21 LSA Bhuvaneshwari

Available online at www.jpsscientificpublications.com

Life Science Archives (LSA)

ISSN: 2454-1354

Volume – 1; Issue - 3; Year – 2015; Page: 157 - 160

©2015 Published by JPS Scientific Publications Ltd. All rights reserved

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 Women’s 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.

Article History Received : 25.04.2015

Revised : 03.05.2015

Accepted :06.05.2015

Key words: Vigna mungo, Indole acetic acid

(IAA), Plant Growth Promoting Rhizobacteria

(PGPB) and Groundnut soil.

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 Women’s 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 L-

tryptophan 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

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 plant’s

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.

2. Materials and Methods

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

subsequently serially dilute to 10-2

– 10-10

. 0.1 ml

of suspension was taken from the dilutions 10-3

,

10-7

and 10-9

and spreaded over the surface of

sterile Nutrient agar medium. The plates were

incubated at 37±2ºC for 24 - 48 hrs.

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 (37ºC) 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 Salkowski’s

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

Salkowski’s reagent (Kuang-Ren et al., 2003).

2.6. Germination test

Germination test was performed to

determine the effect of IAA producing isolate on

plant growth by plate assay.

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



method. Purified IAA sample was compared with

standard IAA on TLC chromatograms.

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

Figure – 2: Germination of seeds after soaking

with the culture

Indole acetic acid (IAA) production is a

major property of rhizosphere bacteria that

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).

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.

Table – 1: Cup assay for plant growth

Soaking

Hours

No: of

seeds

used

No: of

seeds

germinated

% of

Germination

Root

Length

(cm)

Shoot

length

(cm)

No. of

Leaves

Control 15 15 100 2.5 0.3 2

2 15 13 92.31 6 4.2 2

4 15 13 92.31 3 3.7 2



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.

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

1) Ahmad, F., I. Ahmad and M.S. Khan.

2008. Screening of free living rhizospheric

bacteria for their multiple plant growth

promoting activities. Microbiology

Research, 163: 173 –181.

2) Ajay Kumar, Amit Kumar, Shikha Devi,

Sandip Patil, Chandani Payal and Sushila

Negi. 2012. Isolation, screening and

characterization of bacteria from

rhizospheric soils for different plant

growth promotion (PGP) activities: an in

vitro study. Recent Research in Science

and Technology, 4(1): 01 – 05.

3) Arshad, M and W.T. Frankenberger. 1993.

Microbial production of plant growth

regulators. In: Blaine, F., Meeting, Jr.

(Eds.), Soil Microbial Ecology. Marcel and

Dekker, Int., New York, pp: 307-347.

4) Mohite, B. 2013. Isolation and

characterization of indole acetic acid

(IAA) producing bacteria from

rhizospheric soil and its effect on plant

growth Journal of Soil Science and Plant

Nutrition, 13(3): 638 – 649.

5) 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:

1670 – 1680.

6) Janardan Yadav, Jay Prakash Verma and

Kavindra Nath Tiwari. 2013. Effect of

plant growth promoting Rhizobacteria on

seed germination and plant growth

Chickpea (Cicer arietinum L.) under in

Vitro Biological Forum — An

International Journal, 2(2): 15 - 18.

7) Savita Kerkar, Laxmi Raiker, Anil Tiwari,

Shanmugam Mayilra and Syed Dastager.

2012. Biofilm associated indole acetic acid

producing bacteria and their impact in the

proliferation of biofilm mats in solar

salterns. Biologia, 67(3): 454 – 460.

8) Madhuri, M and A. Sahasrabudhe. 2011.

Screening of Rhizobium for indole acetic

acid production. Annuals of Biological

Research, 2(4): 460 – 468.

21 LSA Bhuvaneshwari

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