9(1&2): 213-216, 2015 (Supplement on Rice)

N
EFFECT OF PLANT GROWTH PROMOTING RHIZOBACTERIA ON
GERMINATION AND GROWTH OF RICE (ORYZA SATIVA L.)
Save Nature to Survive

QUARTERLY

A. PRADHAN AND B.B.MISHRA*

Department of Microbiology, College of Basic Science & Humanities,
Orissa University of Agriculture and Technology, Bhubaneswar - 751 003, Odisha, INDIA
e-mail: bb_mishra58@yahoo.com
ABSTRACT

INTRODUCTION
Plant growth promoting rhizobacteria (PGPR) are able to exert a beneficial effect
upon plant growth. The effects of PGPR on plant growth can be mediated by
direct or indirect mechanisms (Glick, 1995). The direct effects have been most
commonly attributed to produce or change the concentration of plant growth
regulators like Indole acetic acid, Gibberellic acid, Cytokinins and Ethylene,
Asymbiotic N2 fixation (Boddey and Dobereiner, 2000). These PGPR also affect
growth by indirect mechanisms such as antagonism against phytopathogenic
microorganisms by production of Siderophores (Scher and Baker, 1982),
Antibiotics and Cyanide, Solubilization of mineral phosphates and other nutrients
(de Freitas et al., 1997). Treatments with PGPR increase germination percentage,
seedling vigor,emergence, plant stand, root and shoot growth, total biomass of
the plants, seed weight, early flowering, grains, fodder and fruit yields etc
(Ramamoorthy et al., 2001). The enhancement of plant growth by PGPR indicates
their potential as biofertilizers in the field of agriculture. It was found that
inoculation of rice seedlings with Bacillus sp. significantly increased the number
and length of root & shoots and dry weight (Biswas et al., 2000). To assess this
hypothesis, the present investigation was under taken to screen the bacterial
isolates from the rhizospheric soil of Rice plant from Sundarban, West Bengal,
India based on Plant Growth Promoting Characters and the potential PGPR isolate
was tried with rice plant and pot experiment was conducted to evaluate the
efficacy of isolate to increase the germination (%), root length, shoot length and
other growth related characters under in- vitro & in-vivo conditions. The objectives
of this study were screening of bacterial isolates based on plant growth promoting
characters and trial on rice plant by using potential PGPR isolates.

MATERIALS AND METHODS

Sample collection & Bacterial isolation
Soil sample was collected from the rhizosphere region of Rice plant from
Sundarban, India and intact root system was dug out and the rhizospheric soil
sample was carefully taken in plastic bags and stored at 40C. A total of ten bacterial
isolates were isolated from the rhizospheric soil sample by serial dilution
technique.
Screening of isolates for different plant growth promoting characters: In vitro
The bacterial isolates were screened for Plant growth promoting activities and the
isolates were streaked on Pikovaskayas agar medium and the colonies formed
on the plates with clear halos were considered as positive for Phosphate
solubilization (Pikovaskya et al., 1948). These isolates were also tested for auxin
production IAA-like substances. Briefly, test bacterial cultures were inoculated in
the nutrient broth with tryptophan (1mg/ml) incubated at 35 2 0C for 7 days.
213

Plant growth promoting rhizobacteria (PGPR)

have gained worldwide importance and have
been identified in influencing the growth &
yield of crop plants. On account of that, an
attempt was made in the present investigation
to study PGPR activities of bacteria isolated
from rhizospheric region of Rice plant from
Sundarban, India. A total of 10 bacteria were
isolated from the soil sample and amongst them
the four designated as D 28, D12, T1 & T8
were selected for study of PGPR traits like
production of IAA, Ammonia, HCN, Nitrate,
Siderophore, Phosphate solubilization and
biochemical characterization. All the isolates
were able to produce IAA, Siderophore,
Nitrate, Ammonia & Phosphate solubilization.
The most potential among the four D28 & D12
were tried with Rice plant to determine effect
on germination and plant growth. A significant
increase in root length and shoot length at
P<0.05 significant level was also observed.
The bacterial isolates were characterized by
biochemical attributes and were identified on
the basis of PIBWin online software. The
present study therefore suggests that the use of
PGPR isolate D 28 & D12 as inoculants
biofertilizers might be beneficial for rice
cultivation as they enhanced growth of rice
and induced Plant growth promoting
characters.

KEY WORDS
PGPR
Oryza sativaL.
Siderophore
IAA, HCN

Received :
Revised :
Accepted :

27.01.2015
13.02.2015
10.04.2015

*Corresponding author

A. PRADHAN AND B. B. MISHRA

Cultures were centrifuged at 3000 rpm for 30 min. 2mL of the

supernatant was mixed with 2 drops of orthophosphoric acid
and 4 ml of Salkowskis reagent (50 ml, 35% perchloric acid;
1 ml 0.5 FeCl3). Development of a pink colour indicates IAA
production (Loper and Schroth, 1986). Bacterial isolates were
tested for the production of ammonia in peptone water. Freshly
grown cultures were inoculated in 10 ml peptone water in
each tube and incubated for 48 h at 35 2C. Nesslers
reagent (0.5 ml) was added in each tube. Development of
brown to yellow colour was a positive test for ammonia
production (Cappuccino and Sherman, 1992).Siderophore
production was checked on solid CAS universal blue agar
plates. Actively growing cultures were spot inoculated on the
CAS blue agar plate and incubated at 35 20C for 72 h.
Formation of yellow-orange halo around the colony indicated
production and release of the siderophores on the agar plate
(Schwyn and Neilands, 1986). Isolates were further screened
for their HCN producing abilities. Bacterial cultures were
streaked on nutrient agar medium containing 4.4 g per liter of
glycine. A Whatman filter paper No. 1 soaked in 0.5% picric
acid solution (in 2% sodium carbonate) was placed inside the
lid of a plate. Plates were sealed with parafilm and incubated
at 35 2 0C for 4 days (Castric et al.,1975).

sterilized seeds of rice were inoculated in broth culture for 30

minutes (ISTA, 1993). Germination tests were carried out by
the paper towel method and PGPR-treated seeds and control
were seeded onto paper towels. For pot experiment, rice
seedlings were soaked in inoculum for 30 minutes and were
sown at 2 cm depth in pot containing 250g soil. A control was
also maintained without inoculated seed.
Harvesting of the plants and Statistical analysis
After 47 days of seed sowing, rice plants were harvested
through separating of plants from soil. The plants were washed
and Germination percentage, height of Plant (cm plant-1), root
length (cm plant-1)were recorded. Data was analyzed statistically
by one way ANOVA. The statistical significance of difference
in germination and growth of rice was assessed using SPSS
16.0 software. All tests were conducted in triplicate and the
significance of differences between mean values with (p<
0.05) level of significance was evaluated by DMRT (Duncans
Multiple Range Test).

RESULTS AND DISCUSSION

Isolation, Identification and Screening of Plant growth

Identification, biochemical characterization & enzymatic

promoting activities of the bacterial isolates

activities of bacterial isolates

Seed germination test

Among the four bacterial isolates, D28 & AD12 are more
potential and it was tested for seed germination and plant
growth under lab and field conditions. Rice seedlings (Oryza
sativa), collected from Seed technology, O.U.A.T and were
surface sterilized with 0.1% HgCl2 for 2 min and rinsed with
sterile distilled water for ten times. Bacterial isolates were grown
in respective broth on shaking incubator (180 rpm) at 28
2C for 24 h. Cell densities in the suspension were adjusted to
a final density of approximately 108CFU seed1.The surface

A total of ten bacterial isolates were isolated from the

rhizospheric region of Oryza sativa (Rice) plant from West
Bengal, Sundarban, India. Among ten bacterial isolates only
four D12, D28, T1 & T8 showed all positive results for PGPR
activities and biochemical identification. The bacterial isolates
were screened for phosphate solubilization. Each of the isolates
was streaked on medium which is selective medium for
phosphorus solubilizers. On Pikovskaya medium, all isolates
showed the development of sharp halo zones and D28 showed
maximum zone of 22mm (Table1). Similar observations have
been reported by S. Ngomle et al., 2014, who stated that
microorganisms capable of producing a clear zone due to P
solubilization in the surrounding medium were selected as
potential phosphate solubilizers and clear zones around the
colonies indicated the capacity of phosphate solubilization
on Pikovskaya medium. It was also observed that increasing
in the incubation time, increases the zone size. Similar
observations have been reported by Naik et al., 2012, who
stated that in phosphate solubilizing fungi, highest phosphorus
solubilization efficiency and phosphorus solubilization index
after 120th hour of incubation was seen as compare to 96th

Table 1: PGPR activities by the bacterial isolates

Table 3: Enzymatic activities by the bacterial isolates

The potential bacterial isolates which showed PGPR activities

were further characterized by biochemical tests like Oxidase,
MR-VP, Indole, Citrate, Urease, Mannitol motility, Esculin
hydrolysis, nitrate production and enzymatic activities like
amylase, gelatinase & caesinase. fermentation of various sugars
and this helped in the bacterial identification up to the genus
level (Gupta et al.,2000) by Bergeys manual of Determinative
bacteriology (Holt et al.,1994)and PIBWin online software
(Bryant et al.,2004 ).

Isolates code Nitrate Ammonia IAA Siderophore

Phosphate

Isolates code

Casein

Gelatin

Starch

D12
D28
T1
T8

+
+
+
+

D12
D28
T1
T8

+
+
+
-

+
+
+

+
+
+

+
+
+
+

+
+
+
+

+
+
+
-

+
+
+
+

(22mm)
(21mm)
(18mm)
(11mm)

(21mm)
(22mm)
(20mm)
(18mm)

Table 2: Biochemical activities by the bacterial isolates

Isolates code

Indole

MR

VP

Citrate

Urease

Mannitol

Esculin

Oxidase

Catalase

D12
D28
T1
T8

+
-

+
+
+

+
+
+
+

+
+
+

+
+
+
+

+
+
+
+

+
+
+
-

+
+
+

214

EFFECT OF RHIZOBACTERIA ON RICE

Table 4: Sugar utilization by the bacterial isolates

Isolates code

Fructose

Glucose

Raffinose

Salicin

Sorbitol

Sucrose

Inositol

Xylose

Lactose

D12
D28
T1
T8

+
+
+

+
+
+

+
+
-

+
-

+
+
-

+
+

+
+
+

+
+
-

+
+
-

Control

D28

D12

Control

D28

D12

Fig.1Effect on Germination & Growth of Rice in Germination Paper and Pot

hour of incubation. The four bacterial isolates were also able
to produce Siderophore and D12 showed maximum zone of
22mm (Table 1). Similar observations have been reported by
Loper and Henkels, 1997, who stated that iron is an essential
growth element for all living organisms including plant
pathogens. Growth promotion may be attributed to other
mechanisms such as production of plant growth promoting
hormones in the rhizosphere. The bacterial isolates D28, D12
& T1 also produced plant growth promoting hormone i.e.
IAA. The ability of bacteria to produce phytohormone like
Auxin i.e. IAA in the rhizosphere depends onthe availability
of precursors and uptake of microbial IAA by plant (Arshad

and Frankenberger,1991). The two bacterial isolates D28 &

D12 also exhibited strong production of ammonia which is
another important trait of PGPR and taken up by plants as a
source of nitrogen for their growth (Ahmad et al., 2008). These
four potential isolates were further tested for Biochemical&
Enzymatic identification (Table 2&3). The bacterial isolates
showed fermentation of various sugars (Table 4). The bacterial
isolate were characterized by biochemical attributes and were
identified as D12 (Bacillus tequilensis), D 2 8 (Bacillus
licheniformis), T1 (Bacillus amyloliquefaciens), T8 (Bacillus
cereus) on the basis of PIBWin online software (Table 5).
Seed germination test
In this study, an increase in the plant growth by seed
bacterization has been demonstrated. It is a well-established
fact that overall plant growth and root development influenced
by improved phosphorous nutrition (Jones et al., 1994). A
large number of evidence suggests that PGPR enhance the
growth, seed emergence and crop yield (Herman et al., 2008).

Table 5: Identification of bacterial isolates by PIBWin online software

Isolates code
Identification
Matching %
D12
D28
T1
T8

Bacillus tequilensis
Bacillus licheniformis
Bacillus amyloliquefaciens
Bacillus cereus

94%
80%
75%
76%

Table 6: Effect of plant growth promoting rhizobacteria on seed germination and growth of rice seedlings
Isolate code Seed germination (%) Shoot length
Root length
Seed germination
Shoot length
(Germination paper)
(Germination paper) (Germination paper) (%) (Pot)
(Pot)
Control
D28
D12

89
97.6
95.3

10.0 0.21a
14.2 0.50c
12.5 0.23b

9.30.72a
13.00.41b
11.80.20b

8.3
9.6
9.3

22.71.50a
35.40.66c
31.40.66b

Root length
(Pot)
14.01.69a
17.90.36b
16.60.76b

*Values are the mean SEM& differ significantly as per DMRT by LSD (P <0.05).Mean values in each column with same superscript (s) do not differ significantly
as per DMRT.

215

A. PRADHAN AND B. B. MISHRA

In this study, we investigated the effectiveness of PGPR isolates

on seed germination percentage and growth of rice seedlings.
To see the effect of PGPR isolates on rice seedlings, Seed
germination was also increased when seeds were pretreated
with D28 & D12 which positively increase the germination of
rice. The PGPR isolates significantly increased the root& shoot
length of rice seedlings (Table 6). Highest root (17.9 cm) &
shoot elongation (35.4 cm) was recorded when seeds were
pre-treated with D28 isolate in pot & 13cm root length and
14.2 cm shoot length in germination paper.All statistical data
are shown as mean SEM. These results suggest that Plant
growth Promoting Rhizobacteriais able to induce the
production of IAA, solubilization of phosphorus, production
of siderophore & thereby improving growth of plants.

solubilizing rhizobacteria enhance the growth and yield but not

phosphorus uptake of canola (Brassica napus L.). Biol. Fertil. Soils.24:
358-364.

The use of PGPR as inoculants biofertilizers is acompetent

approach to replace chemical fertilizers and pesticides for
sustainable rice cultivation in India. Further investigations,
including effectiveness of PGPR inoculants under greenhouse
and field conditions are required to clarify the role of PGPR as
biofertilizers that exert beneficial effects on plant growth and
development.

ISTA. 1993. Proceedings of the international Seed Testing Association,

International Rules for Seed Testing. Seed Science Technology. 21:
2530.

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