1. No category

Vol. 2 (3), pp. 168-174, October, 2014. ©
Global Science Research Journals
http://www.globalscienceresearchjournals.org/
Global Journal of Agriculture and Agricultural Sciences
Full Length Research Paper
Use of N and P biofertilizers together with phosphorus
fertilizer Improves growth and physiological attributes
of chickpea
Moinuddin1, Tariq Ahmad Dar*2, Sajad Hussain2, M. Masroor Akhtar Khan2, Nadeem Hashmi2,
Mohammad Idrees2, Mohammad Naeem2, Akbar Ali2
1
Women’s College, Botany Section, Aligarh Muslim University, Aligarh-202002, U.P., India
2
Department of Botany, Aligarh Muslim University, Aligarh-202002, U.P., India
Accepted 10 October, 2014
Abstract
Leguminous crops grow poorly in phosphorus deficient soils. A two factor factorial experiment was
-1
conducted in the net-house to investigate the effect of graded levels of P fertilizer (0, 30 and 60 kg P ha
or P0, P30 and P60, respectively) along with Rhizobium (BNF) and/or phosphate solubilizing bacteria
(PSB) on growth and physiological parameters of chickpea. Phosphorus was applied as basal dose,
while seeds were treated with respective biofertilizer(s) before sowing according to the treatments [BF 0
(control), BNF, PSB and BNF+PSB]. As per the main effects, P60 proved superior or equivalent to P30,
while among the biofertilizer treatments, BNF+PSB gave the greatest values or was equal to PSB
regarding most growth and physiological parameters. Generally, interaction between P levels and
biofertilizer treatments was significant. 30 kg P ha-1 applied with N and P biofertilizers (P30 × BNF+PSB)
was the cost-effective interaction for most of the parameters studied. Compared to P 60 applied alone
(P60 × BF0), P30 × BNF+PSB resulted in greater dry matter production (10.36%), PN(22.96%),
leghemoglobin content of root nodule (47.53%) and nitrate reductase activity (16.67%). In addition, P30 ×
BNF+PSB was statistically equal to P60 × BNF+PSB regarding dry matter production, net
photosynthesis, leghemoglobin content of root nodule and leaf nitrate reductase and carbonic
anhydrase activities. Thus, compared to P60, application of P30 × BNF+PSB proved to be more profitable
to attain the enhanced values of crop growth attributes and physiological parameters required for the
improved crop yield and quality.
Key words: Cicer arietinum, growth and physiological attributes, phosphorus levels, Pseudomonas striata,
Rhizobium ciceri, phosphorus-deficient soil
INTRODUCTION
Chickpea (Cicer arietinum L.) is the third most widely
grown grain legume in the world after bean and soybean
(Soltani et al., 2006). It occupies an important role in
*Corresponding Author: Tariq Ahmad Dar, Plant Physiology Section,
Department of Botany, Aligarh Muslim University, Aligarh, 202 002 India
E-mail: [email protected], Tel: +91-8755643233
human nutrition due to its high protein content (17-23%)
and because of being a good source of carbohydrates,
minerals and trace elements (Namvar and Sharifi, 2011).
Millions of people of developing countries, who cannot
afford the costlier animal protein, consume chickpea in
large amounts in their diet (Huisman and Poel, 1994). It is
also used as feed for livestock and has a significant role
in farming systems (Singh, 1997).
Glob. J. Agric. Agric. Sci.
169
Phosphorus is (P) the second limiting plant nutrient after
nitrogen (Rudresh et al., 2005). Soils usually contain a
high amount of total P, but its availability to plant is very
low. As a result, costly phosphatic fertilizers are applied
to soil for leguminous crops, which require large amounts
P for proper growth and development (Singh, 1983).
Photosynthesis and stomatal conductance are reduced
by P deficiency (Guidi et al., 1994). On the contrary,
increased P supply has been reported to increase
photosynthesis (Gao et al., 1989). In leguminous crops, P
promotes nodulation, dinitrogen fixation and efficient
partitioning of photosynthates between source and sink
(Giaquenta and Quebedeaux, 1980).
Biofertilizers are living microorganisms, which when
applied through seed or soil treatment, promote growth by
increasing the supply or availability of nutrients to the host
plant (Stephens and Rask, 2000; Moin Uddin et al., 2014). In
plants, they also increase the content of growth hormones
such as IAA and GA, leading to enhancement in the growth
of plants (Asad et al., 2004; Selvakumar et al., 2009). These
biofertilizers include the N2 fixing, phosphate solubilizing and
plant growth promoting microorganisms (Mahdi et al., 2010).
Rhizobium bacteria (BNF) through biological N2 fixation,
meet about 80-90% of total N requirements of legumes
(Verma, 1993). Phosphate solubilizing microorganisms
play a key role in the plant metabolism and crop
productivity. They are known to increase P uptake and
overall P-use efficiency resulting in better growth and
higher yield of crop plants (Alagawadi and Gaur, 1988;
Rudresh et al., 2005). The combined inoculation of
Rhizobium and phosphate solubilizing bacteria has been
reported to increase the nodulation, growth and yield
parameters in chickpea (Sattar and Gaur, 1987;
Alagawadi and Gaur, 1988; Rudresh et al., 2005). As a
matter of fact, supply of N and P biofertilizers along with
inorganic P fertilizer could play an important role in
manifestation of improved nutrient uptake and enhanced
crop yield and quality of chickpea in a cost-effective
manner (Moin Uddin et al., 2014). The present results are
the part of this experiment aimed at exploring the effect of
graded levels of P applied with N and P biofertilizers on
growth attributes, physiological parameters, nutrient
uptake, yield and quality of chickpea.
MATERIAL AND METHODS
Pot culture
The pot experiment was conducted on chickpea in a nethouse under natural conditions at the Botany
Department, Aligarh Muslim University, Aligarh (India)
using N and P deficient soil. Climatic conditions, soil
characteristics and seed treatment with N and P
biofertilizers regarding the pot experiment have earlier
been reported (Moin Uddin et al., 2014). Soil was basally
dressed with three P levels viz. 0, 30 and 60 kg P ha-1
(P0, P30 and P60, respectively), while four biofertilizer
treatments (0, BNF, PSB, and BNF+PSB) were applied to
the seeds prior to planting. Each treatment was replicated
four times. Each experimental pot carried six healthy
plants.
Growth attributes
Growth attributes were determined at 90 days after planting
(DAP). Two randomly selected plants were uprooted carefully
from each treatment-replicate to measure growth attributes
(plant height, number of branches and leaves per plant, and
fresh and dry weight per plant) and leghemoglobin content in
the root-nodules. The uprooted plants were washed thoroughly
with tap water to remove the adhering dust (from the shoot) and
soil particles (from the root). The root-nodules of the plants
were again washed with distilled water and stored fresh in
polythene bags for the estimation of leghemoglobin content.
The clean plants (above ground shoots) were blot-dried and
o
measured for plant fresh weight. They were dried at 80 C for 24
h, recording the dry weight of the plants thereafter.
Photosynthetic Parameters and Transpiration Rate
Photosynthetic parameters net photosynthesis (PN),
stomatal
conductance
(gs)
and
internal
CO2
concentration) and transpiration rate (E) were measured
at 90 DAP on cloudless day at 1000-1100 hours using
the youngest fully expanded leaves with the help of an
Infra Red Gas Analyzer (IRGA, Li-Cor 6400 Portable
Photosynthesis System, Lincoln, Nebraska, USA).
Activity of nitrate reductase (NR) and carbonic
anhydrase (CA)
The NR (E.C. 1.6.6.1) activity in the leaves was
determined by the intact tissue assay method (Jaworski
1971). The enzyme activity was expressed as nM NO2 g-1
leaf FW h-1. The activity of CA (E.C. 4.2.1.1) was
measured in the leaves using the method described by
Dwivedi and Randhawa (1974). The enzyme activity was
-1
-1
expressed as μM CO2 kg leaf FW s .
Leghemoglobin content
Leghemoglobin (Lb) content in the fresh-stored nodules
was determined as described by Sadasivam and
Manickam (2008). The Lb content in the fresh nodules
was calculated using the following formula: Lb content
(mM) = [(OD 556 – OD 539)/23.4] × 2D.
Where OD 556 and OD 539 represent absorbance
values recorded at 556, 539 nm, respectively, and D is
the initial dilution.
Statistical Analysis
Statistical analyses of the data were carried out
according to randomized block design. All the parameters
were subjected to analysis of variance (ANOVA), using
Tariq et al.
Table 1: Effect of graded levels of phosphorus applied with N and P biofertilizer treatments on growth parameters of chickpea
Treatments
Height per plant Number
of Number of leaves Fresh weight per Dry weight per
(cm)
branches per plant
per plant
plant (g)
plant (g)
Main effects of phosphorus (P)
b
b
b
c
c
P0
24.84
4.0
54.4
2.54
0.835
a
a
a
b
b
P30
25.91
4.3
56.1
2.86
0.870
a
a
a
a
a
P60
26.52
4.3
56.1
2.99
0.905
Main effects of biofertilizers (BF)
c
b
c
BF0
23.83
4.0
52.7
2.43
0.760
b
b
b
BNF
25.85
4.3
55.2
2.47
0.867
b
a
a
BPF
26.34
4.3
55.4
3.14
0.926
a
a
a
BNF+BPF
27.00
4.3
58.8
3.14
0.927
Effects of interaction (P × BF)
bc
e
e
d
P0 × BF0
23.00
4.0
52.1
2.30
0.679
b
de
e
d
P30 × BF0
23.50
4.0
53.0
2.40
0.752
ab
de
de
c
P60 × BF0
25.00
4.0
53.0
2.60
0.849
b
d
e
c
P0 × BNF
24.95
4.0
54.3
2.30
0.851
a
d
e
c
P30 × BNF
26.40
4.5
55.0
2.35
0.855
a
c
d
b
P60 × BNF
26.20
4.5
56.3
2.75
0.896
ab
d
b
P0 × BPF
25.50
4.0
54.3
2.65d
0.901
a
cd
a
a
P30 × BPF
26.13
4.5
55.3
3.35
0.937
a
c
a
a
P60 × BPF
27.40
4.5
56.7
3.43
0.939
ab
c
c
b
P0 × BNF+BPF
25.90
4.0
57.0
2.90
0.908
a
a
a
a
P30 × BNF+BPF
27.60
4.5
61.0
3.35
0.937
a
b
b
a
P60 × BNF+BPF
27.50
4.5
58.5
3.17
0.937
Values followed by the same letter in a column section are not significantly different according to Fisher’s Least Significant Difference
-1
-1
(LSD) at p<0.05. P0: no phosphorus application (control), P30: 30 kg P ha , P60: 60 kg P ha , BF0: no biofertilizer application (control),
BNF: biological N fertilizer (Rhizobium ciceri); BPF: biological phosphorus fertilizer (Pseudomonas striata).
LSD (P<0.05)
Height per plant
Number of branches per plant
Number of leaves per plant
Fresh weight per plant
Dry weight per plant
Phosphorus (P)
0.829
0.235
0.753
0.071
0.014
Biofertilizer
NS
NS
0.870
0.082
0.016
P × Biofertilizer
1.657
NS
1.507
0.142
0.028
Table 2. Effect of three P levels with respect to four biofertilizer treatments on photosynthetic parameters of chickpea
Treatments
PN(µM
-2
-1
CO2 m s )
Internal
CO2
concentration
(ppm)
Stomatal
conductance
-2 -1
(µM m s )
Transpiration
-2 -1
mol m s )
rate
(m
Main effects of phosphorus (P)
b
b
b
b
P0
5.23
313
237
6.48
a
a
a
a
P30
5.88
320
254
7.08
a
a
a
a
P60
5.62
326
256
7.02
Main effects of biofertilizers (BF)
c
c
d
BF0
4.86
320
236
5.26
b
c
c
BNF
5.54
320
239
7.18
a
b
b
BPF
5.95
320
257
7.41
a
a
a
BNF+BPF
5.95
321
264
7.60
Effects of interaction (P × BF)
bc
c
f
P0 × BF0
4.54
305
227
5.19
b
c
f
P30 × BF0
4.89
320
237
5.23
b
bc
e
P60 × BF0
5.14
334
243
5.37
b
cd
d
P0 × BNF
5.10
314
220
6.54
a
bc
b
P30 × BNF
5.95
320
240
7.48
ab
b
b
P60 × BNF
5.58
325
258
7.52
ab
b
d
P0 × BPF
5.67
315
253
7.05
a
b
a
P30 × BPF
6.35
320
255
7.80
ab
b
c
P60 × BPF
5.82
325
262
7.37
ab
b
d
P0 × BNF+BPF
5.60
320
249
7.15
a
a
a
P30 × BNF+BPF
6.32
321
283
7.82
a
b
a
P60 × BNF+BPF
5.93
322
260
7.83
-1
-1
P0: no phosphorus application (control); P30: 30 kg P ha ; P60: 60 kg P ha . BF0: No biofertilizer application (control); BNF:
Biological N fertilizer (Rhizobium ciceri); BPF: Biological phosphorus fertilizer (Pseudomonas striata).
LSD (P<0.05)
Net photosynthesis
Internal CO2 Conc.
Stomatal conductance
Transpiration rate
Phosphorus (P)
0.239
6.742
5.000
0.060
Biofertilizer
0.276
NS
6.000
0.069
P × Biofertilizer
0.479
NS
11.000
0.120
170
Glob. J. Agric. Agric. Sci.
171
Table 3. Effect of three phosphorus levels with respect to four biofertilizer treatments on biochemical parameters of chickpea.
Treatments
Nitrate reductase activity
Carbonic anhydrase activity
Leghemoglobin
-1
-1
-1
-1
-1
(n mol NO2 g FW h )
(m mol CO2 Kg FW s )
content (mg g )
Main effects of phosphorus (P)
b
b
c
P0
247
1105
2.51
a
a
b
P30
272
1133
2.93
a
a
a
P60
276
1143
3.03
Main effects of biofertilizers (BF)
b
ab
d
BF0
238
1106
2.14
a
a
c
BNF
273
1131
2.86
a
a
a
BPF
273
1136
3.02
a
a
a
BNF+BPF
277
1135
3.28
Effects of interaction (P × BF)
c
ab
d
P0 × BF0
231
1100
2.06
bc
a
cd
P30 × BF0
237
1102
2.12
b
a
c
P60 × BF0
246
1117
2.23
b
ab
c
P0 × BNF
252
1100
2.36
a
a
b
P30 × BNF
278
1142
3.00
a
a
a
P60 × BNF
289
1152
3.23
b
a
c
P0 × BPF
250
1110
2.38
a
a
a
P30 × BPF
287
1142
3.30
a
a
a
P60 × BPF
283
1155
3.37
b
a
a
P0 × BNF+BPF
257
1110
3.24
a
a
a
P30 × BNF+BPF
287
1146
3.29
a
a
a
P60 × BNF+BPF
286
1150
3.31
-1
-1
P0: no phosphorus application (control); P30: 30 kg P ha ; P60: 60 kg P ha .
BF0: No biofertilizer application (control); BNF: Biological N fertilizer (Rhizobium ciceri); BPF: Biological phosphorus fertilizer
(Pseudomonas striata).
LSD (P<0.05)
Nitrate reductase activity
Carbonic anhydrase activity
Leghemoglobin content
Phosphorus (P)
6.913
27
0.090
two-factor factorial procedure. Fisher’s least significant
difference (LSD) was used to test the significance at the 5%
probability level. The data were analyzed using SPSS-17
(SPSS Inc., Chicago, IL, USA).
RESULTS
Growth Parameters
As per the main effects of P, progressive application of P
enhanced the plant fresh and dry weight gradually, with P 60
proving the best. For rest of the growth attributes, P 30 and
P60 gave statistically equal values. However, P30 as well as
P60 proved invariably better than P0 (control) for all the
growth characteristics studied (Table 1). Considering the main
effects of biofertilizer treatments, the maximal values of the
growth attributes were generally shown by BNF+BPF and/or
BPF compared to the control (BF0), which always gave the
poorest results. Interaction of inorganic P fertilizer and
biofertilizer was significant for most of the growth parameters. In
general, P30 × BPF+BNF resulted in the highest values.
However, for fresh and dry weight per plant, the maximum
values were attained with P60 × BPF, which was statistically at
par with P30 × BPF and/or P30 × BPF+BNF.
Photosynthetic Parameters and Transpiration Rate
Main effects of P application resulted in enhanced values of
net photosynthetic rate (PN), stomatal conductance (gs) ,
Biofertilizer
7.983
29
0.110
P × Biofertilizer
13.826
53
0.180
internal CO2 concentration and transpiration rate (E)
compared to the control (P0) (Table 2). Level P30 resulted
in the highest extent of PN; while P60, being statistically at
par with P30, gave the highest stomatal conductance,
internal CO2 concentration and transpiration rate.
Treatment P30 surpassed the P0 by 12.42, 2.24, 7.17, and
9.26% in PN, internal CO2 concentration, gs and E
respectively. As per the main effects of biofertilizer
treatments, BNF+BPF resulted in the highest PN, gs, and
E, with BNF+BPF excelling the control (BF 0) by 22.42,
11.86, and 44.49%, respectively. Interaction P30 × BPF
and P30 × BNF+BPF enhanced the PN to the highest
extent. In addition, P30 × BNF+BPF was most
advantageous interaction for ‘gs’ and ‘E’. It exceeded the
control (P0 × BF0) by 29.24, 28.64, and 50.67% for ‘PN’,
‘gs’ and ‘E’, respectively. The effect of biofertilizer
treatments and that of interaction between biofertilizer
treatments and P levels was not significant regarding the
internal CO2 concentration.
Activity of Nitrate Reductase (NR) and Carbonic
Anhydrase (CA)
In relation to the main effects of phosphorus, P60 as well
as P30 accounted for the greatest extent of NR and CA
activities. Level P30 exceeded P0 by 10.12 and 2.53% in
NR and CA activity, respectively (Table 3). As for the
main effects of biofertilizer treatments, BNF+BPF was
Tariq et al.
equal to BNF and BPF, registering the highest levels of
NR and CA activities compared to BF0. BNF+BPF
resulted in 16.38 and 2.62% increase in the NR and CA
activity, respectively, over no biofertilizer application
(BF0). Several of the P × biofertilizer interactions,
including P30 × BNF+BPF and P60 × BNF+BPF, resulted
in statistically equal values for NR and CA activities,
exceeding the lowest interaction (P0 × BF0) significantly.
Leghemoglobin Content
This study revealed a significant effect of phosphorus and
N & P biofertilizers on Leghemoglobin content of root
nodules (Table 3). Regarding the main effects of P, level
P30, equaled with P60, exhibited the highest
leghemoglobin content in the root nodules. It excelled the
control (P0) by 16.73%. In consideration with the main
effects of biofertilizer treatments, BNF+BPF resulted in
the maximum content of leghemoglobin, exceeding the
control (BF0) by 53.27%. Interaction P60 × BPF, which
was statistically equal to P30 × BNF+BPF and several
other interactions, resulted in the highest leghemoglobin
content. P60 × BPF and P30 × BNF+BPF exceeded the
lowest interaction (P0 × BF0) by 63.59 and 59.71%,
respectively.
DISCUSSION
Growth Parameters
Growth of plant organs depend on proper supply of
mineral nutrients, including N and P that play important
role in growth and many other physiological processes of
plants (Moorby and Besford, 1983). Of the
macronutrients, P is required in large amounts
specifically by legumes for their proper growth and
development (Wan et al., 1991). Hence, the role of P in
promoting the chickpea growth attributes, as observed in
this study (Table 1), was expected. In this connection, the
present results resemble with those obtained by different
workers regarding chickpea (Yahiya and Samiullah,
1995; Bahadur et al., 2002; Pathak et al., 2003).
In line with our investigation, there was observed a
positive effect of dual inoculation (BNF+BPF) on the
growth of chickpea (Sonoboir and Sarawgi, 2000)
Soybean (Fatima et al., 2006), Lentil (Kumar and
Chandra, 2008) and black gram (Vigna mungo L.)
(Selvakumar et al., 2009). The investigations employing
biofertilizers to improve growth attributes of chickpea
(Saraf et al., 1997; Dutta and Prohit, 2009; Nishita and
Joshi, 2010; Namvar and Sharifi, 2011) and other
leguminous crops (Naeem and Khan, 2005; Fatima et al.,
2006; Selvakumar et al., 2009; Selvakumar et al., 2012)
also substantiate the present findings in this regard.
172
Photosynthetic Parameters and Transpiration Rate
In this study, P30 proved to be the optimum P level,
exhibiting the greatest values for all the photosynthetic
parameters as well as for transpiration rate (E) (Table 2).
These results are confirmed by the findings of other
researchers, who observed a positive effect of P
application on several of these parameters regarding
soybean (Fredeen et al., 1990), groundnut (Hossaini and
Hamid, 2007) and cluster bean (Burman et al., 2009).
Increased PN (due to P application could be due to the
prompt and adequate supply of carbon dioxide to the
mesophyll cells of the leaves that is evident by the Penhanced internal CO2 concentration and stomatal
conductance. Similarly, the P-improved ‘E’ recorded in
the plants supplied with P30/P60 could also be expected
because P application also enhanced the ‘gs’ in the
present study (Table 2). In line with our investigation,
significant decrease in ‘PN’ and ‘gs’ was reported due to P
deficiency in sunflower and soybean (Guidi et al., 1994).
Besides, there was observed enhanced photosynthesis
as a result of P supply to tobacco (Gao et al., 1989).
In this study, BNF+BPF proved to be the most
beneficial biofertilizer treatment for ‘PN’, ‘gs’ and
transpiration rate. Not many references are available on
the effect of biofertilizers on photosynthesis and the
related
parameters.
However,
enhancement
in
photosynthetic efficiency of green gram (Vigna radiata)
due to N biofertilizer, as noted by Sharma (2001), could
be considered in line with the present results in this
regard. Interaction P30 × BNF+BPF, equaled by certain
other interactions, enhanced the net photosynthesis, ‘gs’
and ‘E’ to the greatest extent. Accordingly, this interaction
resulted into the highest yield of chickpea reported
elsewhere (Moin Uddin et al., 2014).
Activity of Nitrate Reductase (NR) and Carbonic
Anhydrase (CA)
The activity of NR in plants is influenced by different
growth conditions including not only the environmental
factors such as light and temperature, but also by the
application of mineral fertilizers, particularly P (Oaks,
1985). The presence of P in the nutrient solution has
earlier been reported to induce greater nitrate
assimilation in corn (de Magalhaes et al., 1998) and
Phaseolus vulgaris L. (Gniazdowaska et al., 1999). In this
regard, our results are in agreement with those of Naeem
and Khan (2005) in the case of Cassia tora.
Carbonic anhydrase is known to have its important role
in photosynthesis, which is obvious by its presence in all
photosynthesizing tissues (Taiz and Zeiger, 2006). It
catalyzes the reversible hydration of CO2, thereby
increasing its availability to the photosynthetic enzyme
RuBisCO (Badger and Price, 1994). The improvement in
CA activity in this study (Table 3) could be as a result of
Glob. J. Agric. Agric. Sci.
173
adequate availability of N and P at the site of their
metabolism, owing to the application of P and the N and
P biofertilizers. A plausible cause for the enhancement of CA
activity due to application of inorganic P and P- biofertilizer
might the positive influence of P availability to plants or the de
novo synthesis of CA as argued by (Okabe et al. 1980).
Leghemoglobin Content
In accordance with this investigation (Table 3), the
increase in leghemoglobin content of the root nodules
might be due to the improved availability of P to the root
nodules due to combined application of inorganic P
fertilizer and N and P biofertilizers (P30 × BNF+BPF).
Similar to our results, there was observed a positive
effect of P application on nodule leghemoglobin content
in Lablab purpureus (Santhaguru and Hariram, 1998) and
Cassia tora (Naeem and Khan, 2005). In conformity with
our study, there was noted beneficial effect of inorganic P
fertilizer as well as that of N and P biofertilizers on
leghemoglobin content in chickpea by Dutta and Prohit
(2009).
The greatest content of leghemoglobin in root nodules
(N2 fixation) and maximum level of PN and the activities of
CA and NR in the leaves in this study might be the
reason for the enhanced yield and quality of chickpea
reported elsewhere (Moin Uddin et al., 2014).
CONCLUSION
Phosphorus application improved all the growth and
physiological attributes studied compared to no P
application (P0), with P30 and P60 being statistically equal
in most cases. Application of N and P biofertilizers
increased the values of most of the parameters studied
significantly compared to no biofertilizer application (BF0).
Of the biofertilizer treatments, BNF+BPF resulted in the
highest values almost invariably. P30 × BNF+BPF was the
most profitable interaction between inorganic P levels
and biofertilizer treatments for most growth and
physiological attributes as it gave the greatest values;
hence, it could be realized as the optimum combination of
inorganic P level and biofertilizer treatment for chickpea
growth and metabolism.
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