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Research Journal of Biotechnology Vol. 11 (3) March (2016)
Res. J. Biotech
108
Isolation, screening and characterization of PGPR
isolated from rhizospheric soils of Pigeonpea
Tiwari Ashish, Devi Shikha, Singh Nand Kumar and Sharma Shivesh*
Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad, UP, INDIA
*shiveshsharmamnnit@gmail.com; ssnvsharma@gmail.com
Abstract
Plant Growth Promoting Rhizobacteria (PGPR) is
group of bacteria that colonize roots of plant and help
in plant growth and disease suppression by various
direct and indirect mechanisms. PGPRs are
recognized as efficient soil microbes as bio-fertilizers
for enhancing the growth of several crops and
controlling soil borne pathogens. Hence, the present
investigation was carried out to isolate, screen and
characterize the PGPR from the rhizosphere soil of
pigeonpea growing in different areas of Bijarkala and
Kumarganj. A total of nine bacteria were isolated
with the occurrence percentage of 1.92% to 98.1%
from pigeonpea rhizosphere here and characterized
for various plant growth promoting activities.
In the present study, six isolates produced indole-3-
acetic acid in the range of 56 to 97µg/ml and only
single isolate (PN13) displayed phosphate solubilizing
activity in Pikovskaya agar. In addition, six isolates
were found to be positive for siderophore production
and also showed antifungal activity against Fusarium
udum. Catalase production was shown by almost all
the isolates and production of HCN was detected in
only single isolate i.e. PN11.Out of nine isolates,
seven isolates were exhibited in vitro plant growth
promotion activities and indicated that these isolates
may be exploited as biofertilizers and microbial
inoculants for pigeon pea crop as they enhanced plant
growth via diverse mechanisms and offered an
attractive strategy to replace synthetic fertilizers and
pesticides.
Keywords: Biofertilizers, HCN, microbial inoculants and
PGPR.
Introduction
Plant growth promoting rhizobacteria (PGPR) are the group
of beneficial bacteria that enhance plant growth and
biocontrol by wide variety of mechanisms1. In the context
of increasing global concern for food and environmental
quality, the utilization of PGPR for minimizing chemical
inputs in agriculture practices is a potentially important
issue2. Pigeonpea (Cajanus cajan) is a drought tolerant
plant, grown on rain fed and semi-arid region of Indian
subcontinent. Pigeonpea crop can grow under poor soil
conditions and tolerate water scarcity. It is nutritious, high
protein legume crop used as a source of dietary protein.
India alone accounts for about 80% of total production of
pigeonpea. Nowadays, modern agriculture relies on
excessive use of chemical fertilizers and pesticides to
increase crop production which caused severe adverse
effect on soil health and environment3. However, use of
PGPR in agriculture in order to enhance the growth of plant
via circulating the nutrients in the soil is an ecofriendly
strategy to minimize the need of synthetic fertilizers as
much as possible2, 4.
PGPRs can be defined as beneficial bacterial strains that
colonize the roots of plant for plant growth stimulation and
biocontrol potential5.They can affect plant growth by
promoting plant-microbe symbiosis, competition for
colonization space and nutrients and decreasing the
activities of plant pathogens6,7. PGPR stimulates plant
growth and biocontrol by various direct and indirect
mechanisms. Direct mechanism of PGPR includes
facilitating resource acquisition i.e. solubilisation of
phosphate, nitrogen fixation, iron acquisition by
siderophore and modulating proper level of plant hormones
like auxins, cytokinins and gibberellins and lowering the
level of ethylene by production of ACC deaminase
enzyme8,9. Indirect mechanisms of PGPR include
suppression of fungal, bacterial and nematode pathogens by
the production of various enzymes and compounds likewise
chitinase, protease, cellulase, antibiotics, HCN, ammonia
and volatile organic compound (VOCs) etc4.
Several other mechanisms of indirect growth promotion
and biocontrol by PGPR include antagonistic activity,
quorum sensing, signal interference, inhibition of biofilm
formation, increasing mineral nutrient solublization,
systemic acquired resistance and induced systemic
resistance8. PGPR have been isolated and screened from
rhizospheric soil of diverse crops to enhance growth, seed
emergence, crop yield and production10. PGPR can be used
as agricultural inputs with plant growth promoting
attributes and as biological control to reduce plant diseases
in various crops11. PGPR have been commercialized as
microbial bioinoculants or biofertilizers to increase crop
production12. PGPR offers an attractive strategy for
replacement and reduction of heavy application of chemical
pesticides and fertilizers13. Hence, the present work was
aimed to isolate, screened and characterize the PGPR from
rhizospheric soils of pigeonpea which can be utilized in
future for increasing growth and yield of plants.
Material and Methods
Collection of soil sample: Rhizosphere soil samples of
pigeonpea (Cajanus cajun) plant were collected from
Research Journal of Biotechnology Vol. 11 (3) March (2016)
Res. J. Biotech
109
different areas of Bijarkala and Kumarganj. The root
system along with bulk soil is removed from 0-20 cm depth
with the help of augur and rhizospheric soil was obtained
by shaking method and subsequent brushing of remaining
root system soil14. The samples were carefully collected
and transferred in sterile polythene bags and labelled
properly.
Isolation of bacterial isolates: Bacteria were isolated from
the rhizospheric soil samples by serial dilution technique
on nutrient agar (NA) plates and incubated at 28±2 0C for
72 hrs. After incubation period, NA plates were observed
for morphological appearances and number of bacterial
colonies. Bacterial isolates having different morphological
appearance on agar plates were selected and maintained on
nutrient agar slants and 50% glycerol at -80°C. All the
isolates were morphologically characterized as per method
described in Bergey’s manual of determinative
bacteriology15.
Characterization of bacterial isolate for different
plant growth promoting activities
IAA production: Indole acetic acid (IAA) production was
quantitatively estimated by Salkowski method16. Bacterial
cultures were grown on Luria broth liquid medium at 36±2
°C. The cultures in the flask showing dense milky white
growth were tested for purity. Fifty milliliter of Luria
Bertani (LB) broth containing 0.1% DL tryptophan were
inoculated with 500 µl of 24 h old bacterial cultures and
incubated in refrigerated incubator shaker at 30±0.1°C at
180 rpm for 48 h in dark. Fully grown bacterial cultures
were centrifuged at 10,000 rpm for 10 min at 4°C.
Estimation of IAA production in the supernatants was done
using colorimetric assay. One millilitre (1 ml) of
supernatant was mixed with 100 ml of 10 mM
orthophosphoric acid and 2 ml of the Salkowski reagent (1
ml of 0.5 M FeCl3 in 50 ml of 35% HCIO4) at 28±2 oC for
30 min. Development of pink colour in test tubes at the end
of the incubation indicated IAA production17.
Quantification of IAA was measured by the pink colour
absorbance at 530 nm after 30 min in UV/Vis
spectrophotometer.
Phosphate solubilisation: A loop full of fresh bacterial
cultures was streaked on the centre of agar plates modified
with Pikovskaya agar with insoluble tricalcium phosphate
(TCP) and incubated for 120 h at 28±2°C 18. The presence
of halo zone around the bacterial colonies indicated
positive phosphate solubilization ability. The solubilization
zone surrounding the developed bacterial colony was
calculated as phosphate solubilisation index (PSI).
PSI = A/B×100
where A = total diameter of colony and halo zone and B =
Colony diameter.
Siderophore and HCN production: Qualitative
estimation of siderophore production by the bacterial
isolates was determined by adapting the method Schwyn
and Neiland19 on chrome azurol sulphonate (CAS)
assay19. Production of siderophore was determined by the
development of orange halo zone around bacterial colonies.
In addition, all the bacterial isolates were screened for the
HCN production by adapting the method of Lorck20.
Colour change of the filter paper from deep yellow to
reddish-brown colour indicated production of HCN21.
Catalase activity: Bacterial cultures were grown in
nutrient agar medium for 48 h at 28°C. 48 hr old bacterial
colonies were added with 2-3 drops of hydrogen peroxide
(3%) on a clean glass slide and mixed using a sterile tooth
pick. The evolution of oxygen as effervescence indicated
catalase activity 22.
Antifungal activity: All the isolates were screened for
antagonistic effects against F. udum by using dual plate
assay with potato dextrose agar (PDA) plates23.The 5 mm
diameter mycelia disc of F. udum culture was placed at the
centre of PDA plate. A loopful of 48h old bacterial culture
grown in agar medium was streaked 2 cm away from the
edge of each plate and perpendicular to the fungi. Plates
with only fungus culture were used as control and
incubated at 25°C for 5-7 d.
The percent inhibition (PI) of radial growth of fungus was
calculated by the formula:
(R-r)
PI = --------- X 100
R
where PI = Percent inhibition; R = Radial growth of
pathogen in control plate and r = Radial growth of the
fungal colony interacting with antagonistic bacteria.
Results and Discussion
PGPR colonize roots of plant and exert beneficial effects
on plant growth and development by diverse mechanisms.
The precise mechanism by that efficient PGPR enhanced
plant growth and productivity is not clearly established,
though many hypothesis like phosphate solubilisation,
production of phytohormones, promotion of the mineral
nutrient uptake and suppression of soil borne pathogens are
typically believed to be involved6, 24-26. In the present study,
PGPR were isolated from pigeonpea rhizosphere and
characterized for various plant growth promotion activities.
A total of nine bacteria were successfully isolated from the
rhizospheric and non rhizospheric soils of pigeonpea and
designated as PN10, PN11, PN12, PN13, PN14, PN15,
PN16, PN17 and PN18 (Table 1).
Five bacteria were isolated from rhizospheric soil of
pigeonpea with occurrence percentage (OC%) ranging
between 3.57% to 93.8% whereas four bacteria were
isolated from non rhizospheric soil with OC% ranging
Research Journal of Biotechnology Vol. 11 (3) March (2016)
Res. J. Biotech
110
between 1.92% to 98.07%. Amongst all, the most dominant
bacterial isolates were found to be PN14 and PN16 with
98.07% and 93.8% respectively (Fig. 1).
The microscopics observation such as gram staining, shape
and motility of bacterial isolates are illustrated in table 2.
Eight isolates were found to be gram negative and rod
shaped while PN11 was gram positive and cocci shaped.
Seven isolates were motile and PN11 and PN16 were non
motile.
IAA production and phosphate solubilisation: IAA is
usually considered to be the most vital phytohormone that
functions as important signal molecule in the regulation of
plant growth and development processes. It has been
reported that more efficient auxin producers are commonly
associated with rhizosphere soil as comparison to bulk
soil27. In the present study, isolates PN10, PN11, PN13,
PN14, PN15, PN17 and PN18 induced the production of
IAA in the presence of tryptophan (Table 3). IAA
production was found in the range of 56 to 97 µg/ml.
Among all isolates, PN14 was found to produce high
amount of IAA i.e. 97 µg/ml. Additionally, PN14, PN15
and PN13 were found to be medium producers of IAA as
compared to weak producers isolates PN10, PN11, PN17
and PN18. Moreover, production of IAA by PGPR isolates
may vary from different species and strains and was
additionally influenced by substrate availability, growth
stage and culture conditions28.
Phosphate solubilization: Phosphorus is the second most
important nutrient, next to nitrogen (N) required for growth
of plants. A greater portion of phosphorus in soil is in the
form of insoluble phosphates and cannot be used directly
by the plants29. In the present study, only PN13 isolate was
found to give clear zone on Pikovskaya agar containing
insoluble mineral phosphate such as tri-calcium phosphate
(Table 3).Moreover, this isolate was also found to be
medium producer of IAA. Several studies disclosed that
higher concentration of phosphate-solubilizers is usually
found in the rhizosphere in comparison to bulk soil.
Siderophore, HCN and catalase production: Siderophore
production by rhizobacteria acts as biocontrol mechanism
under iron limiting condition. Rhizobacterial strains
produce diverse range of siderophore like catechol,
rhizobactin, citrate and anthranalate that have a higher
affinity for iron30. Out of nine bacterial isolates, six isolates
PN10, PN11, PN13, PN14, PN15 and PN18 showed
positive activity for siderophore production and it is
depicted by the development of orange halos surrounding
the bacterial colonies in blue agar medium (Table 3).
Besides this, there are various mechanisms of biocontrol
including production of HCN and antibiotics, lytic enzyme
secretion and elicitation of induced systemic resistance
(ISR) in plant31. In addition, HCN and catalase production
is another important trait of rhizobacteria that indirectly
influences growth of plants by playing essential role in the
biological control of plant pathogens or acting as an
inducer of plant resistance32.
In our study, production of HCN was detected in only
single isolate i.e. PN11 (Table 3). Furthermore, this isolate
also displayed siderophore production. Catalase production
was shown by almost all the isolates (Table 3). It has been
reported that rhizobacteial isolates showing catalase
activity must be highly resistant to chemical, mechanical
and environmental stress27.
Antagonistic activity of bacterial isolates against F.
udum: Antifungal activity of all the isolates was tested
against F. udum on potato dextrose agar (PDA) plates using
dual culture technique23. In the present study, six isolates
(PN10, PN11, PN13, PN14, PN15 and PN18) showed
inhibition activity against F. udum. Varied level of
inhibition percentage was recorded in six isolates and
maximum inhibition was recorded in bacterial isolate PN14
(Table 3). In our study, six isolates which inhibit the
growth of F. udum were able to produce siderophore which
confirmed that siderophore produced by these isolates
functions as suppressor to the growth of F. udum.
Table 1
Description of bacterial isolates
S. N.
Isolate code
Location
Soil sample
1.
PN10
Bijarkala
Rhizospheric soil
2.
PN11
Rhizospheric soil
3.
PN12
Rhizospheric soil
4.
PN13
Non-rhizospheric soil
5.
PN14
Non-rhizospheric soil
6.
PN15
Kumarganj
Rhizospheric soil
7.
PN16
Rhizospheric soil
8.
PN17
Non-rhizospheric soil
9.
PN18
Non-rhizospheric soil
Research Journal of Biotechnology Vol. 11 (3) March (2016)
Res. J. Biotech
111
Fig. 1: Dominance (OC%) of bacterial isolates in the rhizosphere of pigeonpea crop.
Table 2
Microscopic observation of bacterial isolates.
Isolates
Gram reaction
Shape
Motility
PN-10
Gram –ve
Rod
Motile
PN-11
Gram +ve
Cocci
Non-motile
PN-12
Gram –ve
Rod
Motile
PN-13
Gram –ve
Rod
motile
PN-14
Gram –ve
Rod
Motile
PN-15
Gram –ve
Rod
Motile
PN-16
Gram +ve
Rod
Non-motile
PN-17
Gram –ve
Rod
Motile
PN-18
Gram –ve
Rod
Motile
Table 3
Characterization of bacterial isolates for PGP attributes.
Isolates
IAA productiona
Siderophorep
roductiona
HCNa
Phosphate
solubilizationa
Antagonism
assay against
F. udumb
Catalase
production
Pink
colouration
Concentration
(µg/ml)
CAS
assay
Halo
size
(cm)
Halo
formation
PSI
PN-10
+
56
++
1.60
-
-
-
+
positive
PN-11
+
66
+
-
+++
-
-
+
positive
PN-12
-
-
-
-
-
-
-
-
positive
PN-13
++
75
++
1.40
-
+
2.8
+
positive
PN-14
+++
97
++
1.35
-
-
-
++
positive
PN-15
++
79
++
1.15
-
-
-
+
positive
PN-16
-
-
-
-
-
-
-
-
positive
PN-17
+
56
-
.30
-
-
-
-
positive
PN-18
+
62.63
++
1.10
-
-
-
+
positive
a - = no production + = low production; ++ = moderate production and +++ = strong production
b + = low inhibition (< 25 %); ++ = moderate inhibition (25% to 35%); +++ = strong inhibition (>35%)
In conclusion, the results suggest that seven isolates
exhibited plant growth promotion activities and were found
to produce siderophores, IAA, HCN and catalase,
solubilized insoluble phosphorus and also showed
antagonistic activity against F. udum. Therefore, these
isolates may be exploited as biofertilizers and microbial
inoculants for pigeon pea crop as they enhanced plant
growth via diverse mechanisms and offered an attractive
strategy to replace synthetic fertilizers and pesticides.
Acknowledgement
The present work is the result of DBT project
(BT/PR469/AGR/05/545/2011) entitled, “Harnessing
PGPRs from Indo Gangetic Plain Region of Uttar Pradesh
3.57
32.14
64.28
1.92
98.07
6.17
93.8
13.33
86.66
0
20
40
60
80
100
120
PN10
PN11
PN12
PN13
PN14
PN15
PN16
PN17
PN18
Occurence %
Research Journal of Biotechnology Vol. 11 (3) March (2016)
Res. J. Biotech
112
for Growth Promotion and Disease Suppression in Rice and
Pigeon Pea” funded by Department of Biotechnology
(DBT), Government of India. The authors greatly
acknowledge Director, MNNIT Allahabad for providing
necessary facilities to the execution of the present study.
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