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Rock phosphate-enriched organic fertilizer with phosphate-solubilizing

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Article in Communications in Soil Science and Plant Analysis · November 2018

DOI: 10.1080/00103624.2018.1538374

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ISSN: 0010-3624 (Print) 1532-2416 (Online) Journal homepage: http://www.tandfonline.com/loi/lcss20

Rock phosphate-enriched organic fertilizer with

phosphate-solubilizing microorganisms improves
nodulation, growth, and yield of legumes

Allah Ditta, Muhammad Imtiaz, Sajid Mehmood, Muhammad Shahid Rizwan,

Fathia Mubeen, Omar Aziz, Zheng Qian, Raina Ijaz & Shuxin Tu

To cite this article: Allah Ditta, Muhammad Imtiaz, Sajid Mehmood, Muhammad Shahid Rizwan,
Fathia Mubeen, Omar Aziz, Zheng Qian, Raina Ijaz & Shuxin Tu (2018): Rock phosphate-
enriched organic fertilizer with phosphate-solubilizing microorganisms improves nodulation,
growth, and yield of legumes, Communications in Soil Science and Plant Analysis, DOI:
10.1080/00103624.2018.1538374

To link to this article: https://doi.org/10.1080/00103624.2018.1538374

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COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS
https://doi.org/10.1080/00103624.2018.1538374

Rock phosphate-enriched organic fertilizer with

phosphate-solubilizing microorganisms improves nodulation,
growth, and yield of legumes
Allah Dittaa,b, Muhammad Imtiazc,d, Sajid Mehmoode, Muhammad Shahid Rizwanf,
Fathia Mubeend, Omar Azize, Zheng Qiane, Raina Ijazg, and Shuxin Tue
a
Department of Environmental Sciences, Shaheed Benazir Bhutto University, Dir (U), Pakistan; bSchool of Biological
Sciences, The University of Western Australia, Perth, Australia; cSchool of Environmental Science and Engineering,
Guangzhou University, Guangzhou, China; dSoil and Environmental Biotechnology Division, National Institute for
Biotechnology and Genetic Engineering, Faisalabad, Pakistan; eCollege of Resources and Environment, Huazhong
Agricultural University, Wuhan, China; fCholistan Institute of Desert Studies, The Islamia University of Bahawalpur,
Bahawalpur, Pakistan; gDepartment of Horticulture, The University of Poonch Rawalakot, Azad Kashmir, Pakistan

ABSTRACT ARTICLE HISTORY

Phosphorus availability from rock phosphate (RP) can be increased via com- Received 20 May 2018
posting with organic fertilizer and phosphate-solubilizing microorganisms Accepted 2 October 2018
(PSMs) in the soil. Incubation and pot experiments were conducted to
KEYWORDS
investigate the comparative efficacy of RP-enriched organic fertilizer with Chemical phosphatic
PSMs vs. diammonium phosphate (DAP) as a control to enhance the produc- fertilizers; legumes;
tion of legumes. In incubation studies, RP-enriched organic fertilizer with phosphate-solubilizing
PSM2 significantly increased soil aggregate stability (37%) and P-release microorganisms;
(2.35 folds) as compared to control, whereas, in pot study, the number and phosphorus; RP-enriched
dry weight of nodules plant−1, shoot length, number of pods plant−1, and organic fertilizer
grain yield of chickpea increased by 23%, 13%, 17%, 15%, and 15% with
RP-enriched organic fertilizer + PSM2 compared to control. Similarly, nitrogen
and phosphorus contents in straw (11 and 42% respectively) and grains (11
and 13% respectively) and protein contents (16%) of chickpea were also
increased. A similar trend was observed regarding the growth and yield
parameters of lentil. The use of RP-enriched organic fertilizer with PSM
could produce results similar to that of DAP for the optimum production of
legumes.

Introduction
The soils with low fertility contains lower concentrations of available nutrients, especially macronutrients
like phosphorus (P), usually due to drastic low (acidic) and high pH (alkaline) in soils, resulting in lower
crop yields (Li et al. 2017). Although P is abundantly present in soils, it is not easily available for plant
uptake due to the formation of complexes with soil constituents in acidic (Fe and Al) and alkaline soils
(Ca and Mg) (Takahashi and Anwar 2007). To meet P demands for proper plant growth, approximately
three-fourths of P applied should be in soluble forms through chemical fertilizers. Therefore, frequent
applications of phosphatic fertilizers are required to achieve optimum crop yields, and this practice
makes it a financial burden and economically not feasible for poor farmers (Aziz et al. 2006). In the case
of leguminous crops, the situation becomes more adverse as these crops require more P than other crops
(Gitari and Mureithi 2003). Moreover, with rapid developments in the agriculture sector, high-yielding

CONTACT Muhammad Imtiaz m.imtiazpk92@hotmail.com School of Environmental Science and Engineering, Guangzhou
University, Guangzhou 510006, China; Allah Ditta allah.ditta@sbbu.edu.pk Department of Environmental Sciences, Shaheed
Benazir Bhutto University, Sheringal, Dir (U), Khyber Pakhtunkhwa 18000, Pakistan
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lcss.
© 2018 Taylor & Francis
2 A. DITTA ET AL.

varieties with continuous uptake of nutrients and no returns to the soil in the form of organic matter
convert and degrade the cultivated fertile soils into poor fertility soils (Shu, Zhang, and Bao 2013).
Although crop growth rapidly increases with the application of chemical fertilizers, it severely
deteriorates the soil quality via destruction of microflora and fauna and ultimately results in degraded
soils. Chemical phosphatic fertilizers are produced by reactions of an inorganic acid with high-quality
phosphatic ore in the form of phosphate rock at high temperature, which makes it costly and
undesirable for a sustainable environment (Aziz et al. 2006). Therefore, the need of the hour is to
find an economically feasible and eco-friendly way to restore the fertility of these degraded soils.
Rock phosphate (RP) is a natural P source and could be used as an alternative to chemical
phosphatic fertilizers, but it is only soluble under acidic conditions and is not applicable to alkaline
soils (Caravaca et al. 2004). Recently, RP solubilization with the use of microbes having phosphate-
solubilizing ability is gaining more attention (Gyaneshwar et al. 2002; Vassilev, Vassileya, and
Nikolaeva 2006). In this regard, phosphate-solubilizing bacteria could be utilized for the solubiliza-
tion of RP as bacterial solubilization is more efficient than fungi and could easily be utilized for
industrial application (Yadav et al. 2017). RP along with phosphate-solubilizing bacteria could be
utilized as P source in alkaline soils. However, the efficiency of bacteria to solubilize RP could be
enhanced by providing an easily available nutrient source for microbes such as organic fertilizer
produced through composting. It has been well known that combined application of RP, phosphate-
solubilizing bacteria, and organic fertilizer resulted in better agronomic efficiency than chemical
phosphatic fertilizers when applied in equal amounts of total P2O5 basis (Yadav et al. 2017).
Recently, interest has been developed in compound fertilizers composed of suitable phosphate-
solubilizing bacteria, organic matter, and RP.
Additionally, the organic matter in the form of organic fertilizers improves soil physicochemical
properties and increases the amount of active organic carbon, thereby improving soil quality and soil
vitality (Biau et al. 2012). However, sole application of organic fertilizers cannot support food
production from poor/less fertile soils for the world population which is increasing at an alarming
rate. The major reason is the lack of adequate amount of available nutrients and slow availability to
the crop plants (Khaliq, Abbasi, and Hussain 2006). Preparation of organic fertilizer from agricul-
tural wastes is a very old practice in Asian countries as it improves soil physicochemical properties,
supplies nutrients to the crop plants and soil microbes, reduces solid waste, and ultimately restores
the environmental pollution (Barzegar, Yousefi, and Daryashenas 2002). Certain organic acids, such
as humic and fulvic acids, etc., are released during composting, increasing the solubility of RP.
Moreover, organic matter complexes and various metallic cations reduce P fixation in soil. It has
been found that inoculation of appropriate microbes can improve the quality of the organic fertilizer
and shorten the maturity period (Chen, Lin, and Yang 2007). Therefore, organic fertilizer that
produced through composting may serve as an ideal medium to increase the efficiency of RP and can
be employed for direct use of RP along with phosphate-solubilizing bacteria in alkaline soils as an
economical and eco-friendly phosphatic fertilizer.
Based on these assumptions, incubation (under laboratory condition) and pot studies (wire house
condition) were conducted with objectives to find out the comparative efficacy of RP-enriched
organic fertilizer with phosphate-solubilizing microorganisms (PSM1 and PSM2) in improving the
productivity of chickpea and lentil in comparison to chemical phosphatic fertilizer (control).
Moreover, the effect of RP-enriched organic fertilizer along with phosphate-solubilizing bacteria
on soil aggregate stability and P-release pattern and the microbial population at the end of incuba-
tion studies (28 days) was investigated and compared with control. The same treatments were tested
in pot study under wire house conditions.

Materials and methods

In a laboratory experiment, two pre-isolated bacterial strains Bacillus thuringiensis serovar ostriniae
(PSM1) and Bacillus sp. Cp-h60 (PSM2) (previously reported as KAP5 and KAP6, respectively) were
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 3

used (Baig et al. 2012). PSM2 had high auxin production and phosphate-solubilizing activities
(753.0 µg mL−1) but lower ACC-deaminase activity (1.53 µmol mL−1) compared with PSM1 which
had 698.5 µg mL−1 P-solubilizing and 1.85 µmol mL−1 ACC-deaminase activities.

Compost and RP-enriched organic fertilizer analysis

RP-enriched organic fertilizer was prepared according to Ditta et al. (2015). It was analyzed for
carbon contents using loss-on-ignition method (Nelson and Sommers 1996), total nitrogen (N)
contents by Kjeldahl distillation apparatus (Jackson 1962), vanadomolybdo-phosphoric yellow color
method was used for total P determination (Jackson 1973), and Olsen method was used for available
P (US Salinity Laboratory Staff 1954). Simple compost was analyzed by using the same standard
methods. All the analyses were performed using four replications. Moreover, pH and carbon to
nitrogen and phosphorus ratios were also calculated.

Incubation studies
Before conducting the pot trial, a series of incubation studies were conducted under axenic conditions
to investigate the effect of RP-enriched organic fertilizer and PSM on physicochemical and biological
properties of soil and P-release pattern. The details of these studies are as follows: An incubation study
was conducted to find out the effect of different combinations of RP, RP-enriched organic fertilizer, and
PSM on the P-release pattern and soil aggregate stability for 28 days under controlled laboratory
conditions. The RP-enriched organic fertilizer was applied at 0.2 g kg−1 soil (60–62 kg P2O5 ha−1).

P-release pattern
P-release pattern from different combinations of RP, RP-enriched organic fertilizer, and PSM was
investigated for 4 weeks under controlled laboratory conditions, using plastic beakers 500 mL with
250 g soil. The soil used in the study was analyzed for physicochemical properties as given in the
following sections. The RP-enriched organic fertilizer was applied at 0.2 g kg−1, containing 30 mg
P2O5 kg−1. Each sample was taken after an interval of 1 week. A beaker with 250 g soil that did not
receive any treatment was considered as the control. The treatments were arranged using a
completely randomized design in triplicate. All of the treatments were mixed thoroughly, distilled
water was added to moist soil, and all the beakers were incubated at 28 ± 1°C for 4 weeks. To find
out the available P for the plant growth released from each treatment was assessed after every 7 days,
and the soil from each treatment was analyzed by using Olsen method (US Salinity Laboratory Staff
1954). A standard curve was prepared by measuring the absorbance of standard solutions of P
(KH2PO4) from 1 to 5 ppm at 882 nm by using spectrophotometer (Watanabe and Olsen 1965).

Soil aggregate stability (%)

Wet sieving method was used to determine soil aggregate stability (Kemper and Rosenau 1986).
Different treatments in triplicate were mixed in soil by hand, arranged in completely randomized
design, and were incubated for 28 days at 30°C. After 28 days, all the crumbled soils were passed
through the sieve (8.0 mm), followed by wet sieving (1.0 mm) for 5 min in degassed distilled water
(180–250 g water kg−1 soil). The soil material remained in the opening of the sieve was then dried,
weighed, and expressed as the percent stable aggregate fraction.

Pot experiment
A pot trial was conducted during 2013 to evaluate the comparative efficacy of different combination of
RP (0.11 g kg−1; containing 28% P2O5) and RP-enriched organic fertilizer (0.2 g kg−1) with PSMs in
4 A. DITTA ET AL.

improving the productivity of chickpea (var. Noor-2009) and lentil (var. Punjab Masoor-2009) compared
to recommended chemical phosphatic fertilizer (30 mg P kg−1 soil). In all the treatments, the same
amount of P was added. The experiment comprises six treatments: T1 = control (P as diammonium
phosphate [DAP]), T2 = RP, T3 = DAP + compost + PSM1, T4 = DAP + compost + PSM2, T5 = RP-
enriched organic fertilizer + PSM1, and T6 = RP-enriched organic fertilizer + PSM2. The experiment was
conducted in a wire house at ambient light and temperature. The recommended rate of nitrogen and
potassium (NK) (12.5 N and 12.5 K mg kg-1 soil) and rhizobia was applied in all the treatments using
completely randomized design with six replications.

Inoculation procedure
The inoculum of each strain was prepared by growing in 250 ml Erlenmeyer flasks containing National
Botanical Research Institute’s Phosphate (NBRIP) medium [glucose, 10.0 g: calcium phosphate
(Ca3(PO4)2), 5.0 g; magnesium chloride (MgCl2.6H2O), 5.0 g; magnesium sulfate (MgSO4.7H2O),
0.25 g; potassium chloride (KCl), 0.2 g; and ammonium sulfate ((NH4)2SO4), 0.1 g L−1] (Nautiyal,
1999), for PSM and for rhizobia, N-free medium (For 500 cm3, 0.05 g ferrous chloride (FeCl3.6H2O), 2 g
dipotassium phosphate (K2HPO4), 0.25 g MgSO4.7H2O, 10 g glucose, 1 g calcium carbonate (CaCO3),
and 7.5 g agar). The flasks were incubated at 28 ± 1°C for 48 h in the orbital shaking incubator (Model
OSI-503 LD; Firstek Scientific, Taiwan) at 100 rev min−1. Chickpea and lentil seeds were surface sterilized
using ethanol (70%) for 2 min and with NaClO (5%) for 5 min, followed by three times washing with
sterilized distilled water. For inoculation, surface sterilized seeds of chickpea and lentil were coated with a
suspension of the strains, sugar solution (15%), and peat plus clay (1:1 w/w). The control was treated with
sterilized peat, sterilized broth, and sugar solution containing plus clay. Inoculated seeds were placed
under shade for drying.
The inoculated seeds were sown in plastic pots with 25 cm diameter and 23 cm height. Eight kilograms
of dried and sieved (2 mm) soil that were collected from the research area of the Institute of Soil and
Environmental Sciences (ISES), University of Agriculture Faisalabad (UAF), Pakistan, were filled in each
pot. Respective treatments were thoroughly mixed manually in the soil before sowing of inoculated
chickpea and lentil seeds. After proper germination, one plant in each pot was maintained till maturity.
A pre-soil analysis was shown that the soil used in the current study was alkaline (pH = 7.6) in nature, sandy
clay loam in texture, and low in organic matter (0.67%). Electrical conductivity of soil extract was 0.34 S m−1
with total N = 0.056%; available P, 8.6 mg kg−1; and extractable K, 110 mg kg−1 (US Salinity Laboratory Staff
1954). At the flowering stage (80 days after sowing), the plants were harvested in triplicate and nodules were
excised manually. The nodules were placed on filter paper to absorb any moisture and then the fresh and
dry weight was recorded using an electrical balance. The other three replications were allowed to grow until
maturity and were harvested after 120 days after sowing for growth and yield parameters. Nitrogen (N)
content was determined by the Kjeldahl method (Jackson 1962). Briefly, plant samples were ground to a
fine powder using a Wiley’s mill, and a known quantity of the ground material was digested by Wolf
method using H2SO4 and H2O2. Later, the digested samples were distilled using Kjeldahl apparatus, and N
was determined by titration with standard H2SO4 (Jackson 1962). For phosphorus, the digested samples
(5 mL) were mixed in Barton reagent (10 mL), and total volume was made 50 mL. The samples were kept
for half an hour for color development, and phosphorus contents were measured by a spectrophotometer
(Shimadzu, Japan) at λ 420 nm using standard curve (Jackson 1962). For the calculation of crude protein
contents, N concentration was multiplied by the conversion factor of 6.25 (AOAC 1990).

Statistical analysis
Analysis of variance (ANOVA) was calculated using computer software Statistix 8.1 (Analytical
Software, Tallahassee, Florida). The treatment means and standard error of means (SEMs) were
calculated using Microsoft® excel version 2016 (Microsoft Corp., Pullman, WA). The means were
compared by least significant difference (LSD) test at P < 0.05 (Steel, Torrie, and Dicky 1997).
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 5

Results
Soil aggregate stability improves the soil structure which ultimately enhances the water-holding
capacity of the soil and the availability of the nutrients to the plants. Figure 1 contains the results
about soil aggregate stability and combinations of RP and RP-enriched organic fertilizer with PSMs
under controlled laboratory condition. RP-enriched organic fertilizer along with PSM2 exhibited the
maximum soil aggregate stability (37%), followed by RP-enriched organic fertilizer with PSM1,
whereas the minimum soil aggregate stability was found where only RP was applied.
The release of P was substantially increased with the application of RP-enriched organic fertilizer
with PSM compared to the alone application of RP (Figure 2). Among all the treatments, a slow and
continuous increase in plant-available P (Olsen P) resulted in RP-enriched organic fertilizer with
PSM2, followed by RP-enriched organic fertilizer with PSM1. Application of RP alone showed the
minimum release of plant-available P. In case of DAP application, the P-release was increased in the
first 2 weeks and later on was decreased. After the fourth week of the incubation, the available P was
increased about 9.8 times higher when treated with RP-enriched organic fertilizer with PSM2 than
control, while RP-enriched organic fertilizer with PSM1 increased eight times as compared to the
alone application of RP.
The number of nodules plant−1 and fresh and dry weight of nodules of chickpea and lentil
were significantly enhanced with the application of RP-enriched organic fertilizer with PSMs
compared to the alone application of RP and recommended fertilizer (control) (Table 1).
Application of DAP + compost + PSM2 resulted in a maximum number of nodules plant−1
which were 23% and 14% more in chickpea and lentil, respectively, as compared to the control.
The same treatment exhibited the maximum fresh and dry weight of nodules (g) in both
chickpea and lentil plants, whereas the minimum number of nodules plant−1 was observed
with the application of RP alone.
RP-enriched organic fertilizer with PSMs also enhanced the growth and yield of both plants (Tables 2
and 3). The results about shoot lengths show that the application of DAP + compost + PSM2 exhibited
the maximum (17%) shoot length in chickpea plants, while 19% shoot length was increased in lentil when
treated with RP-enriched organic fertilizer + PSM2. On the other hand, application of RP alone exhibited
the minimum shoot length. A similar trend was also observed in 100-grain weight of chickpea and lentil.
Again, the application of RP-enriched organic fertilizer + PSM2 and DAP + compost + PSM2 treatments

40
Soil aggregate stability (%)

35

30

25

20

15

Figure 1. Impact of different combinations of RP, RP-enriched organic fertilizer, and DAP with PSMs on soil aggregate stability (%).
The error bars show standard error of means (SEMs) where n = 3.
6 A. DITTA ET AL.

45 Control (P as DAP)
Rock phosphate
DAP + compost + PSM1
40 DAP + compost + PSM2
RP-enriched organic fertilizer + PSM1
RP-enriched organic fertilizer + PSM2
35
P-released (mg kg-1)
30

25

20

15

10

0
7 14 21 28
Incubation time (days)

Figure 2. Impact of different combinations of RP, RP-enriched organic fertilizer, and DAP with PSMs on P-release in incubation
study under laboratory conditions.
The error bars show standard error of means where n = 3.

Table 1. The chemical analysis of raw organic fertilizer and composted rock phosphate-enriched organic fertilizer.
Parameters Raw organic fertilizer Composted RP-enriched organic fertilizer (50:50 w/w) Composted organic fertilizer
Carbon (g kg−1) 345 ± 3.2† 215 ± 5.5 255 ± 6.6
Nitrogen (g kg−1) 13.8 ± 0.1 15.3 ± 0.3 22.1 ± 0.3
Total P (g kg−1) 3.5 ± 0.06 123.0 ± 4.4 3.7 ± 0.9
Olson P (mg kg−1) 340 ± 10 2380 ± 30 450 ± 20
C:N 25.00 ± 0.34 14.05 ± 0.63 11.53 ± 0.18
C:P 98.57 ± 1.63 9.19 ± 0.18 54.26 ± 0.96
pH 5.94 ± 0.06 5.43 ± 0.03 5.46 ± 0.04
†Shows the standard error of means (SEMs) where n = 4.

Table 2. Impact of different combinations of RP, RP-enriched organic fertilizer, and DAP with PSMs on the number of nodules
plant−1 and fresh and dry weight of nodules plant−1 (g).
Number of nodules Fresh weight of nodules Dry weight of nodules
plant−1 (g) plant−1 (g) plant−1
Different combinations of RP, compost, and PSM Chickpea Lentil Chickpea Lentil Chickpea Lentil
Control (P as DAP) 54.3 c† 25.7 ab 2.31 bc 1.25 ab 0.69 c 0.27 bc
Rock phosphate 44.3 d 14.0 c 2.15 c 1.24 b 0.62 d 0.24 c
DAP + compost + PSM1 60.3 b 23.3 b 2.45 ab 1.28 ab 0.76 ab 0.29 ab
DAP + compost + PSM2 70.3 a 29.7 a 2.51 a 1.30 a 0.79 a 0.31 a
RP-enriched organic fertilizer + PSM1 66.7 a 25.7 ab 2.40 ab 1.29 ab 0.74 b 0.27 b
RP-enriched organic fertilizer + PSM2 69.0 a 27.7 ab 2.42 ab 1.30 ab 0.76 ab 0.29 ab
LSD at P = 0.05 4.58* 5.01* 0.17* 0.063* 0.041* 0.021*
†Means followed by the same letters in a column are not different at P < 0.05 according to least difference (LSD) test.
*Significant at P < 0.05.

proved to be better treatments for grain yield (g pot−1), and the increase was recorded about 15% and
31%, respectively, as compared to control when treated with said treatments. The application of
DAP + compost + PSM2 exhibited the maximum biological yield (fresh biomass) in both chickpea
(21.84 g pot−1) and lentil (11.36 g pot−1) plants. The maximum nitrogen (0.59%) and protein contents
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 7

Table 3. Impact of different combinations of RP, RP-enriched organic fertilizer, and DAP with PSMs on shoot, root length (cm), and
root dry weight (g).
Shoot length (cm) Root length (cm) Root dry weight (g) plant−1
Different combinations of RP, compost, and PSM Chickpea Lentil Chickpea Lentil Chickpea Lentil
Control (P as DAP) 42.7 c† 39.3 c 10.8 c 5.0 a 2.1 a 0.91 b
Rock phosphate 35.7 d 36.0 d 4.2 d 4.5 b 2.0 b 0.82 d
DAP + compost + PSM1 48.0 b 41.0 bc 10.9 b 5.0 a 2.1 a 0.88 c
DAP + compost + PSM2 51.7 a 47.3 a 11.1 a 5.2 a 2.2 a 0.92 b
RP-enriched organic fertilizer + PSM1 46.7 b 43.3 b 10.9 b 5.0 a 2.1 a 0.88 c
RP-enriched organic fertilizer + PSM2 48.3 b 48.7 a 11.0 ab 5.1 a 2.2 a 1.06 a
LSD at P = 0.05 3.20* 2.87* 0.13* 0.36* 0.075* 0.036*
†Means followed by the same letters in a column are not different at P < 0.05 according to least difference (LSD) test.
*Significant at P < 0.05.

(0.38%) in the straw of chickpea and lentil plants were recorded when treated with DAP + comp-
ost + PSM2 which were 11% and 20% more as compared to the control (Table 4). The treatment
DAP + compost + PSM2 accumulated the maximum nitrogen and protein contents in grains of chickpea
and lentil; however, the effect of the application of DAP or RP-enriched organic fertilizer with PSM2 was
statistically nonsignificant in lentil. The maximum P contents in the straw of chickpea (0.22%) and lentil
(0.21%) were recorded when treated with DAP + compost + PSM2 which were 42% and 56% higher as
compared to control. The maximum P contents in grain of chickpea (0.35%) and lentil (0.32%) were
recorded when treated with RP-enriched organic fertilizer + PSM2 and with DAP + compost + PSM2,
respectively, which were 13% and 23% higher as compared to the control.

Discussion
The chemical analysis of the finished product, i.e., RP-enriched organic fertilizer, showed an improve-
ment in the availability of N and P (Table 5). The production of organic acids during composting by
the microbes (PSMs) might be a reason to decrease the pH of the material being composted, i.e., RP
and compost, and resulted in an increased release of P from RP-enriched organic fertilizer (Rashid
et al. 2004). According to Jansson, Olsson, and Pettersson (1988), it may also be due to the production
of enzymes and chelating agents (organic acids) by the microbes which play a critical role in the
mineralization of organic P present in the compost. Similarly, reduction in pH of materials being
composted might also be due to the production of a large amount of carbon dioxide during
composting, which forms carbonic acids after combining with water (Bangar et al. 1989; Chien 1979).
Soil aggregate stability is an indirect measurement of soil aggregate resistance against physical
stresses, i.e., ability to maintain soil structure, etc. Higher aggregate stability shows that the soil
has a more water-holding capacity which ultimately increases the nutrient availability by the
crop plants. It also increases carbon storage ability of soil via protection of organic molecules

Table 4. Impact of different combinations of RP, RP-enriched organic fertilizer, and DAP with PSMs on the number of pods plant−1,
100-grain weight (g), grain yield (g pot−1), and fresh biomass (g pot−1).
Number of pods 100-grain weight Grain yield Fresh biomass
plant−1 (g) (g pot−1) (g pot−1)
Different combinations of RP, compost, and PSM Chickpea Lentil Chickpea Lentil Chickpea Lentil Chickpea Lentil
Control (P as DAP) 37.3 b† 19.3 c 11.6 ab 2.99 b 8.0 e 3.42 e 21.28 a 9.72 de
Rock phosphate 35.0 bc 10.7 d 11.5 b 2.64 c 7.6 f 3.12 f 19.56 b 9.48 e
DAP + compost + PSM1 38.7 b 30.0 b 11.7 ab 3.13 a 8.4 d 4.32 d 21.72 a 10.16 cd
DAP + compost + PSM2 44.0 a 36.3 a 11.9 a 3.19 a 8.8 c 4.96 a 21.84 a 11.36 a
RP-enriched organic fertilizer + PSM1 36.7 b 32.3 b 11.6 ab 3.15 a 9.2 b 4.64 c 21.40 a 10.48 bc
RP-enriched organic fertilizer + PSM2 43.3 a 36.0 a 11.8 ab 3.17 a 9.2 a 4.80 b 21.64 a 11.04 ab
LSD at P = 0.05 2.12* 3.12* 0.41* 0.11* 0.18* 0.14* 1.0* 0.56*
†Means followed by the same letters in a column are not different at P < 0.05 according to least difference (LSD) test.
*Significant at P < 0.05.
8 A. DITTA ET AL.

Table 5. Impact of different combinations of RP, RP-enriched organic fertilizer, and DAP with PSMs on N and P in straw and grains
(%) of chickpea and lentil.
Different combinations of N in straw (%) N in grain (%) P in straw (%) P in grain (%) Seed protein (%)
RP, compost, and PSM Chickpea Lentil Chickpea Lentil Chickpea Lentil Chickpea Lentil Chickpea Lentil
Control (P as DAP) 0.53 b† 0.30 d 2.32 d 1.74 c 0.13 c 0.09 cd 0.31 a 0.25 c 14.50 d 10.90 c
Rock phosphate 0.45 c 0.26 e 2.16 e 1.35 d 0.07 d 0.07 d 0.17 b 0.17 d 13.52 e 8.46 d
DAP + compost + PSM1 0.56 ab 0.32 cd 2.45 cd 1.84 b 0.18 b 0.17 b 0.33 a 0.26 bc 15.31 cd 11.52 b
DAP + compost + PSM2 0.59 a 0.38 a 2.70 a 1.90 a 0.22 a 0.21 a 0.35 a 0.32 a 16.85 a 11.85 a
RP-enriched organic 0.57 ab 0.35 bc 2.55 bc 1.86 ab 0.20 b 0.16 b 0.32 a 0.28 b 15.96 bc 11.60 ab
fertilizer + PSM1
RP-enriched organic 0.59 a 0.37 ab 2.64 ab 1.90 a 0.20 ab 0.17 b 0.35 a 0.29 b 16.52 ab 11.85 a
fertilizer + PSM2
LSD at P = 0.05 0.044* 0.029* 0.14* 0.051* 0.025* 0.032* 0.05* 0.027* 0.82* 0.31*
†Means followed by the same letters in a column are not different at P < 0.05 according to least difference (LSD) test.
*Significant at P < 0.05.

against physical stresses. Availability of more carbon in soil increases the organic matter which
increases seed germination and ultimately better yield (Jastrow and Miller 1997; Le Bissonnais
1996; Lynch and Bragg 1985). The maximum soil aggregate stability resulted in the treatment
where RP-enriched organic fertilizer + PSM2 were applied (Table 5). The reason behind this
phenomenon might be an increase in the supply of soluble carbon, which increases the organic
matter in the soil and ultimately more soil aggregates will be formed. Similar findings were
observed with the application of composted organic material and mycorrhizal inoculation
(Caravaca et al. 2002).
On the other hand, the adequate nutrient supply is essential for optimum utilization of nutrients and
ultimately for the crop yield (Baligar, Fageria, and He 2001). The growth and yield parameters were
significantly improved in chickpea and lentil when treated with RP-enriched organic fertilizer + PSM2 as
compared to control and application of RP alone. It might be due to the positive effect of PSM
inoculation on the nodules, which increases the supply of N to the crop plants and ultimately growth
and yield of both the legumes. It might also be due to the increase in the supply of P by the P-solubilizing
activity of PSMs via the production of organic acids and proton extrusion which indirectly increased the
nodulation and ultimately more N was fixed (Glick, Penrose, and Li 1998; Surange et al. 1997). Moreover,
the organic material served as a better source of carbon and nutrients for the microbes (indigenous and
inoculated) as well as for the crop plants, and source of continuous supply of essential nutrients
(Figure 2) to the crop plants, and ultimately led to improve the growth and yield (Nevens and Reheul
2003; Verma, Penfold, and Marschner 2013). Better nutrient supply to the plants also improved N and P
contents in straw and grain samples of both chickpea and lentil (Table 4).
Abiotic and biotic stresses produce ethylene, and it causes stress on the plant root growth (Arshad
and Frakenberger 2002; Suganuma, Yamauchi, and Yamamoto 1995). Ethylene stress could be
ameliorated via the application of microbes with ACC-deaminase activity. The microbes with this
activity are able to reduce the ethylene production as these can convert ACC into ammonia and α-
ketobutyrate and ultimately reduce ethylene stress (Glick, Penrose, and Li 1998). The PSMs used in
the study had higher ACC-deaminase activity which might have reduced this stress in roots and
ultimately increase root growth as clear from the data in Table 2. Similar results were found in alfalfa
(Ma, Charles, and Glick 2004) and in mung bean (Shaharoona, Arshad, and Zahir 2006).
The N, P, and protein contents of straw and grain in chickpea and lentil were improved with the
application of RP-enriched organic fertilizer. It might be due to an increase in the supply of nutrients
through composted RP-enriched organic fertilizer and better nodulation and root growth via ACC-
deaminase activity of PSMs applied (Yang, Kloepper, and Choong-Min 2009). Similar improvement
in N, P, and K was also reported by various researchers around the world (Ditta and Khalid 2016;
Ditta et al. 2018; Farooq et al. 2018; Salimpour et al. 2010; Shahzad et al. 2008; Zabihi et al. 2011).
 COMMUNICATIONS IN SOIL SCIENCE AND PLANT ANALYSIS 9

Regarding the comparative efficacy of both the strains used in the study, PSM2 (Bacillus sp. Cp-
h60) performed better which might be due to its high colonization ability as clear from Figure 1 and
high auxin production activity (Baig et al. 2012) compared to PSM1 (Bacillus thuringiensis serovar
ostriniae). Other traits like high P-solubilizing and ACC-deaminase activities of the strain PSM2
(Bacillus sp. Cp-h60) might have helped in the better acquisition of nutrients through increased
solubilization and ameliorating the ethylene stress.

Conclusions
Application of RP-enriched organic fertilizer with PSM2 resulted in the maximum productivity of
chickpea and lentil via improving nodulation, growth, and yield parameters compared with chemical
phosphatic fertilizers (control). RP-enriched organic fertilizer with PSM2 could provide a sustainable
source of P for optimum production of legumes in alkaline soils. However, more field experiments
with different crops are required to warrant a successful performance.

Acknowledgement
We are also thankful to Dr Khurram S. Baig for giving us bacterial strains used in the study.

Funding
Financial support for this study was provided by the Higher Education Commission of Pakistan, under the
Indigenous PhD Fellowship Program (PIN #: 074-2995-AV4-029).

Competing interests
None of the authors have any competing of interest.

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