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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 3; Year 2015; Page: 157 - 160

Research Article

STUDIES ON PRODUCTION AND CHARACTERIZATION OF INDOLE

ACETIC ACID USING RHIZOBACTERIA ISOLATED FROM
GROUNDNUT SOIL
E. Bhuvaneshwari* and K. Jagathy,
PG and Research Department of Microbiology, Sri Akilandeswari Womens College, Wandiwash,
Tiruvannamalai District, Tamil Nadu, India.
Abstract
Plant Growth Promoting Rhizobacteria (PGPB) is considered to promote plant growth directly or
indirectly. PGPB can exhibit a variety of characteristics responsible for influencing plant growth. The
common traits include production of plant growth regulators (auxin, gibberellin, ethylene etc.), siderophores,
HCN and antibiotics. Indole acetic acid (IAA) is one of the most physiologically active auxins. IAA is a
common product of L-tryptophan metabolism by several microorganisms including PGPR. To study the
effect of IAA producing rhizospheric isolates on plant growth, cup assay was performed. Vigna mungo
(black grams) seeds were used for seed coating. The seeds were surface sterilized by immersing in 95%
ethanol for 30 sec and mercury chloride (0.2%) for 3 min. Then further to remove traces of mercury chloride,
the disinfected seeds were washed 5 times by sterile distilled water. 0.1 ml of overnight grown culture was
applied on seed surface for seed coating. Seeds were dried and sowed into sterile plate until the seeds start
germinating. The uncoated seeds were used as control. The germinated seeds were transferred to pots and
grown for 7 days. The growth was checked by measuring their root length, shoot length and the number of
leaves. The IAA produced by rhizospheric bacteria displayed appreciable activity. The shoot length, root
length and the number of leaves obtained were higher compared to control. It is concluded that presence of
such growth promoting rhizoflora accountable for the beneficial effects on Vigna mungo growth and yield.
Key words: Vigna mungo, Indole acetic acid
(IAA), Plant Growth Promoting Rhizobacteria
(PGPB) and Groundnut soil.

Article History
Received : 25.04.2015
Revised : 03.05.2015
Accepted :06.05.2015
1. Introduction

Plant Growth Promoting Rhizobacteria

(PGPB) is considered to promote plant growth
directly or indirectly. PGPB can exhibit a variety
of characteristics responsible for influencing plant
growth. The common traits include production of
*Corresponding author: E. Bhuvaneshwari,
PG and Research Department of Microbiology, Sri
Akilandeswari Womens College, Wandiwash, Tamil
Nadu, India.

plant growth regulators (auxin, gibberellin,

ethylene etc.), siderophores, HCN and antibiotics
(Arshad and Frankenberger, 1992). Indole acetic
acid (IAA) is one of the most physiologically
active auxins. IAA is a common product of Ltryptophan metabolism by several microorganisms
including PGPR (Ahmad et al., 2008; Ajay Kumar
et al., 2012).
Indole-3-acetic acid (IAA) is the main
auxin in plants, controlling many important
physiological processes including cell enlargement
and division, tissue differentiation, and responses

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E. Bhuvaneshwari / Life Science Archives (LSA), Volume 1, Issue 3, Page 157 - 160, 2015

158

to light and gravity. Bacterial IAA producers

(BIPs) have the potential to interfere with any of
these processes by input of IAA into the plants
auxin pool. In the recent years, scientists have
diverted their attention towards exploring the
potential of beneficial microbes and the use of
plant growth promoting rhizobacteria (PGPR) for
sustainable agriculture has increased tremendously
in various parts of the world. Plant growth
promoting rhizobacteria (PGPR) are group of
bacteria that actively colonize plant roots region
and increase plant growth and yield. Various
species
of
bacteria
like
Pseudomonas,
Azospirillum,
Azotobacter,
Klebsiella,
Enterobacter,
Alcaligenes,
Arthrobacter,
Burkholderia, Bacillus and Serratia have been
reported to enhance the plant growth. PGPR
enhance plant growth either by direct or indirect
mechanisms. The direct growth promoting
mechanisms
involve
nitrogen
fixation,
solubilization of minerals, production of
phytohormones and the indirect approach occurs
when PGPR lessen or prevent the deleterious
effects of plant pathogens on plants. With these
background, the present study was focused on
isolating rhizospheric bacteria, to screen the
isolates and characterize the selected isolate by
biochemical analysis and also to produce IAA,
extract and partially purify the compound. On
application orientation, the germination test was
carried out for promoting growth.

subsequently serially dilute to 10-2 10-10. 0.1 ml

of suspension was taken from the dilutions 10 -3,
10-7and 10-9 and spreaded over the surface of
sterile Nutrient agar medium. The plates were
incubated at 372C for 24 - 48 hrs.

2. Materials and Methods

Germination test was performed to

determine the effect of IAA producing isolate on
plant growth by plate assay.

2.1. Sample collection

The first step in rhizobacteria isolation was
collection of rhizosphere soil close to the roots of
groundnut, Vandavasi (soil at 1 3 mm from the
root and the soil adhering to the root). To collect
the soil fraction, the root was normally shaken
vigorously and soil still adhering to roots was
collected as the rhizosphere soil. Rhizobacteria
were isolated by the suspension soil in water, and
serially diluted and plated in Nutrient agar media.
2.2. Isolation of bacteria
One gram of soil sample was suspended in
10 ml of sterile distilled water to make 10 -1
dilution. From 10-1 suspension, 1 ml of sample
was transferred to 9 ml of sterile water and

2.3. Maintenance of Pure cultures

The isolated bacteria were sub-cultured
from serially diluted plates and it was maintained
in Nutrient agar slants (Isolate - 1 to Isolate -12)
for screening of isolates.
2.4. IAA production from potent isolate
For IAA production, IAA production
medium (300 ml) was prepared, autoclaved and
inoculated with the selected isolate. The medium
was incubated at room temperature (37C) in
orbital shaker for 72 - 96 hours. The culture broth
was filtered using Whatman filter paper and the
filtrate was centrifuged at 10,000 rpm for 15 min.
Two ml of supernatant was mixed with 2 drops of
orthophosphoric acid and 4 ml of Salkowskis
reagent (50 ml of 35% perchloric acid and 1 ml of
0.5 M FeCl3).
2.5. Extraction and partial purification of IAA
Extraction and partial purification of
Indole acetic acid (IAA) was performed using
Thin layer chromatography (TLC) slide and
Salkowskis reagent (Kuang-Ren et al., 2003).
2.6. Germination test

3. Results and Discussion

The selected PGPR isolates were screened
for indole production. Among the 12 isolates, only
one isolate showed positive for indole production.
The results of biochemical characterization tests
revealed that the selected PGPR isolate was Gram
positive, Indole positive, Methyl red negative,
Voges Proskauer negative, Citrate positive,
Oxidase positive, Catalase positive, H2S negative,
Carbohydrate utilization: sucrose negative,
maltose negative, galactose positive, fructose
positive. Partial purification of the IAA dried
extract from selected isolate was done by TLC

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E. Bhuvaneshwari / Life Science Archives (LSA), Volume 1, Issue 3, Page 157 - 160, 2015

method. Purified IAA sample was compared with

standard IAA on TLC chromatograms.

159

stimulate and facilitate plant growth. The present

work deals with isolation, characterization and
identification of indole acetic acid producing
bacteria from the rhizosphere soil. The present
study was carried out for IAA production from
rhizobacteria. Twelve isolates were selected and
among 12 isolates, only one was selected since it
showed indole positive reaction. A similar study
was carried out for IAA production from
rhizobacteria, Out of ten Indole acetic acid
producing isolates, five were selected as efficient
producers. Optimization of indole acetic acid
production was carried out at different cultural
conditions of pH and temperature with varying
media components such as carbon and nitrogen
source,
tryptophan
concentration.
Partial
purification of IAA was done and purity was
confirmed with Thin layer chromatography (TLC).
Subsequently, effect on plant growth was tested by
pot assay, the Vigna mungo seeds germination was
efficient and the shoot length, root length and the
number of leaves obtained was observed. The root
length (6 mm) was higher compared to shoot
length (4.2 mm) and the leaves produced. In
conclusion, the study suggests the IAA producing
bacteria as efficient biofertilizer inoculants to
promote
plant
growth
(Madhuri
and
Sahasrabudhe, 2011; Savita Kerkar et al., 2012;
Mohite, 2013; Janardan Yadav et al., 2013).

TLC of ethyl acetate extract showed pink

colour spot at the Rf corresponding to the
authentic IAA (0.57). It confirmed IAA producing
potential of rhizospheric isolates.
The Vigna mungo (Black grams) was soaked with
culture and the germination was observed after 24
hours (Figure 1 and Figure 2).

Figure 1: Before soaking

The property of synthesizing IAA is

considered as effective tool for screening
beneficial microorganisms suggesting that IAA
producing bacteria have profound effect on plant
growth. Inoculation with IAA producing bacteria
induces the proliferation of lateral roots and root
hairs. Fatima et al. (2009) also showed that
germination rate, roots, shoot growth of plant were
increased by IAA and PGPR.

Figure 2: Germination of seeds after soaking

with the culture

Indole acetic acid (IAA) production is a

major property of rhizosphere bacteria that
Table 1: Cup assay for plant growth
Soaking No: of
Hours
seeds
used
Control
15
2
15
4
15

No: of
seeds
germinated
15
13
13

% of
Germination
100
92.31
92.31

Root
Length
(cm)
2.5
6
3

Shoot
length
(cm)
0.3
4.2
3.7

No. of
Leaves
2
2
2

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E. Bhuvaneshwari / Life Science Archives (LSA), Volume 1, Issue 3, Page 157 - 160, 2015

Therefore, the selected PGPR isolate was studied

for its effect on plant growth under controlled
conditions. There was a significant increase in the
germination process .The isolate was found much
effective to show potential increase. Data obtained
from seed germination demonstrated positive
effect on plant growth and thus can be considered
as plant growth promoter.

5)

4. Conclusion
In conclusion, the IAA produced by
rhizospheric bacteria displayed appreciable
activity. The IAA produced by the bacteria was
one of the plant growth promoting hormone. The
presence of plant growth promoting hormone
compounds was confirmed by Thin layer
chromatography (TLC). Overall, the 12 isolates
were obtained and among the 12 isolates, only one
isolate was indole acetic acid producing isolate.
The
PGPR
isolate
was
biochemically
characterized. The shoot length, root length and
the number of leaves obtained in PGPR treatments
were higher when compared to control. It is
concluded that presence of such growth promoting
rhizoflora accountable for the beneficial effects on
Vigna mungo growth and yield.
5. References

6)

7)

8)

160

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Cattelan, A. J., P.G. Hartel and J.J.
Fuhrmann. 1999. Screening of plant
growth promoting Rhizobacteria to
promote early soybean growth. Soil
Science Society of American Journal, 63:
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Janardan Yadav, Jay Prakash Verma and
Kavindra Nath Tiwari. 2013. Effect of
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Chickpea (Cicer arietinum L.) under in
Vitro
Biological
Forum

An
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Madhuri, M and A. Sahasrabudhe. 2011.
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