Phosphorus release capacity of soluble P fertilizers and insoluble rock phosphate in response to phosphate solubilizing bacteria and poultry manure and their e ff ect on plant growth promotion and P utilization e ffi ciency of chilli ( Capsicum annuum L . )

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at the start of the experiment (day 0).However, the P released tendency from SSP and DAP declined during incubation and at the end 82 and 79 % of P initially present had been lost from the mineral pool.Integrated use of PSB and PM with RP in 1/2 RP + 1/2 PM + PSB treatment stimulated P mineralization by releasing a maximum of 25 mg P kg −1 that was maintained at high levels without any loss.Application of PSB tended to decrease pH showing an acidifying effect on soil.In the greenhouse experiment, RP alone or RP + PSB was not able to generate any significant impact on plant while DAP displayed the superiority over the remaining treatments.Combined use of RP, PM and PSB in 1/2 RP + 1/2 PM + PSB resulted in the growth, yield 1 Introduction Phosphorus (P) is the second important key plant nutrient after nitrogen (N) which strongly 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 of plants (Shenoy and Kalagudi, 2005;Khan et al., 2009Khan et al., , 2014)).On average, soils contain very little total P of 0.02-0.5 % (w / w) (Fernandez et al., 2007) and only 0.1 % of this P is available to plant (Zou et al., 1992).Phosphorus is thus added to the soils as soluble P fertilizers, a part of which (1 %) is utilized by plants and the rest is rapidly converted into insoluble complexes (Mehta et al., 2014) by entering into the immobile pools through precipitation reaction with highly reactive Al 3+ and Fe 3+ in acidic, and Ca 2+ in calcareous soils (Khan et al., 2009).These metal ion complexes precipitated about 80 % of added P fertilizer hence, the recovery efficiency of P throughout the world is not more than 20 % of applied P (Qureshi et al., 2012).Considering the low recovery of applied and native P and high cost of chemical phosphatic fertilizers besides increasing concern of environmental degradation (Aziz et al., 2006;Khan et al., 2014), it has become imperative to find multidimensional and viable solutions to tackle the problem.
In this regard, two management options can be workout simultaneously for efficient utilization of P fertilizers i.e. (i) to increase the recovery and solubility of applied P more ecological but nevertheless efficient P sources, such as use of indigenous rock phosphates (RPs) in our agriculture inputs system.Interest in the use of RPs as alternative P sources has been increased in recent times due to their relative low costs and their utilization potentials (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 in the market (Cordell et al., 2009;Beardsley, 2011;Ekelöf et al., 2014).Application of RPs directly to the soils has yielded some positive results under acidic soils but the efficacy of such material is almost negligible in neutral and alkaline soils (Begum et al., 2004).However, there are reports indicating that Syrian RP was is an effective P fertilizer for rape plants (Brassica napus L.) in alkaline soil (pH = 7.72; Habib et al., 1999) and in an acidic Lilysoil grown maize (pH = 3.95; Alloush and Clark, 2001).Therefore, efforts have been made and in progress to find out suitable ways to improve the solubility and efficiency of indigenous RPs.
Numerous studies have been conducted to evaluate the efficiency of different amendments used to increase the availability and solubility of P from native and applied sources including RP.Among these, organic amendments including animal manures, plant residues, and green manures (Alloush, 2003;Toor, 2009;Aria et al., 2010;Adesanwo et al., 2012), composting (Nishanth andBiswas, 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 efficiency of P. In addition, combined application of water soluble P fertilizers with RP is another management strategy for increasing the efficiency of RP-P.Mashori et al. (2013) conducted a pot experiment on maize to examine the relative performance of RP, SSP, RP + SSP with and without FYM and reported that RP + SSP (25 + 75 %) with FM (10 t ha −1 ) (RP + SSP + FYM) increased maize growth, dry matter, leaf P content and P uptake followed by 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 Figures

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Full complex P compounds (Wani et al., 2007;Khan et al., 2014).In particular, Psolubilizing bacteria (PSB) are reported to play a significant role in increasing 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, application of organic manures with phosphatic fertilizers is considered another possible means of mobilizing P because of the acidic environment generated during decomposition of the manures (Nishanth and Biswas, 2008).These organic manures increase the density of 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, 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 super phosphate (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 huge foreign exchange involved in importing raw material for manufacturing P fertilizers or P fertilizers directly imported, it is imperative to explore the possible utilization of indigenous RPs and the ways to increase the efficiency of other P fertilizers by applying different management strategies.Effect of PSB or organic manures on the efficiency of both soluble and insoluble P fertilizers with regard to plant growth and yield had been studied and seemed to be a topic of interest in these days.However, effect of these combinations on P release capacity (mineralization) of both soluble and insoluble P sources especially RPs is given little attention.Therefore, the present study was planned to examine the effect of poultry manure (PM) and PSB Introduction

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Full with soluble P fertilizers (SSP and di-ammonium phosphate, DAP) and insoluble rock phosphate (RP) on P release capacity and their subsequent effect on growth, yields, P-uptake and P utilization efficiency (PUE) of chilli (Capsicum annuum L.) grown under greenhouse conditions.

Soil sampling/collection
Surface bulk soil (0-15 cm) from a field under long-term wheat-maize management system in the Faculty of Agriculture, 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 (not more than two weeks' time).A sub-sample of about half kg 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 K 2 Cr 2 O 7 in concentrate sulphuric acid followed by titration with ferrous-ammonium sulphate (Nelson and Sommers, 1982).Total N was determined by 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 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 (COOCH 3 NH 4 ) (Simard, 1993).The bulk density (BD) was determined from undisturbed soil cores taken from the upper horizon (0-15 cm) at about five locations Figures

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Full from the field.Bulk density of the soil was calculated on a volume basis (Blake and Hartge, 1986).

Collection of added amendments/materials
The different amendments used in this study were rock phosphate (RP), single super phosphate (SSP), di-ammonium phosphate (DAP), poultry manure (PM) and P-solubilizing bacteria (PSB).Rock phosphate was collected from Land Resources Research Institute (LRRI), NARC Islamabad, Pakistan.Major reserves of this RP in the country are found in lagarban region of Hazara division positioned in the North East of Pakistan (Mashori et al., 2013).According to Memon (2005), this RP contains average 25.8 % P 2 O 5 along with 6 % MgO.Di ammonium phosphate and SSP were purchased from the local market while PM was collected from the local farms located near by the university campus.A composite sample of well-rotted PM was air dried, crushed into smaller particles by hand pressing, homogenized, and passed through 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.The total N and total P contents of PM were 2.53 and 1.64 %, respectively.The bio-power powder of PSB was provided by the Nuclear Institute of Biology and Genetic Engineering (NIBGE), Faisalabad, Pakistan.The PSB used in this study was a mixture of Pseudomonas, Azospirillum, and Agrobacterium strains at a 2 : 2 : 1 ratio.

Experimental procedures and details -incubation study
The fresh soil samples already stored in the refrigerator was taken and transferred into a glass jar.The soil sample was pre-incubated at 25 • C for one week prior to actual incubation to stabilize the microbial activity.A known weight of soil (30 g oven dry weight basis) from this sample was taken and transferred into a 100 mL capacity jars.
Moisture content of soil was adjusted to 60 % of its water holding capacity (WHC) by Phosphorus from all the treatments or sources was applied on equivalent basis i.e. at the rate of 90 mg P kg −1 soil generally recommended as the optimum P rate for chilli under the environmental conditions of 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, soil was properly mixed and weight of each jar was recorded.Jars were covered with parafilm that was perforated with a needle to ensure natural gas exchange.All the amended jars were kept in an incubator at 25 for a total of 60 days.Jars in the incubator were arranged according to the completely randomized design.Soil moisture was checked/adjusted after 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 available P and pH of the soil.Triplicate samples from each treatment was drawn from the incubator at 0, 5, 15, 25, 35, 60 days and analyzed for extractable P by AB-DTP extraction method (Soltanpour and Workman, 1979).Twenty (20) g soil was weighed in 125 mL Erlenmeyer flasks and 20 mL of extraction solution was added (1 : 2).The soil available P was measured by ammonium heptamolybdate (Murphy and Riley, 1962)  different amendments.The soil pH was determined using a glass electrode on 1 : 2.5 (v/v) soil / water suspensions.

Experimental procedures and details -greenhouse experiment
To complement the incubation study, a greenhouse experiment was conducted in pots with chilli (Capsicum annuum L.) used as a test crop.Seedling nursery of chilli was grown by making nursery beds in the greenhouse during the last week of April 2013.
Chilli seeds of variety "Pusa Jawla" were sown separately on ridges.All the necessary culture practices were carried-out when needed.Thoroughly processed soil (passed through a 4 mm sieve) was placed in cleaned earthen pots (38 cm height and 18 cm width) of 12 kg soil capacity.There were twelve treatments including a control i.e.
(unfertilized control); RP full, SSP full; DAP full; PM full; with four replications to form a total of 48 treatment combinations.Pots were arranged according to a completely randomized design.Addition of different amendments were made according to the methods/procedures followed by the incubation study.However, PSB was grown in LB broth to lag phase, containing about 10 8 CFU mL −1 , applied to respective treatments by dipping roots of chilli plants in inoculum up to 20 min.On attaining 5-8 leave stage, 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 vegetative stage (just before flowering) for measuring growth traits of 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.Introduction

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Full For the determination 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-mesh sieve in a Micro Wiley Mill.The total P was determined after digestion in a triple acid mixture (HClO 4 , H 2 SO 4 , and HNO 3 in the 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 any.Soil organic matter, total N, available P, K and pH of soil from each treatment were determined according to methods described earlier for Table 1.

Statistical analysis
All data from incubation experiment were statistically analyzed by multifactorial analysis of variance (ANOVA) using the software package Statistix 8.1.Least significant differences (LSD) 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, time interval and their interactions.Data of the greenhouse experiment was analyzed by one way analysis of variance and LSD is given to indicate significance variations among different treatments.A probability level of P ≤ 0.05 was considered significant for both experiments.Introduction

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P release capacity (mineralization) of added amendments
Phosphorus release capacity of soil amended with RP varied between 6.0-11.5 mg kg −1 significantly (P ≤ 0.05) higher than the control but lower than the remaining treatments (Table 2).Application of PSB with RP in RP + PSB did not show any remarkable effect on P solubilization or P mineralization except that a significant increase was noticed on 25 and 60 days of incubation.Soil amended with the soluble P fertilizers i.e.SSP and DAP displayed the highest P mineralization of 73.3 and 68.5 mg kg −1 immediately after their application at day 0.However, this mineral P significantly (P ≤ 0.05) decreased with subsequent incubation periods and at the end only 14 mg P kg −1 was left in the mineral P pool.In contrast, contents of available P under PM amended soil progressively increased with time and the highest release of 20.2 mg P kg −1 was recorded at day 35 compared to 10.4 mg kg −1 at day 0.However, the mineralization trend of PM-P changed at the end (day 60) and P contents declined to a background level i.e. 9.6 mg kg −1 .Rock phosphate combined with soluble P fertilizers (SSP, DAP) did not show any significant impact on P mineralization.However, the P release capacity under the treatments 1/2 RP + 1/2 DAP + PSB and 1/2 RP + 1/2 SSP + PSB was significantly higher than their sole application throughout the incubation.Similarly, the P mineralization under 1/2 RP + 1/2 PM + PSB showing increasing trend with subsequent incubation periods (showing no losses) a trend normally not common for phosphatic fertilizers.

Effect of different amendments on changes in soil pH
Effect of different P amendments and their combinations on changes in soil pH over 60 days incubation is presented in Table 3. pH at day 0 displayed the actual pH of each amendment had wide variation depended on the source of applied P. 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.pH of both DAP and PM also significantly decreased and at the end (day 60) and the reduction in pH compared to day 0 was 8 %.Among different amendments RP showed the lowest pH.Average across different amendments, the data presented in Fig. 2 indicated that 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, results showed that application of PSB decreased soil pH.Average pH under the treatments RP, 1/2 RP + 1/2 SSP, 1/2 RP + 1/2 DAP, 1/2 RP + 1/2 PM was 7.80 while application of PSB with these four amendments tended to decline pH to 7.72 showing a 8 unit decrease in pH due to PSB.The maximum reduction in pH of about 15 units was recorded in the treatment where PSB was applied with PM.Introduction

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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 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, 54 % increase in root length, 50 % increase in SDW and 8 % increase in RDW.Application of PSB with DAP, SSP and PM displayed 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.Yield and yield characteristics 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 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.Application of RP alone induced significant increase in yields (over control) however, the extent of increase was remarkably higher when PSB was combined with RP.The relative increase in fruit length, no. of fruits, fruit yield and number of seed due to RP + PSB was 18, 34, 14 and 16 %, respectively compared to the RP alone.Between the two synthetic P fertilizers used (SSP, DAP), DAP showed superiority over SSP while PM also exhibited a comparative yields to DAP and SSP.
Integrated use of RP with SSP, DAP and PM (50 : 50) was not comparative to their full dose.However, combined used 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, no. of Introduction

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P content, P-uptake and P utilization efficiency
The P content of plant biomass and fruits of chilli treated with different P sources and combinations was significantly (P ≤ 0.05) higher compared to P content of the control (Table 5).Soil amended with DAP (full) resulted in the highest P content of shoot (1.33 mg plant −1 ) and fruit (1.57mg plant −1 ) as compared to SSP and other P materials.However, fruit P content recorded under PM (full) and 1/2 RP + 1/2 PM + PSB (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 application of PSB with RP (RP + PSB) further increased shoot and fruit P by 6 and 5 %, respectively compared to RP alone.Application of phosphatic fertilizers had significant effect (P ≤ 0.05) on P-uptake of plant biomass and fruit of chilli compared to the control treatment (Table 5).The values ranged between 4.3-15.3mg 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 PM and 1/2 RP + 1/2 PM + PSB showed values (for fruit P-uptake) at par (statistically equivalent) with DAP.
The total P-uptake (shoot + fruit) in the control was 5.5 mg plant −1 that significantly increased to 8.7-31.3mg plant −1 following the application of different P sources.DAP and 1/2 RP + 1/2 PM + PSB exhibited the highest total P-uptake and the difference between the two was non-significant.RP alone was able to significantly increased Puptake (8.7 mg plant −1 ) compared to the control (5.5 mg plant −1 ).The effectiveness of Introduction

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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 % that increased to 6-8 % when RP was combined with either PSB or SSP, DAP or PM.Results indicated a significant improvement in PUE when PSB was combined with P amendments.For example, the PUE under 1/2 RP + 1/2 SSP + PSB, 1/2 RP + 1/2 DAP + PSB, 1/2 RP + 1/2 PM + PSB was 14, 19 and 27 % compared to 8, 7 and 7 % in 1/2 RP + 1/2 SSP, 1/2 RP + 1/2 DAP, and 1/2 RP + 1/2 PM, respectively showing about 2-4fold 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.

P release capacity of added amendments
In order to access the P release capacity of soluble and insoluble P fertilizers and their response to PM and PSB, incubation study of 60 days period 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 P release capacity compared to insoluble RP and organic PM.In most of the cases, except PM and combined treatment of 1/2 RP + 1/2 PM + PSB, there was a general trend of an increase in the extractable P in the first few days of incubation followed by a gradual decrease and a sharp decline thereafter.The Introduction

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Full P mineralization trend (over time) of added P sources observed in this study was in accordance with the other P mineralization studies where it was generally observed that the extractable P from different P sources and combinations significantly declined over time (Begum et al., 2004;Toor, 2009;Toor and Haggard, 2009).This decreasing trend may be ascribed due to rapid conversion of available P into insoluble complexes (Mehta et al., 2014) by entering into the immobile pools through precipitation reaction with highly reactive Al 3+ and Fe 3+ in acidic, and Ca 2+ in calcareous soils (Khan et al., 2009).Aria et al. (2010) reported that the most important changes in the concentration of soluble P from applied P sources occurred during the first 35 days of incubation and then the variation in available was almost constant until the end of incubation.
Addition of RP alone released a maximum of 11.5 mg P kg −1 equivalent to 6 % (by subtracting P of the control soil) of total P applied showing that mineralization capacity of RP-P even under favorable environmental conditions is low and the fertilizer value of this RP (alone) is quite 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 incubtion.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, application of RP alone to an 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).It has been reported that most of the RP deposits in Pakistan are low-grade and unsuitable for manufacturing of commercial P-fertilizers because of their low P content and low reactivity (Memon, 2005).Application of PSB with RP in RP + PSB (T 8 ) showed similar values for most of the incubation periods, however, a significant increase in available P was recorded at day 25 and 60.As a results, the overall effect of RP + PSB was 17 % higher than RP alone showing a solubilizing effect of PSB on RP.Jha et al. (2013) isolated ten PSB strains and tested 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.Introduction

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Full The P released from soluble P fertilizers i.e.SSP and DAP was 73 and 68 mg kg −1 at day 0 that had been disappeared rapidly from the mineral pool.Results indicated that the P unaccounted for at the end (day 60) was 82 and 79 %, respectively to that recorded at day 0.This disappearance of P may be attributed to the P fixation by soil that tended to the rapid conversion of applied P via sorption and precipitation reactions to unavailable-P as reported earlier (Toor, 2009;Khan et al., 2009).Rock phosphate combined with soluble P fertilizers (SSP, DAP) did not indicate any significant impact on P mineralization showing that combination of soluble P fertilizers with RP did not have any 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 (compacted) with SSP and MAP (mono-ammonium phosphate).However, RP when combined with PM in 1/2 RP + 1/2 PM released significantly higher P compared to full RP treatment (80 %) and 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 Ca 2+ and Mg 2+ phosphates.
Effect of PSB on 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 alone (RP + PSB, T 8 ), 12 % increase under 1/2 RP + 1/2 SSP + PSB (T 9 ) compared to 1/2 RP + 1/2 SSP (T 5 ), 18 % increase under 1/2 RP + 1/2 DAP + PSB (T 10 ) compared to 1/2 RP + 1/2 DAP (T 6 ), and 28 % increase under 1/2 RP + 1/2 PM + PSB (T 11 ) compared to 1/2 RP + 1/2 PM (T 7 ).These results suggested 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 Figures

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Full + 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 solubilize the tested RPs.Similar effects of bio-and organic fertilizers on RP availability and P fertilizers 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 has been attributed to the processes of acidification, chelation, exchange reactions, and the production of organic acids (Chen et al., 2006;Ekin, 2010).Application of PSB decreased soil pH showing acidifying effect of PSB on soil pH.The maximum reduction in pH of about 15 units was recorded in the treatment where PSB was applied with PM.Our results were in accordance with the previous observations of Aira 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
To complement the incubation study, a greenhouse experiment was conducted in pots with chilli (Capsicum annuum L.) used as a test crop to examine the effect of different P sources supplemented with PM and PSB on the growth, yield, P-uptake and P-utilization efficiency of added amendments.RP alone had little effect on plant growth but the response of RP + PSB over RP was: no effect on shoot length, 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 on releasing P either from RP or from native soil P thereby increased plant growth.Among 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 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 Figures

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Full attributes was lower.The possible reasons for this discrepancy is not understood however, in addition to supply P to plants the additional beneficial effects of PM on soil physiochemical characteristics, root proliferation and plant nutrient uptake may affect growth and yield of plant grown in PM amended soil.Results of present study indicated that 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 almost negligible.Application of PSB with RP, SSP, DAP and PM or their combinations displayed a remarkable improvement in the growth and yield of chilli.Especially the treatment receiving 1/2 RP + 1/2 PM + PSB generated growth and yield comparable to that recorded under full DAP showing that such combinations can save almost 50 % of chemical P fertilizer.The higher response of plant to PSB might be due to 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 that enhance growth and yield of plants (Ekin, 2010).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 decomposition of organic manures (Nishanth and Biswas, 2008) and then additional beneficial effects of PSB to 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 to 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 order: DAP > PM > SSP > RP.The total P-uptake under PM was significantly higher than the SSP (31 %) but lower than the DAP ( 20 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 use of PSB with PM had dominating effect in increasing plant P-uptake than its application with other P materials.The overall PSB effect (group effect) displayed that total P-uptake under the treatments supplemented with PSB was 23.4 mg plant −1 compared to 13.2 mg plant −1 under the treatments without PSB showing a relative increase in P-uptake by PSB was 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 (r 2 = 0.64) (determined at the end of the experiment at day 60) showing that P-uptake by plants is associated with the mineralization capacity of added P amendments.Similarly, effect of added amendments on increasing root mass may also affected the P-uptake as significant correlation existed between the two (r 2 = 0.71).The increasing effect of P mineralization and plant root mass/density on P-uptake due to PSB and organic amendments had 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, and it was four-to five-fold compared 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 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 Introduction

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Full under SSP (ii) reduction of Ca 2+ , Al 3+ and Fe 3+ in activity 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 P fertilizer.However, the PUE of RP, SSP and DAP was remarkably increased when these sources were combined either with PM or PSB.Among different combinations, 1/2 RP + 1/2 PM + PSB showed the significant contribution by increasing PUE to 27 % equivalent to that recorded under full DAP treatment.This finding highlighted the importance of RP as P source when combined with organic and microbial amendments.The increased in PUE may result in increased dry matter yield (DMY), fruit yield and greater P accumulation as significant correlations existed between PUE and DMY (r 2 = 0.93), fruit yield (r 2 = 0.97), PUE and shoot and fruit P concentrations (r 2 = 0.86, r 2 = 0.93), and PUE and shoot and fruit P uptake (r 2 = 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 release of organic acids from these amendments in the root rhizosphere can complex Fe and Al in soils, can reduce 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 available forms to be used by plants.

Conclusions
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 (at day 60) about 80 % of P initially present had been disappeared from the system showing that the P recovery in the soil mineral pool was 20 % of applied Introduction

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Full demonstrating that application of RP directly to the soil having neutral pH did not show any positive effect on overall P mineralization.However, use of PSB and PM with RP in 1/2 RP + 1/2 PM + PSB treatment released a substantial amount of available P (25 mg kg −1 ) that maintained at high levels (without any loss) till the end of incubation (day 60) showing that combination of PSB and PM with RP may be a feasible option for releasing P for a longer period.This correlates well with the fact that two-thirds of the total P demand of most of the crops is met during the first one-third of their growth period.When these amendments were applied to chilli under greenhouse conditions, DAP exhibited the highest growth, yield and P-uptake.RP was able to increase many components of plants compared to the control but not as effective as SSP, DAP and PM.Combination of RP with SSP and DAP in 50 : 50 proportion did not show any significant effect on P mineralization and subsequent plant growth and Puptake.However, treatment receiving 1/2 RP + 1/2 PM + PSB showed a remarkable effect and induced growth, yields, P-uptake comparable to that recorded under full DAP treatment.The P utilization efficiency of chilli supplemented with 1/2 RP + 1/2 PM + PSB was statistically at par with that recorded under full DAP treatment (27 and 29 %).This combination (1/2 RP + 1/2 PM + PSB) hold a lot of promise as an efficient alternative to conventional P fertilizers especially its effectiveness for the utilization of RP.However, the results need to be confirmed under field conditions and the economic feasibility of its application needs to be worked out.Introduction

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Full

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  Full  Full  Full  Full and Environmental Sciences, Faculty of Agriculture, The University of Poonch, Rawalakot Azad Jammu and Kashmir, Pakistan fertilizers and (ii) to replace the expensive chemical P fertilizers by novel, cheaper, Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | using spectrophotometer.At each sampling time, the remaining ten g soil from each jar was taken and used for measuring the changes in pH in response to Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | P kg −1 in the control.Both soluble P fertilizers i.e.SSP and DAP displayed the highest P release capacity of 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 Introduction Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | %). Application of PSB with different P sources Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | P fertilizers.RP alone or RP + PSB released a maximum of only about 12 mg P kg −1 Discussion Paper | Discussion Paper | Discussion Paper | Nishanth, D. and Biswas, D. R.: Kinetics of phosphorus and potassium release from rock phosphate and waste mica enriched compost and their effect on yield and nutrient uptake by wheat (Triticum aestivum), Bioresour.Technol., 99, 3342-3353, 2008.Olsen, S. R. and Sommers, L. E.: Phosphorus, in: Methods of Soil Analysis Part 2 Chemical and Microbiological Properties, edited by Page, A. L., Miller, R. H., and Keeney, D. R.Discussion Paper | Discussion Paper | Discussion Paper | Wani, P. A., Khan, M. S., and Zaidi, A.: Synergistic effects of the inoculation with nitrogen fixing and phosphate-solubilizing rhizobacteria on the performance of field grown chickpea, J. Plant Nutr.Soil Sci.170, 283-287, 2007.Wickramatilake, A. R. P., Kouno, K., and Nagaoka, T.: Compost amendment enhances the biological properties of Andosols and improves phosphorus utilization from added rock phosphate, Soil Sci.Plant Nutr., 56, 607-616, 2010.Zapata, F. and Zaharah, A. R.: Phosphorous availability from phosphate rock and sewage sludge as influenced by the addition of water soluble phosphate fertilizer, Nutr.Cycl.Agroecosyst., 63, 43-48, 2002.Zou, K., Binkley, D., and Doxtadar, K. G.: New methods for estimating gross P Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper | Discussion Paper |

Table 1 .
The initial physical and chemical characteristics of soil used in the incubation study.

Table 2 .
Phosphorus release capacity i.e. mineralization potential of soluble P fertilizers and insoluble rock phosphate in response to phosphate solubilizing bacteria and poultry manure applied to a soil incubated under controlled laboratory conditions at 25 • C over 60 days periods.
RP = rock phosphate; SSP = single super phosphate; DAP = di-ammonium phosphate; PM = poultry manure; PSB = phosphate solubilizing bacteria; full means application of full dose of these amendments at the rate of 90 mg P kg −1 soil.Introduction

Table 3 .
Changes in pH of the soil supplemented with soluble P fertilizers and insoluble rock phosphate along with phosphate solubilizing bacteria and poultry manure and incubated under controlled laboratory conditions at 25• C over 60 days periods.
RP = rock phosphate; SSP = single super phosphate; DAP = di-ammonium phosphate; PM = poultry manure; PSB = phosphate solubilizing bacteria; full means application of full dose of these amendments at the rate of 90 mg P kg −1 soil.Introduction

Table 4 .
Effect of soluble P fertilizers and insoluble rock phosphate applied alone or in combination with phosphate solubilizing bacteria and poultry manure on the growth and yield characteristics of chilli (Capsicum annuum L.) grown in pots under greenhouse conditions at Rawalakot Azad Jammu and Kashmir.
RP = rock phosphate; SSP = single super phosphate; DAP = di-ammonium phosphate; PM = poultry manure; PSB = phosphate solubilizing bacteria; full means application of full dose of these amendments at the rate of 90 mg P kg −1 soil.Introduction

Table 5 .
Effect of soluble P fertilizers and insoluble rock phosphate applied alone or in combination with phosphate solubilizing bacteria and poultry manure on P content and Puptake of chilli (Capsicum annuum L.) grown in pots under greenhouse conditions at Rawalakot Azad Jammu and Kashmir.