J. Env. Bio-Sci., 2015: Vol.

29 (2):383-387
(383) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

THE COMBINED USE OF CHEMICAL FERTILIZERS WITH MULTI -TRAIT

PSEUDOMONAS FLUORESCENS TO ENHANCE BELL PEPPER GROWTH
Anurag Yadav1*, Kusum Yadav2 and Shelly Gupta3
1*
Department of Microbiology, College of Basic Science & Humanities, S.D. Agricultural University, S.K. Nagar, Gujarat
2
Department of Biochemistry, University of Lucknow, Lucknow, Uttar Pradesh
3
Department of Microbiology, Division of Life Sciences, S.B.S. (PG) Institute of Biomedical Sciences and Research,
Balawala, Dehradun
[Corresponding author E-mail1*: anuragyadav123@gmail.com]

Received: 25-07-2015 Accepted: 06-11-2015

An experiment was conducted at the S.B.S. (PG) Institute of Biomedical Sciences and Research, Uttarakhand, India to study the
effect of co-treatment of Pseudomonas fluorescens Tf5 with five different chemical fertilizers on bell pepper (Capsicum annuum
var annuum L., California wonder) plants. The experiment was based on complete randomized block design pattern with five
replications. The rhizosphere of fenugreek (Trigonella foenum-graecum L.) plant was screened for efficient plant growth promoting
fluorescent pseudomonad biovars. Of the total 139 bacterial isolates with fluorescent colonies, ten biovars of P. fluorescens were
identified. These isolates were screened for plant growth promoting properties. Growth promoting traits like IAA production,
phosphate solubilization and ammonia solubilization were used in screening. The most efficient biovar (P. fluorescens Tf5) was
selected to check its effect on bell pepper growth. Treatments of sodium nitrate, diammonium phosphate, potash, superphosphate
and urea with or without P. fluorescens Tf5 treatment were provided to bell pepper plants. Results show that with chemical
fertilizer treatment the highest (72.28 cm) and the lowest (33.07 cm) plant length (shoot and root) was obtained from Diammonium
phosphate (DAP) and control, respectively. The highest (5.68 g) and lowest (2.53 g) plant dry weigh was recorded with DAP and
control respectively. Application of P. fluorescens Tf5 along with chemical fertilizers significantly enhanced plant height fresh,
weight and dry weight. In a separate experiment spectrophotometric analysis showed that enhancing the fertilizer concentration
negatively affects P. fluorescens Tf5 growth. Maximum bacterial growth retardation, in the form of optical density (OD) drop, was
observed with urea treatment.

Bell pepper (Capsicum annuum) L. is one of the main
commercial and export-oriented crop of India and is known for
its tangy taste, low calorific value and is a good source of
vitamin A, C and E. Like any other crop the bell pepper
production in India widely relies on the use of chemical
fertilizers. Chemical fertilizers are known to increase plant
growth, provide drought tolerance and resistance to frost to
plant. They are also known to help plants resist an attack of
pests and diseases.
biofertilizers have proven to be inexpensive and ecofriendly
way of crop improvement with no harmful effects on human
populations. Therefore, it is becoming increasingly relevant to
moderate the use of synthetic fertilizers by co-treating plants
with growth promoting bacteria3-4. But lesser is known about
the impact of chemical fertilizers on bell pepper. This study
has been undertaken with an objective to study the effect of
co-treatment of P. fluorescens Tf5 with chemical fertilizers on
bell pepper growth.

But from past few decades, the unprecedented application of MATERIAL AND METHODS
fertilizer has resulted in disturbance of soil nature and
The study was conducted at Balawala region of Dehradun,
associated microflora. Since chemical fertilizer application is
India (30° 25'49" N, 78°11'26" E) in SBSPGI, campus polyhouse
a costly input, every effort needs to be taken to enhance their
during March to May, during which the region received average
efficiency1. Moreover, the use of chemical fertilizers has
rainfall of 42.6 mm and relative humidity of 57% with average
reached to the level that beyond which no further crop yield
temperature of 21.8°C.
enhancement is possible2. Now-a-days, certain species of
In situ effect of fertilizer concentrations on bacterial
microorganisms are being used as biofertilizers as an
growth in growth medium: The In situ bacterial growth effect
alternative to chemical fertilizers. In their brief tenure of use,
by chemical fertilizer treatment was noticed using nutrient
NAAS Rating (2016)-4.20
 THE COMBINED USE OF CHEMICAL FERTILIZERS WITH MULTI - TRAIT
broth as a growth medium. The fertilizer concentrations @ 4
mg, 8 mg, 12 mg, 16 mg, and 20 mg, respectively per 100 mL
broth. Ten mL of broth from each of the fertilizer concentrations
was transferred in culture tubes. Bacterial suspension was
prepared by inoculating a loopful of culture in 10 mL sterile
distilled water. The culture tubes containing nutrient broth with
varying fertilizer concentrations were inoculated with 0.1 mL
of P. fluorescens Tf5 suspension. The tubes were incubated
at 27°C for 24 h. After incubation the optical density of
inoculated nutrient broth was measured using
spectrophotometer at 550 nm wavelength.
Bacterial isolation and identification: The young fenugreek
plants were carefully uprooted and the rhizosphere soil attached
to the roots was collected and serially diluted. The dilutions
were spread on King's - B agar plates, incubated for 24 h at
28°C and developed fluorescent colonies were observed for
fluorescence4. The isolates were identified with the help of
Bergey's manual of determinative bacteriology6. Of the total
139 bacterial isolates with fluorescent colonies, ten were
identified as P. fluorescens. Out of them, P. fluorescens Tf5
was selected for further study because this biovar displayed
best plant growth promoting traits among all other isolates
(Table-1). This biovar was IAA, HCN and catalase producer,
phosphate solubilizer and possessed antifungal properties.
Seed germination: Healthy seeds of bell pepper of variety
California wonder were obtained from local market. The seeds
were immersed in distilled water and soaked in 0.5 % (w/v)
solutions of sodium nitrate, potash, diammonium phosphate,
potash, urea and superphposphate solutions, respectively, for
7 hours. The soaked seeds were taken out and washed with
distilled water and sowed in the Petri plates containing water
agar medium.
Experimental design and setup: A study was laid out in
completely randomized design with five replications for each
treatment. The soil was collected from the nearby forest from
Balawala Dehradun and mixed with acid washed and
neutralized sand. The ratio of sand and soil (60:40) was made
by their volumes. The soil was tested as per the available
standard methods (Table-2)7. Each pot was uniformly filled
with 8 kg of soil and treated with different combinations of P.
fluorescens Tf5 and chemical fertilizers (0.2 g per pot). The
bacterial inoculum was prepared by incubating bacteria at 30ºC
(384)
for 24 h to obtain a concentration of 1 x 109 colony forming
units (cfu) mL-1. After germination the plants were removed
from agar plates and their roots were dipped in the bacterial
suspension for five minutes and planted in pots. The pots were
watered daily and holes were made at the base of the pot to
prevent water logging.
Plant harvest and analysis: The plants were carefully
removed from pots on the 90th day of transplantation without
causing any damage. The adhering soil particles were removed
by washing gently with water and the water droplets were
removed by blotting with the filter paper. Then these plants
were used for the biometric observations of plant height, fresh
weight and dry weight. Plants were weighed immediately to
know their fresh weight. The plant dry weight was estimated
by drying plant parts at 700C for 72 h and weighing them
immediately thereafter.
Statistical analysis: The data generated in the experiment
was analyzed using Analysis of Variance (ANOVA) followed
by post hoc test using SPSS® software. Means were taken at
p ≤ 0.05 level of significance.
RESULTS AND DISCUSSION
In situ bacterial screening for PGPR traits: Of the ten
isolates used in screening six were HCN producers, seven ≤
IAA producers, six P solubilizes, four with antifungal properties,
and one each with celllulase and chitinase activity (Table-1).
The isolate Pseudomonas fluorescens Tf5 demonstrated
maximum plant growth promotion attributes among all other
isolates.
In situ effect of fertilizer concentrations on bacterial
growth in growth medium: There was a continuous drop in
optical density (OD) with enhancement in fertilizer
concentrations (Fig.-1). The minimum OD (0.187) was noted
with urea and maximum (OD 0.554) with potash applied at 20
mg and 4 mg concentrations, respectively. The urea application
lowered the P. fluorescens Tf5 population, causing drop in OD
by reducing pH of the medium. Urea reacts with water to form
ammonia causing pH drop8-9. It was also observed that with an
increase in fertilizer concentration the OD of solution decreased,
suggesting that higher fertilizer concentrations have negative
effect on P. fluorescens Tf5 growth.
 (385) YADAV, YADAV AND GUPTA

Tabel-1. Screening of P. fluorescens biovars for plant

growth promoting traits.
Biovar HCN IAA production NH3 P Antifungal Catalase Cellulase Chitinase
production pg mL-1 production solubilization activity* activity activity activity

Tf1 - - - +++ - +++ - -

Tf2 + 15.64 - - - ++ + ++

Tf3 + - - + - + - -

Tf4 - - - + - ++ - -

Tf5 + 43.60 - +++ 17.83 ++ - -

Tf6 - - - + - + - -

Tf7 + - - - 23.33 +++ - -

Tf8 - - - ++ - +++ - -

Tf9 +++ - - - - ++ - -
Figure-1 . Effect of chemical fertilizer concentrations on
Tf10 - 18.50 - - - ++ - -
optical density of P. fluorescens Tf5. (OD of
control = 0.587). Vertical bars represent standard *Zone of inhibition (%) (+) positive, (-) negative test, IAA-Indole acetic acid
error.

Seed germination: There was variable effect of fertilizer
treatments on bell pepper seed germination (Table-3).
Application of nitrogenous fertilizers like, urea, DAP and sodium
nitrate significantly reduced germination of bell pepper seeds.
This effect was due to pH reduction in the growth medium as
a result of ammonia formation thorough urea hydrolysis8-9.
Plant growth: Except DAP, other two nitrogenous fertilizers
demonstrated moderate effect on yields. One study shows
≤ that input of nitrate based fertilizer increases nitrate
accumulation in vegetables much more than did ammonium
based fertilizers, thus helping in better plant growth10. But in
our results nitrate based growth enhancement with sodium
nitrate was much lesser than DAP. The significant differences
were found in measured plant growth parameters between
inoculated and plant control (Table-4). The application of DAP
provided consistently good results in all the aspects of plant
growth, which was fairly higher than control. DAP fertilizer is
known to be a good source of plant nutrition. It is highly water
soluble in nature and dissolves quickly in soil to release plant
usable phosphate and ammonium. In our results, DAP
application provided maximum plant growth perhaps due to
the release of dual nutrition sources (N and P) in soil. When
P. fluorescens Tf5 was inoculated with fertilizers all the
treatments performed even better results. This growth
enhancement was may be due to increased availability of P
to plant and release of IAA by P. fluorescens Tf5. Application
of some microbes, including arbuscular mycorrhiza fungi
(AMF), is known to enhance P solubilization, IAA production
and HCN production in rhizosphere, thus resulting in higher
crop yield. The principle mechanism for increased availability
of inorganic P appears to be through the action of organic
acids synthesized by inoculants11-12.
Plant length: Previous studies on bell peppers have shown
that increasing the nitrogen fertilizer application increases plant
growth13. Our results indicate that five different fertilizer
treatments significantly enhanced the height of bell pepper
plants (Table-4). The highest (54.62 cm) and the lowest (23.56
cm) shoot lengths were obtained from DAP and control,
respectively. Similar trend was observed with plant root length
observations. The increase in plant height, root length was
even more pronounced when fertilizer treated pots were co-
treated with P. fluorescens Tf5. Maximum shoot length was
observed with DAP treatment (62.91 cm) and minimum with
control (31.24 cm). Similarity, maximum root length was with
DAP treatment and minimum with inoculated control.
Plant fresh weight: DAP treatment yielded maximum shoot
fresh weight (13.4 g), whereas a minimum value was obtained
with control (9.82 g). When co-treated with bacterial inoculant
the shoot fresh weight increased further, reaching the maximum
value (20.91 g) with DAP treatment. The fresh shoot weight
enhancement was also observed with bioinoculant treated
control (14.79 g). Likewise, the maximum root fresh weight
was observed with DAP treatment (5.46 g) and minimum with
 THE COMBINED USE OF CHEMICAL FERTILIZERS WITH MULTI - TRAIT (386)

Table-2. Physico-chemical properties of Table-3. Effect of different fertilizers on bell pepper seed
experimental soil. germination.

Means with the same letter in each column are not significantly
different (P 0.05)
Table-4. Effect of chemical fertilizers and P. fluorescens Tf5 treatment on bell pepper growth

≤T

(
Means with the same letter in each column are not significantly different (P ≤ 0.05) K

control (2.01 g). Also, a positive growth effect within each previous observations, further growth enhancement was C

inoculation treatment was recorded where maximum root weight observed when the chemical fertilizer treated plants were co- C
reached the value 8.49 g with DAP treatment and minimum treated with bioinoculant. Root dry weight was found maximum L
(2.83 g) with control. with DAP treatment (1.83 g) and minimum with control (0.69
g). With bioinoculant co-treatment the maximum root dry weight
Plant dry weight: Plant dry weight was approximately a
was obtained with DAP (2.50 g) and minimum with control
magnitude lower than fresh weight of similar treatment. It
(1.10 g). Bacterial treatment was responsible for net dry weight
showed almost similar patterns of growth with treated pots as
enhancement, which can be proved by net plant growth
in case of fresh weight observations. The maximum shoot dry
enhancement in inoculated control.
weight, obtained with DAP treatment (5.54 g), was
approximately two times higher than control (2.68 g). Like
(387) YADAV, YADAV AND GUPTA

CONCLUSION IBH Publ. Co.

3.
The overall analysis of the study concludes that out of five
chemical fertilizers used in the study, DAP application 4.
enhanced bell pepper growth to the maximum. The treatment
of P. fluorescens Tf5 with chemical fertilizers further enhanced 5.
the bell pepper growth. The co-application of P. fluorescens
Tf5 might have facilitated plant growth by increasing the supply 6.
of P, IAA and HCN. Thus amending the soil with lower quantity
of chemical fertilizer and co-application of P. fluorescens Tf5 7.
Adesemoye, A.O., Torbert, H.A. and Kloepper, J.W. (2009).
Microbial Ecol. 58(4): 921.
Kumar, S., Pandey, P. and Maheshwari, D.K. (2009). European
J. Soil Biol. 45(4): 334.
King, E.O., Ward, M.K. and Raney, D.E. (1954). The J. Laboratory
& Clinical Med. 44(2): 301.
Bergey, D.H. and Holt, J., (2000). In: Bergey's manual of
determinative bacteriology. 9 ed. Philadelphia: Williams & Wilkins.
Norman, A.G., (1965). In: Methods of Soil Analysis. Part 2.

can help to reduce the fertilizer use in bell pepper plants. Chemical and Microbiological Properties. Agronomy Monograph
9.2. Madison, WI: American Soc. of Agro., Soil Sc. Soc. of America.
ACKNOWLEDGEMENT 8. Zasoski, R. J. , R.D.M. (1992). J. Pl. Nut. 15: 977.
9. Bremner, J.M. and Krogmeier, M.J. (1989). In: Proceedings of the
The authors are thankful to Department of Microbiology, Division
National Academy of Sciences of the United States of America.
of Life Sciences, S.B.S. (PG) Institute of Biomedical Sciences 86(21): 8185.
& Research, Balawala and Dehradun for granting permission 10. Wang, Z. and Li, S. (2004). J. Pl. Nut. 27(3): 539.
to carry out the research. 11. Souchie, E.L., et al. (2006). Anais da Academia Brasileira de
Ciências. 78(1): 183.
REFERENCES
12. Rodriguez, H. and Fraga, R. (1999). Biotechnol. Adv. 17(4-5):
1. Tumbare, A.D. and Bhoite, S.U. (2002). Indian J. Agri. Sci. 72(2). 319.
109. 13. Bhuvaneswari, G., et al. (2014). Int. ltrs. Nat. Sc. 13: 81.
2. Ahmed, S., (1995). In: Agriculture-fertilizer interface in Asia-
Issues of growth and sustainability. New Delhi: Oxford and