011) are considered
beneficial for improving the P efficiency. In addition, the combined
application of water-soluble P fertilizers with RP is another option to
increase the efficiency of RP P. Mashori et al. (2013) used maize as a test
crop in a pot experiment to examine the relative performance of RP, single
superphosphate (SSP) and RP+SSP with and without farm yard manure (FYM). They reported that RP+SSP (25+75 %) with
FYM (10 t ha-1) (RP+SSP+FYM) increased
maize growth, dry matter, leaf P content and P uptakeleaf P content and P uptake; the next highest increase was seen in the treatment receiving RP+SSP (50+50 %).
Soil microorganisms have generally been found effective in making P available
to the plants from both inorganic and organic sources by solubilizing and
mineralizing complex P compounds (Wani et al., 2007; Khan et al., 2014). In
particular, P-solubilizing bacteria (PSB) are reported to play a significant
role in increasing the P efficiency of both native and applied P and
improving the growth and yield of various crops (Khan et al., 2009). It is
generally accepted that the mechanism of P solubilization by PSB is
associated with the release of low-molecular-weight organic acids (Goldstein,
1995; Kim et al., 1997), which through their hydroxyl and carboxyl groups
chelate the cations bound to phosphate, thereby converting it into soluble
forms (Kpomblekou and Tabatabai, 2003; Chen et al., 2006).
Similarly, the application of organic manure with phosphatic fertilizers is
considered another possible means of mobilizing P because of the acidic
environment generated during the decomposition of the manure (Nishanth and
Biswas, 2008). The different types of organic manure increase the
microorganisms, release acids in the root rhizosphere and may help to
solubilize P and to increase P availability to the plants (Fankem et al.
2006; Hu et al., 2006). In addition, the combined use of RP, soluble P
fertilizers and bacterial inoculation is also considered an option that may
increase the efficiency of both RP and soluble P fertilizers.
Experimentations on this option are not common, however; recently it has been
reported that 50 % of triple superphosphate (TSP) could be substituted
with RP when P-solubilizing bacterial inoculants Enterobactor gegovie, Bacillus pumilus and Bacillus subtilis were
applied with RP to wetland rice both under pot and field conditions
(Rajapaksha et al., 2011).
Keeping in view the considerable expense involved in importing raw material
for manufacturing P fertilizers or in P fertilizers directly imported, it is
imperative to explore the possibility of the utilization of indigenous RPs
and the ways to increase the efficiency of other P fertilizers. The effects
of PSB or organic manure on the efficiency of both soluble and insoluble P
fertilizers with regard to plant growth and yield have been studied and are a
topic of interest today. However, the effect of these combinations on P
release capacity (mineralization) of both soluble and insoluble P sources,
especially RPs, has been given little attention. Therefore, the present study
was conducted to examine the effect of poultry manure (PM) and PSB with
soluble P fertilizers (SSP and diammonium phosphate – DAP) and insoluble
rock phosphate (RP) on P mineralization capacity and their subsequent effect
on the growth, yields, P uptake and P utilization efficiency (PUE) of chilli
(Capsicum annuum L.) grown in a greenhouse.
Materials and methods
Soil sampling and collection
Surface bulk soil (0–15 cm) from a field under a long-term wheat–maize
management system in the Faculty of Agriculture of the University of Poonch,
Rawalakot Azad Jammu and Kashmir, Pakistan, was collected during spring 2013.
The soil used in the experiment was classified as a Humic Lithic Eutrudepts
(Inceptosols). The field-fresh soil was passed through a 2 mm sieve to
eliminate coarse rock and plant material, thoroughly mixed to ensure
uniformity and stored at 4 ∘C prior to use (for no more than 2
weeks). A subsample of about 500 g was taken, air-dried and passed through
2 mm sieve and used for the determination of physical and chemical
characteristics (Table 1). Soil texture was determined by the hydrometer
method. Soil pH was determined in a 1 : 2.5 (w/v) soil–water suspension.
Soil organic carbon was determined by oxidizing organic matter in soil
samples with K2Cr2O7 in concentrated sulfuric acid followed by
titration with ferrous ammonium sulfate (Nelson and Sommers, 1982). Total N
was determined by the Kjeldahl distillation and titration method (Bremner and
Mulvaney, 1982). Available P from soil samples was determined according to
Soil and Plant Analysis Laboratory Manual (Ryan et al., 2001) using
the AB-DTPA method modified by Soltanpour and Workman (1979). Exchangeable K
was determined using a flame photometer following soil extraction with 1 N ammonium acetate
(COOCH3NH4) (Simard, 1993). The bulk density (BD) was determined
from undisturbed soil cores taken from the upper horizon (0–15 cm) at about
five locations in the field. The bulk density of the soil was calculated on a
volume basis (Blake and Hartge, 1986).
Collection of added amendments and materials
The different amendments used in this study were RP, SSP, diammonium
phosphate (DAP), poultry manure (PM) and P-solubilizing bacteria (PSB). Rock
phosphate was collected from the Land Resources Research Institute (LRRI),
NARC Islamabad, Pakistan. Major reserves of this RP are found in the Lagarban
region of Hazara Division in the northeast of Pakistan (Mashori et al.,
2013). According to Memon (2005), the RP contains an average of 25.8 %
P2O5 along with 6 % MgO. Diammonium phosphate and SSP were
purchased from the local market, while PM was collected from local farms
located near by the university campus. A composite sample of well-dried PM
was taken, crushed into smaller particles by hand pressing, homogenized and
passed through a 1 mm sieve before use. Total N in PM was determined by the
Kjeldahl method of digestion and distillation (Bremner and Mulvaney, 1982).
The P content was determined by the vanadomolybdate yellow color using
spectrophotometer. Total N and P in PM were 2.53 and 1.64 %,
respectively. The biopower inoculant of PSB was provided by the National
Institute of Biology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.
The inoculant used in this study was a commercialized product containing K-1
(Pseudomonas stutzeri) as nitrogen fixer, ER-20
(Azospirillum brasilense) as indole acetic acid (IAA)
producer and Ca-18 (Agrobacterium tumefaciens) as phosphate
solubilizer.
Experimental procedures and details – incubation study
The fresh soil samples stored in the refrigerator were taken and transferred
into a glass jar. Soil samples were preincubated at 25 ∘C for 1 week
prior to actual incubation to stabilize the microbial activity. A known
weight of soil (30 g oven-dry weight basis) was taken and transferred into
100 mL capacity jars. Moisture content of soil was adjusted to 60 % of
water holding capacity (WHC) by adding deionized water. There were 12
treatments including the control, i.e., the unfertilized control, RP, SSP,
DAP, PM, 1/2 RP+1/2 SSP, 1/2 RP+1/2 DAP, 1/2 RP+1/2 PM, RP+PSB,
1/2 RP+1/2 SSP+PSB, 1/2 RP+1/2 DAP+PSB, and 1/2 RP+1/2
PM+PSB; six incubation periods (0, 5, 15, 25, 35 and 60 days, after adding
amendments); and three replications. A total of 216 treatment combinations
(experimental units) were used at the start of the experiment.
Phosphorus from all the treatments and sources was applied on an equivalent
basis, i.e., at the rate of 90 mg P kg-1 soil as this is a
recommended optimum P application rate for chilli production in the region.
Nitrogen was also added to each jar (including control) at the rate of
100 mg N kg-1 as urea. The amount of N added as urea was adjusted
after taking into account the amount supplied by DAP and PM. Following the
addition of all amendments, the soil was thoroughly mixed and the weight of
each jar was recorded. Jars were covered with parafilm which was perforated
with a needle to ensure natural gas exchange. All the amended jars were kept
in an incubator at 25 ± 2 ∘C for a total of 60 days. Jars in
the incubator were arranged according to a completely randomized design. Soil
moisture was checked and adjusted every 2 days by weighing the jars; the
required amount of distilled water was added when the loss was greater than
0.05 g. During this process, care was taken not to disturb the soil either
through stirring or shaking.
Soil extraction and analysis
Samples of all treatments incubated for different time intervals were
analyzed for changes in soil-available P and pH. Triplicate samples from each
treatment were taken from the incubator at 0, 5, 15, 25, 35 and 60 days and
analyzed for available P by the AB–DTP extraction method (Soltanpour and
Workman, 1979). Soil (20 g) was weighed in 125 mL Erlenmeyer flasks, and
40 mL of extraction solution was added (1 : 2). The soil-available P was
measured by ammonium molybdate (Murphy and Riley, 1962) using a
spectrophotometer. At each sampling time, the remaining 10 g soil from each
jar was taken and used for measuring the changes in pH in response to
different amendments. The soil pH was determined using a glass electrode in a
1 : 2.5 (v/v) soil–water suspension.
Experimental procedures and details – greenhouse experiment
To complement the incubation study, a greenhouse experiment was conducted in
pots with chilli (Capsicum annuum L.) as a test crop. A seedling
nursery of chilli was grown by making nursery beds in the greenhouse during
the last week of April 2013. Chilli seeds of the variety “Pusa
Jwala” were sown separately on ridges. All the
necessary culture practices were carried out when needed. About 12 kg soil
(passed through a 4 mm sieve) was placed in cleaned earthen pots of 38 cm
height and 18 cm width. There were 12 treatments including a control, i.e.,
the unfertilized control; RP, SSP; DAP; PM; 1/2 RP+1/2 SSP; 1/2
RP+1/2 DAP; 1/2 RP+1/2 PM; RP+PSB; 1/2 RP+1/2 SSP+PSB; 1/2
RP+1/2 DAP+PSB; and 1/2 RP+1/2 PM+PSB (same as used in incubation
study), with four replications to form a total of 48 treatment combinations.
Pots were arranged according to a completely randomized design. The addition
of different amendments was made according to the methods and procedures
followed in the incubation study. However, PSB was grown in LB (lysogeny
broth) to lag phase, containing about
108 CFU (colony-forming units) mL-1, applied to respective
treatments by dipping the roots of chilli plants in inoculum for up to
20 min. On attaining leaf stages 5–8, four healthy and vigorous plants from
the nursery were transplanted into each pot. All pots were equally irrigated
when needed. The soil was moistened with water and maintained at 58 %
water-filled pore space throughout the study.
Plant sampling was done at two stages of development, i.e., one at the
vegetative stage (just before flowering) for measuring the growth traits of
the plant, including shoot length, root length, shoot dry weight, root dry
weight and shoot P contents; the second one at physiological maturity stage
for measuring growth, shoot P contents and yield traits, i.e., fruit length,
number of fruits per plant, number of seeds per fruit, fruit yield and fruit
P contents.
For the determination of plant P content, vegetative tissue of a plant (shoot
+ leaves) and fruits were washed, cleaned and then oven-dried at
70 ∘C for 48 h. The oven-dried samples were ground to pass through
a 1 mm mesh sieve in a Micro Wiley
Mill. The total P was determined after digestion in a triple acid mixture
(HClO4, H2SO4 and HNO3 in a ratio of 1 : 3 : 9).
Total P in the acid digest was determined by the vanadomolybdate phosphoric
yellow-color method (Olsen and Sommers, 1982). The P uptake and P utilization
efficiency was computed according to the methods reported earlier (Abbasi et
al., 2010).
At the end of the experiment, i.e., after the final crop harvest, soil
samples from each pot were taken to examine changes in soil properties. A
composite sample from each pot was collected and air-dried for 2 to 3 days.
The samples were ground and sieved to pass through a 2 mm mesh to remove
rocks and large organic residues if present. Soil organic matter, total N,
available P, K and the pH of the soil from each treatment were determined
according to the methods described in Table 1.
The initial physical and chemical characteristics of soil
used in the study.
Soil properties
Values
Bulk density (g cm-3)
1.32
Sand (g kg-1)
433.9
Silt (g kg-1)
326.0
Clay (g kg-1)
240.1
Textural class
loam
Soil pH (1:2.5H2O)
7.57
Organic matter (g kg-1)
10. 3
Organic carbon (g kg-1)
5.64
Total N (g kg-1)
0.53
NH4+-N (mg kg-1)
8.85
NO3--N (mg kg-1)
7.21
Available P (mg kg-1)
4.70
Available K (mg kg-1)
98.5
Calcium Ca (mg kg-1)
58.7
Magnesium Mg (mg kg-1)
15.5
Iron Fe, (mg kg-1)
17.8
Manganese Mn (mg kg-1)
6.2
Zinc Zn (mg kg-1)
8.4
Copper Cu (mg kg-1)
3.79
CaCO3 content (%)
0.68
Cation exchange capacity (CEC) cmol(+) kg-1 soil
11.9
Statistical analysis
All data from the incubation experiment were statistically analyzed by
multifactorial analysis of variance (ANOVA) using the software package
Statistix 8.1. Least significant differences (LSDs) are given to indicate
significant variations between means of either treatments or time intervals.
Confidence values (P) are given in the text for the significance between
treatments, the time interval and the interactions between treatment and time
interval. Data from the greenhouse experiment were analyzed by one-way
analysis of variance and LSD is given to indicate significant variations
among different treatments. A probability level of P≤ 0.05 was
considered significant for both experiments.
Results
P release capacity (mineralization) of added amendments
Phosphorus release capacity (mineralization) of soil amended with RP varied
between 6.0 and 11.5 mg kg-1, significantly (P≤ 0.05) higher
than the control but lower than the remaining treatments (Table 2). The
application of PSB with RP in RP+PSB did not show any remarkable effect on
P mineralization except that a significant increase was noticed on days 25
and 60 of the incubation. Soil amended with the soluble P fertilizers, i.e.,
SSP and DAP, displayed the highest P mineralization (73.3 and
68.5 mg kg-1) immediately after application (day 0). However, the initial P released was significantly (P≤ 0.05) decreased with
subsequent incubations, and at the end only 14 mg P kg-1 was left in
the mineral P pool. The P released from the PM-amended soil was progressively
increased with time and the highest P concentration of 20.2 mg kg-1
was recorded at day 35, compared to 10.4 mg kg-1 at day 0. However,
this increasing trend changed at day 60, when P contents declined to a
background level, i.e., 9.6 mg kg-1. Rock phosphate when combined with
soluble P fertilizers (SSP, DAP) did not show any significant impact on P
mineralization. However, throughout the incubation, P mineralization of the
1/2 RP+1/2 DAP+PSB and 1/2 RP+1/2 SSP+PSB treatments was
significantly higher than when they were applied alone. Similarly, the P
mineralization under 1/2 RP+1/2 PM+PSB exhibited an increasing trend
with subsequent incubation periods (showing no losses), a trend not normally
common for phosphatic fertilizers.
Mineralization potential (P release capacity) of soluble P
fertilizers and insoluble rock phosphate (RP) in response to
phosphate-solubilizing bacteria (PSB) and poultry manure (PM) applied to a
loam soil incubated under controlled laboratory conditions at 25 ∘C
over a 60-day period.
Treatments
Days after amendment application
0
5
15
25
35
60
LSD (P≤ 0.05)
Extractable (available) P (mg kg-1 soil)
Control
4.7
4.8
5.3
5.7
6.5
4.5
0.44
RP
6.0
7.7
9.9
6.2
11.5
6.2
1.02
SSP
73.3
30.5
21.6
18.8
21.7
13.5
2.25
DAP
68.4
29.4
23.0
19.5
20.5
14.1
2.37
PM
10.4
13.1
18.8
17.7
20.2
9.6
1.16
1/2 RP+1/2 SSP
42.9
21.0
14.6
21.0
5.8
6.9
2.34
1/2 RP+1/2 DAP
43.3
17.3
25.2
13.6
7.9
6.2
2.21
1/2 RP+1/2 PM
11.8
12.8
15.3
13.5
23.0
8.9
3.32
RP+PSB
6.1
6.3
10.4
11.5
11.4
9.8
1.13
1/2 RP+1/2 SSP+PSB
38.2
18.8
18.8
16.0
22.7
11.2
1.80
1/2 RP+1/2 DAP+PSB
44.6
20.9
16.9
16.0
23.1
13.0
2.30
1/2 RP+1/2 PM+PSB
12.7
12.4
16.8
17.5
25.2
24.2
2.21
LSD (P≤ 0.05)
1.23
2.11
1.45
1.11
1.21
1.15
RP: rock phosphate; SSP:
single superphosphate; DAP: diammonium phosphate; PM: poultry manure; PSB:
phosphate-solubilizing bacteria; full dose of P from different sources was
applied at the rate of 90 mg P kg-1 soil.
The overall effect of different amendments on P mineralization (averaged
across incubation timings) is presented in Fig. 1. Results indicated that by
applying 90 mg P kg-1 from different P sources, RP was able to
release only about 8 mg kg-1 compared to 5 mg P kg-1 in the
control. Both soluble P fertilizers, i.e., SSP and DAP, displayed the highest
P release capacity (about 30 mg kg-1). The P mineralization tendency
of soil amended with soluble P fertilizers+insoluble RP did not show any
increasing effect. However, RP when combined with PM in 1/2 RP+1/2 PM
released a significantly higher amount of P compared to the RP treatment
(80 %), and the amount released was equivalent to that recorded under PM
treatment. The effect of PSB on the P release capacity of different P
amendments was significant (P≤ 0.05). The efficiency of RP was
increased by 17 % when PSB was applied with RP (RP+PSB, T8) and
showed a 12 % increase with 1/2 RP+1/2 SSP+PSB (T9) compared
to 1/2 RP+1/2 SSP (T5), an 18 % increase with 1/2 RP+1/2
DAP+PSB (T10) compared to 1/2 RP+1/2 DAP (T6) and a 28 %
increase with 1/2 RP+1/2 PM+PSB (T11) compared to 1/2 RP+1/2
PM(T7).
The P release capacity of different P sources applied alone or in
combination with PSB and PM (average over incubation periods) to a soil
incubated under controlled laboratory conditions at 25 ∘C. The
legend on x axis refers to the different treatments: T0 – control;
T1 – RP; T2 – SSP; T3 – DAP; T4 – PM full; T5 –
1/2 RP+1/2 SSP; T6 – 1/2 RP+1/2 DAP;T7 – 1/2 RP+1/2
PM; T8 – RP+PSB; T9 – 1/2 RP+1/2 SSP+PSB; T10 –
1/2 RP+1/2 DAP+PSB; T11 – 1/2 RP+1/2 PM+PSB. Full dose of P
from different sources was applied at the rate of 90 mg P kg-1 soil.
Changes in pH of the soil (average over incubation periods)
supplemented with different P sources applied alone or in combination with
PSB and PM and incubated at controlled laboratory conditions at
25 ∘C. The legend at x axis refers to the different
treatments: T0 – control; T1 – RP; T2 – SSP; T3 –
DAP; T4 – PM full; T5 – 1/2 RP+1/2 SSP; T6 – 1/2
RP+1/2 DAP;T7 – 1/2 RP+1/2 PM; T8 – RP+PSB; T9 –
1/2 RP+1/2 SSP+PSB; T10 – 1/2 RP+1/2 DAP+PSB; T11 –
1/2 RP+1/2 PM+PSB. Full dose of P from different sources was applied at
the rate of 90 mg P kg-1 soil.
Effect of different amendments on changes in soil pH
The effect of different P amendments and their combinations on changes in
soil pH over 60 days' incubation is presented in Table 3. Soil amended with
DAP, PM and SSP alone or with different combinations showed the maximum pH,
and among all, PM and DAP had the highest pH. However, except RP+PSB, pH of
all the added amendments tended to decline with time. The pH of both DAP and
PM significantly decreased at the end (day 60) and the reduction in pH
compared to day 0 was 8 %. Among the different amendments RP showed the
lowest pH.
Changes in pH of the soil supplemented with soluble P fertilizers
and insoluble rock phosphate (RP) along with phosphate-solubilizing bacteria
(PSB) and poultry manure (PM) and incubated under controlled laboratory
conditions at 25 ∘C over a 60-day period.
Treatments
Days after amendment application
0
5
15
25
35
60
LSD (P≤ 0.05)
pH
Control
7.57
7.74
7.82
7.68
7.6
7.29
0.11
RP
7.57
7.65
7.87
7.76
7.5
7.39
0.08
SSP
7.93
7.91
7.85
7.76
7.86
7.43
0.16
DAP
8.00
7.94
8.00
7.98
7.81
7.34
0.06
PM
8.10
7.93
8.07
8.10
7.96
7.49
0.13
1/2 RP+1/2 SSP
7.90
7.91
7.89
8.07
7.99
7.56
0.08
1/2 RP+1/2 DAP
7.92
7.96
8.01
8.03
7.71
7.62
0.09
1/2 RP+1/2 PM
7.89
7.93
7.78
8.03
7.68
7.66
0.10
RP+PSB
7.52
7.59
7.46
7.47
7.69
7.63
0.09
1/2 RP+1/2 SSP+PSB
7.93
7.91
7.91
7.65
7.65
7.58
0.09
1/2 RP+1/2 DAP+PSB
7.95
7.92
7.93
7.77
7.69
7.63
0.08
1/2 RP+1/2 PM+PSB
7.95
7.87
7.75
7.66
7.59
7.54
0.07
LSD (P≤ 0.05)
0.13
0.08
0.16
0.10
0.10
0.07
* RP: rock phosphate; SSP: single superphosphate; DAP:
diammonium phosphate; PM: poultry manure; PSB: phosphate-solubilizing
bacteria; full dose of P from different sources was applied at the rate of
90 mg P kg-1 soil.
Averaged across different amendments, the data presented in Fig. 2 indicated
that a combination of SSP, DAP and PM with RP significantly increased RP pH
from 7.62 to 7.89, 7.88 and 7.83, respectively. However, the application of
PSB decreased soil pH. Average pH under the treatments RP, 1/2 RP+1/2
SSP, 1/2 RP+1/2 DAP and 1/2 RP+1/2 PM was 7.80, while the application
of PSB with these four amendments tended to result in a decline of pH to
7.72. The maximum reduction in pH of about 15 units was recorded in the
treatment where PSB was applied with PM.
Growth and yield characteristics of chilli
Different P treatments when applied alone or used in different combinations
significantly (p≤ 0.05) increased chilli growth characteristics
compared to the control, i.e., shoot length (7–53 %), root length
(22–113 %), shoot dry weight (SDW, 8–156 %) and root dry weight
(RDW, 12–108 %) (Table 4).
Among different P amendments, growth characteristics were at a maximum in the
treatments under full DAP or DAP, SSP and PM with PSB. RP alone had little
effect on plant growth but the response of RP+PSB over RP was no effect on
shoot length, a 54 % increase in root length, a 50 % increase in SDW
and an 8 % increase in RDW. The application of PSB with DAP, SSP and PM
displayed a significant increase in growth characteristics over treatments
without PSB. The relative increase in shoot length, root length, SDW and RDW
due to PSB over the treatments without PSB (as a group) was 20, 14, 51 and
32 %, respectively.
Effect of soluble P fertilizers and insoluble rock phosphate (RP)
applied alone or in combination with phosphate-solubilizing bacteria (PSB)
and poultry manure (PM) on the growth and yield characteristics of chilli
(Capsicum annuum L.) grown in pots under greenhouse conditions at
Rawalakot Azad Jammu and Kashmir.
Treatments
Shoot length
Root length
Shoot dry wt.
Root dry wt.
Fruit length
No. of seeds
No. of fruits
Fruit yield
(cm)
(cm)
(g plant-1)
(g plant-1)
(cm)
fruit-1
plant-1
(g plant-1)
Control
30.0
8.8
4.8
1.13
6.1
33.7
6.7
3.5
RP
32.0
10.7
5.2
1.26
7.2
38.0
8.7
4.9
SSP
39.0
17.4
8.4
1.33
7.8
50.0
15.3
7.2
DAP
43.3
15.6
11.8
1.76
9.3
49.0
19.9
10.0
PM
31.3
14.2
10.9
1.56
8.5
42.7
13.7
9.9
1/2 RP+1/2 SSP
33.3
14.5
6.4
1.37
7.5
41.3
11.7
5.8
1/2 RP+1/2 DAP
34.2
17.5
7.9
1.42
7.7
48.7
13.0
6.6
1/2 RP+1/2 PM
32.2
16.2
6.9
1.36
7.6
50.0
13.3
6.4
RP+PSB
30.3
16.4
7.8
1.35
8.5
44.0
10.6
5.6
1/2 RP+1/2 SSP+PSB
32.3
16.7
9.5
1.40
8.4
40.3
14.9
7.2
1/2 RP+1/2 DAP+PSB
43.7
15.7
10.2
1.73
8.5
42.3
18.5
8.7
1/2 RP+1/2 PM+PSB
45.8
18.7
12.3
2.35
9.5
50.3
21.2
10.4
LSD (P≤ 0.05)
3.7
1.73
1.81
0.09
0.74
4.10
1.7
0.8
* RP: rock phosphate; SSP: single
superphosphate; DAP: diammonium phosphate; PM: poultry manure; PSB:
phosphate-solubilizing bacteria; full dose of P from different sources was
applied at the rate of 90 mg P kg-1 soil.
Yield and yield component responses of chilli to applied P treatments are
presented in Table 4. Significant differences in fruit length (18–56 %),
number of fruits per plant (45–226 %) fruit yield per plant
(10–194 %) and the number of seeds per fruit (13–50 %) were
observed between the control (no-P) and the rest of the P treatments.
Significant differences in yield components were also recorded among the
sources of P, with DAP (full) and 1/2 RP+1/2 PM+PSB producing the
largest yields. The application of RP alone induced a significant increase in
yields (over the control). However, the magnitude of increase was remarkably
higher when PSB was combined with RP. The relative increase in fruit length,
the number of fruits, fruit yield and the number of seeds with RP+PSB was
18, 34, 14 and 16 %, respectively, compared to the RP alone. Of the two
synthetic P fertilizers used (SSP, DAP), DAP showed superiority over SSP,
while PM also exhibited comparable yields to DAP and SSP.
The integrated use of RP with SSP, DAP and PM (50 : 50) was not comparable
to their full dose. However, the combined use of these amendments with PSB
resulted in yields significantly higher than their application without PSB
and equivalent to or higher than the yields recorded under full P fertilizer
treatments. For example, fruit length, the number of fruits and fruit yield
from the 1/2 RP+1/2 SSP+PSB, 1/2 RP+1/2 DAP+PSB and 1/2
RP+1/2 PM+PSB treatments (as a group) was significantly higher (16, 44
and 40 %, respectively) than their application without PSB. The highest
fruit yields (10.4 g plant-1) and the highest number of fruits per
plant (21.2) were recorded from the 1/2 RP+1/2 PM+PSB treatment,
equivalent to that recorded from the full DAP (10.0 g and 19.9) but
significantly higher than that from full SSP (7.2 g and 15.3).
P content, P uptake and P utilization efficiency
The P content of plant biomass and the fruits of chilli treated with
different P sources and combinations was significantly (P≤ 0.05) higher
compared to the P content of the control (Table 5). Soil amended with DAP
resulted in the highest P content of shoot (1.33 mg plant-1) and fruit
(1.57 mg plant-1) as compared to SSP and other P amendments. However,
fruit P content recorded from the PM and the 1/2 RP+1/2 PM+PSB
treatment (1.54 and 1.51 mg plant-1) were statistically equivalent (at
par) to that recorded under DAP. P content of shoot and fruit under RP was
significantly higher than the control (6 and 77 %), and the application
of PSB with RP (RP+PSB) further increased shoot and fruit P by 6 and
5 %, respectively, compared to RP alone.
Effect of soluble P fertilizers and insoluble rock phosphate (RP)
applied alone or in combination with phosphate-solubilizing bacteria (PSB)
and poultry manure (PM) on P content and P uptake of chilli (Capsicum annuum L.) grown in pots under greenhouse conditions at Rawalakot Azad Jammu
and Kashmir.
Treatments
Shoot P
Fruit P
Shoot P uptake
Fruit P uptake
Total P uptake
(mg g-1)
(mg g-1)
(mg plant-1)
(mg plant-1)
(mg plant-1)
Control
0.78
0.50
3.7
1.8
5.5
RP
0.83
0.89
4.3
4.4
8.7
SSP
1.03
1.30
8.7
9.3
18.0
DAP
1.33
1.57
15.3
15.7
31.3
PM
1.01
1.54
11.0
15.2
26.2
1/2 RP+1/2 SSP
0.89
1.12
5.7
6.5
12.2
1/2 RP+1/2 DAP
0.92
1.08
7.3
7.1
14.4
1/2 RP+1/2 PM
0.90
1.06
6.2
6.8
13.0
RP+PSB
0.88
0.94
6.9
5.3
12.1
1/2 RP+1/2 SSP+PSB
0.98
1.17
9.3
8.4
17.7
1/2 RP+1/2 DAP+PSB
1.10
1.28
11.2
11.2
22.4
1/2 RP+1/2 PM+PSB
1.17
1.51
11.4
15.5
30.1
LSD (P≤0.05)
0.11
0.13
1.81
1.95
2.11
* RP: rock phosphate; SSP: single superphosphate; DAP:
diammonium phosphate; PM: poultry manure; PSB: phosphate-solubilizing
bacteria; full dose of P from different sources was applied at the rate of
90 mg P kg-1 soil.
P utilization efficiency of chilli grown under greenhouse conditions
following the application of different P sources applied alone or in
combination with PSB and PM. The legend at x axis refers to the different
treatments: T1 – RP full; T2 – SSP; T3 – DAP; T4 –
PM; T5 – 1/2 RP+1/2 SSP; T6 – 1/2 RP+1/2 DAP;T7 –
1/2 RP+1/2 PM; T8 – RP+PSB; T9 – 1/2 RP+1/2 SSP+PSB;
T10 – 1/2 RP+1/2 DAP+PSB; T11 – 1/2 RP+1/2 PM+PSB.
Full dose of P from different sources was applied at the rate of
90 mg P kg-1 soil.
The application of phosphatic fertilizers had a significant effect (P≤ 0.05) on the P uptake of plant biomass and the fruit of chilli compared to
the control treatment (Table 5). The values ranged between 4.3 and
15.3 mg plant-1 for shoot and 4.4–15.7 mg plant-1 for fruit
compared to 3.7 and 1.8 mg plant-1 in the control, respectively. Among
different P sources and combinations, DAP exhibited the highest P uptake,
while the PM and 1/2 RP+1/2 PM+PSB treatments showed values (for fruit
P uptake) on par (statistically equivalent) with DAP.
The total P uptake (shoot + fruit) in the control was
5.5 mg plant-1, which significantly increased to
8.7–31.3 mg plant-1 following the application of different P sources.
The DAP and 1/2 RP+1/2 PM+PSB treatments exhibited the highest total P
uptake and the difference between the two was nonsignificant. The RP
treatment alone resulted in a significant increase in P uptake
(8.7 mg plant-1) compared to the control (5.5 mg plant-1). The
effectiveness of P fertilizers with regard to plant P uptake had in the
following order: DAP > PM > SSP > RP. The total P uptake under PM
was significantly higher than the SSP (31 %) but lower than the DAP
(20 %). The application of PSB with different P sources resulted in a
significant (P≤ 0.05) increase in P uptake, i.e., 20 % with RP,
29 % with 1/2 RP+1/2 SSP, 56 % with 1/2 RP+1/2 DAP and
132 % with 1/2 RP+1/2 PM.
The P utilization efficiency (PUE) of added P sources and their combinations
ranged from 4 % by RP to a maximum of 29 % with DAP (Fig. 3). The PUE
of SSP and PM was 14 and 23 %, respectively, showing higher PUE by PM
compared to SSP. The PUE of RP was only 4 % and increased to 6–8 %
when RP was combined with either PSB, SSP, DAP or PM. Results indicated a
significant improvement in PUE when PSB was combined with P amendments. For
example, the PUE of the 1/2 RP+1/2 SSP+PSB, 1/2 RP+1/2 DAP+PSB,
1/2 RP+1/2 PM+PSB treatments was 14, 19 and 27 % compared to 8, 7
and 7 % from the 1/2 RP+1/2 SSP, 1/2 RP+1/2 DAP and 1/2
RP+1/2 PM treatments, respectively, showing about a 2–4-fold increase in
PUE due to PSB. The response of PUE to PSB was more prominent when PSB was
combined with PM compared to its combination with DAP or SSP.
Discussion
P release capacity of added amendments
In order to determine the P release capacity (mineralization) of soluble and
insoluble P fertilizers and their response to PM and PSB, an incubation study
of 60 days was conducted under controlled laboratory conditions. The P
release capacity of different amendments and their combinations varied with
source and timings. Soluble P fertilizers, i.e., SSP and DAP, displayed the
highest mineralization compared to the insoluble RP and organic PM. In most
of the cases (except PM and the combined treatment of 1/2 RP+1/2
PM+PSB), there was a general trend of a rapid mineralization in the first
few days of incubation followed by a gradual decrease and a sharp decline
thereafter. The P mineralization trend (over time) observed in this study was
in accordance with the previous studies, where P mineralization of different
P sources significantly decreased with time (Begum et al., 2004, Toor, 2009;
Toor and Haggard, 2009). This decreasing trend may be ascribed to the rapid
conversion of available P into insoluble complexes (Mehta et al., 2014) by
entering into the immobile pools through precipitation reaction with highly
reactive Ca2+ ions (Khan et al., 2009). The soil under investigation was
slightly alkaline (pH 7.57), noncalcareous (CaCO3 0.68 %) and had a
Ca content of 58.7 mg kg-1. The soil Ca concentration was low, but,
nevertheless, its presence may have contributed towards P fixation or
precipitation. In addition, soil belong to the Chinasi soil series and parent
material is residuum colluvium from shales. Therefore, it is likely that
kaolinite may be a dominant clay mineral present in soil composition that
adsorbs high H2PO4-. The other possibility may be the fixation
of some of the applied or native P on the surface of the clay particles given
the 24 % clay content of the soil used in the study. As organic matter
plays an important role in P solubilization through the acidifying and
chelation mechanisms, the low level of organic matter in our soil may also be
an important factor for the overall low mineralization trend of P observed in
this study.
The addition of RP alone released a maximum of 6 % of the total P
applied, showing that the mineralization capacity of RP P is low even under
favorable environmental conditions and the fertilizer value of this RP
(alone) is negligible. These values were substantially lower than those
reported for North Carolina and Syrian RP applied to an acid Lily soil,
showing P dissolutions of about 27 % after 126 days of incubation.
However, the observed values are in the range reported for Indian RP, i.e.,
6–8 mg kg-1, applied under alkaline conditions (pH 8.5) (Begum et
al., 2004). Similarly, the application of RP alone to slightly alkaline soil
(pH 7.9) at Faisalabad, Pakistan, did not show any significant effect on
bioavailable P contents of the soil (Saleem et al., 2013). These reports
suggest that RP works best in acidic soils, while they show poor efficiency
in neutral and alkaline soils. Under acidic conditions, organic acid anions
with oxygen-containing OH- and COOH-groups, have the ability to
form stable complexes with cations such as Ca2+, Fe2+,
Fe3+ and Al3+ that are commonly bound with phosphate (Jones,
1998). By complexing with cations on the mineral surface, organic acid anions
loosen cation–oxygen bonds of the mineral structure and catalyze the release
of cations to solution (Kpomblekou and Tabatabai, 1994). This is the major
reason why RP is more effective under acidic conditions.
The effect of PSB on the P release capacity of different P amendments was
significant (P≤ 0.05). The application of PSB with RP in RP+PSB
(T8) exhibited an overall 17 % higher mineralization than RP alone,
showing a solubilizing effect of PSB on RP. Jha et al. (2013) isolated 10 PSB
strains and tested them for mineral phosphate solubilization activity of RP
and stated that all these strains could solubilize only 0.02–2.6 % of
the total RP P applied. In addition, Aspergillus niger (a fungus),
used in the industrial production of citric acid, has been recognized as one
of the most effective organisms for RP solubilization (Abd-Alla and Omar,
2001). These results suggest that (i) PSB increased P solubilization of added
P fertilizers either from soluble or insoluble source and (ii) the relative
efficiency of PSB for releasing P was higher with PM compared to soluble or
insoluble P fertilizers. Khan and Sharif (2012) conducted an incubation study
in soil amended with PM, PM+RP and PM+RP+EM (EM: effective
microorganisms) and reported that the extractable P was significantly higher
in the treatments PL+RP+EM and PL+RP compared to PL only. Reddy et
al. (2002) compared the efficiency of three isolates on the solubilization of
RP and reported that all the isolates increased RP P release efficiency by
solubilizing the tested RPs. Similar effects of bio- and organic fertilizers
on RP availability and P fertilizer efficiency had also been reported in
soils incubated for different incubation periods (Aria et al., 2010; Alzoubi
and Gaibore, 2012). The mechanisms involved in the potential of PSB to
solubilize P complexes or insoluble phosphates are well known and have been
attributed to the processes of acidification, chelation, exchange reactions
and the production of organic acids (Chen et al., 2006; Ekin, 2010).
The mineralization of RP P was unaffected when RP was combined with soluble P
fertilizers (SSP, DAP), demonstrating that soluble P fertilizers had no
solubilizing effect on RP. In contrast to our results, Begum et al. (2004)
found a substantial improvement in extractable-P status when RP was combined
with SSP and MAP (monoammonium phosphate). However, RP when combined with PM
in 1/2 RP+1/2 PM released a significantly higher amount of P compared to
the RP alone (80 %) and was equivalent to that recorded under full PM
treatment, showing that the additional P released from RP was associated with
PM. Toor (2009) found a substantial increase in soil solution P following the
application of PM with P fertilizers because of the release of organic acids
during decomposition of the manure and production of carbon dioxide during
organic-matter decomposition that may increase the solubility of Ca2+
and Mg2+ phosphates.
The PSB tended to decrease soil pH, showing an acidifying effect. The maximum
reduction in pH of about 15 units was recorded in the treatment where PSB was
applied with PM. The effect of PSB on soil pH at different time intervals
indicated that, in some cases, the addition of PSB temporarily increased soil
pH more than in other treatments. The general decrease in pH during the
experiment could have arisen from a move back to
equilibrium as well as due to an
increase in microbial activity. Our results were in accordance with the
previous observations of Aria et al. (2010) and Khan and Sharif (2012), who reported a
significant decrease in soil pH after applying PSB.
Growth, yield, P uptake and P utilization of chilli
RP alone had little effect on plant growth, but the response of RP+PSB over
RP was no effect on shoot length, a 54 % increase in root length,
50 % increase in SDW and 8 % increase in RDW. The difference between
the two treatments is attributed to the effect of PSB of releasing P either
from RP or from native soil P, thereby increasing plant growth. Among the
four main P sources used (SSP, DAP, RP and PM), DAP showed superiority over
SSP and PM because of the highest P release capacity shown in the incubation
study. However, the efficiency of SSP for the growth and yield
characteristics of chilli was significantly lower than the DAP and PM for
most of the parameters studied. The P release capacity of SSP was higher than
the PM throughout the incubation while the growth and yield attributes were
lower. The possible reasons for this discrepancy are not understood; however,
in addition to supplying P to plants, the additional beneficial effects of PM
on soil physicochemical characteristics, root proliferation and plant
nutrient uptake may affect the growth and yield of plants grown in PM amended
soil. The results of the present study indicated that the application of RPs
directly to soils had shown positive effects on root dry weight and yield
components of chilli, but the efficacy of RP for most of the growth
characteristics was negligible.
The application of PSB with RP, SSP, DAP and PM or their combinations
displayed a remarkable improvement in the growth and yield of chilli. The
treatment which received 1/2 RP+1/2 PM+PSB produced growth and yield
comparable to that recorded from the full DAP, showing that this mixed
treatment may be able to save almost 50 % of chemical P fertilizer. The
higher response of plant growth to PSB might be due to the mobilization of
available P by the native soil microflora or increased PSB activity in the
rhizosphere following PSB application and consequently by enhanced P
solubilization which enhanced the
growth and yield of plants (Ekin, 2010). The combined application of PSB and
PM with P fertilizers is considered an important management strategy for
mobilizing P, where inert P is expected to be converted into plant-available
forms because of the acidic environment prevailing during the decomposition
of organic manure (Nishanth and Biswas, 2008) and the additional beneficial
effects of PSB on the processes of acidification, chelation, exchange
reactions and the production of organic acids (Chen et al., 2006; Ekin,
2010). These combined effects increased the efficiency of applied materials,
thereby increased the growth and yield of the plant as observed in the
present study. Our results are in accordance with the previous studies
conducted on the use of organic materials and PSB for increasing the
efficiency of applied P fertilizers and their subsequent effect on the growth
and yields of plants (Biswas and Narayanasamy, 2006; Nishanth and Biswas,
2008; Abbasi et al., 2013).
The effectiveness of P fertilizers with regard to plant P uptake was in the
following order: DAP > PM > SSP > RP. The total P uptake under PM
was significantly higher than the SSP (31 %) but lower than the DAP
(20 %). The application of PSB with different P sources resulted in a
significant (P≤ 0.05) increase in P uptake, i.e., 20 % with RP,
29 % with 1/2 RP+1/2 SSP, 56 % with 1/2 RP+1/2 DAP and
132 % with 1/2 RP+1/2 PM. These results indicated that the use of PSB
with PM had a dominant effect on increasing plant P uptake compared to the
application of other P sources. The overall PSB effect (group effect) showed
that total P uptake under the treatments supplemented with PSB was 23.4
compared to 13.2 mg plant-1 under the treatments without PSB, showing
a relative increase of 77 % compared to the treatments without PSB.
Results of our incubation and pot experiment indicated that the total P
uptake by the plants in response to the different amendments was
significantly correlated with P mineralization (r2= 0.64) (determined
at the end of the experiment on day 60), showing that P uptake by plants is
associated with the mineralization capacity of added P amendments. Similarly,
the effect of added amendments on increasing root mass may also have affected
the P uptake as significant correlations were found between these two
parameters (r2= 0.71). The increasing effect of P mineralization and
plant root mass and density on P uptake due to PSB and organic amendments has
also been reported earlier (Lorion, 2004; Nishanth and Biswas, 2008; Abbasi
et al., 2013). Wickramatilake et al. (2010) investigated P release capacity
of RP treated with compost prepared from PM, cattle manure (CM), sewage
sludge (SS) or P-adjusted sawdust (PSD) and reported that the uptake of P
from RP by plants is enhanced by compost, especially PM or CM compost; the
increase was 4–5-fold compared to treatments with no compost addition.
Results of this study showed that PUE of chemical P fertilizers commonly used
in most parts of the world, i.e., SSP and DAP, was low, i.e., 14 and
29 %, respectively. However, this recovery of applied P is in accordance
with the recovery efficiency of P generally reported (20–25 %) (Qureshi
et al., 2012). The organic P sources, i.e., PM, displayed a higher PUE
(23 %) compared to SSP (14 %), although the P mineralization capacity
of SSP was significantly higher than PM. This favorable effect may be
attributed to (i) the increased P uptake by plants through enlarged
proliferation of roots as the root mass of plants under PM was 17 %
higher than the root mass recorded under SSP and (ii) the reduction in the
activity of Ca2+, Al3+ and Fe3+ ions by root-exuded organic
anions, as reported earlier (Toor, 2009).
The PUE of RP and RP+PSB was just 4 and 7 %, respectively, indicating
that RP alone was not able to generate any positive impact as a P fertilizer.
However, the PUE of RP, SSP and DAP was remarkably increased when these
sources were combined with either PM or PSB. Among different combinations,
1/2 RP+1/2 PM+PSB showed the most significant contribution by
increasing PUE to 27 %, equivalent to that recorded under full DAP
treatment. This finding highlighted the importance of RP as a P source when
combined with organic and microbial amendments. The increased PUE may have
resulted in increased dry-matter yield (DMY), fruit yield and greater P
accumulation as significant correlations existed between PUE and DMY
(r2= 0.93), fruit yield (r2= 0.97), PUE and shoot and fruit P
concentrations (r2= 0.86. r2= 0.93), and PUE and shoot and fruit
P uptake (r2= 0.97).
The role of organic amendments or PSB in improving P utilization from applied
P fertilizers has been reported earlier by several researchers (Begum et al.,
2004; Toor, 2009, Abbasi et al., 2013). This positive effect is attributed to
the fact that the release of organic acids from these amendments in the root
rhizosphere can reduce the fixation of applied P, induce greater P
availability in the soil and form phosphor–humic complexes that are easily
assimilated by plants (Toor, 2009). These mechanisms can result in greater
amounts of applied P in forms available to be used by plants.
Conclusions
The results of our incubation experiment indicate that chemical P fertilizers
used in the study, i.e., SSP and DAP, released the highest P at the start of
the experiment, but this mineral P significantly decreased with subsequent
incubation periods. At the end of the experiment (on day 60), about 80 %
of P initially present had disappeared from the system, showing that the P
recovery in the soil mineral pool was 20 % of the total P applied from P
fertilizers. Rock phosphate (RP) alone or RP+PSB released a maximum of
12 mg P kg-1, demonstrating that the application of RP directly to
soil with a slightly alkaline pH did not show any positive effect on overall
P mineralization or P availability. However, the use of PSB and PM with RP in
the combined treatment (1/2 RP+1/2 PM+PSB) released a substantial
amount of P (25 mg kg-1) that remained at high levels (without any
loss) until the end of incubation (day 60), showing that the combination of
PSB and PM with RP may be a feasible option for releasing P from insoluble RP
for a longer period. When these amendments were applied to chilli under
greenhouse conditions, DAP exhibited the highest growth, yield and P uptake.
RP alone was able to increase yield compared to the control but was not as
effective as SSP, DAP and PM. Combinations of RP with either SSP or DAP in a
ratio of 50 : 50 did not show any significant effect on P mineralization
and subsequent plant growth and P uptake. However, the application of PM and
PSB with RP in 1/2 RP+1/2 PM+PSB showed a remarkable effect and induced
growth, yields and P uptake comparable to that recorded under the DAP
treatment. The P utilization efficiency of chilli supplemented with 1/2
RP+1/2 PM+PSB was not statistically different from that recorded from
full DAP treatment (27 and 29 %). The combination of PM and PSB with RP
(1/2 RP+1/2 PM+PSB), therefore, holds a lot of promise as an efficient
alternative to conventional P fertilizers, especially regarding its
effectiveness for the utilization of RP. However, the results need to be
confirmed under field conditions, and the economic feasibility of the
application of this particular combination needs to be
quantified.