VENKATESH

Journal of Scientific & IndustrialBABU et al: INDOLE-3-ACETIC ACID FROM FILAMENTOUS CYANOBACTERIA
Research 581
Vol. 72, Sept - Oct 2013, pp. 581-584

Indole-3-acetic acid from filamentous cyanobacteria: screening, strain

identification and production
S Venkatesh Babu1, B Ashokkumar2, N Sivakumar1, P Sudhakarsamy3 and P Varalakshmi1*
1*
Department of Molecular Microbiology, 2Department of Genetic Engineering, School of Biotechnology, 3School of
Biological sciences, Madurai Kamaraj University (MKU), Madurai 625 021, India

Received 19 March 2013; revised 08 May 2013; accepted 29 May 2013

This study presents Filamentous cyanobacterial isolates for their ability to produce phytohormone indole-3-acetic acid
(IAA) by Salkowski’s colorimetric assay method. Among 15 isolates screened, two of them produced auxin-like secretions
significantly in BG11/ ASN III media containing 4 mg/ml of tryptophan. Based on 16S rDNA sequence analysis, strains that
produce maximum IAA were most closely related to Geitlerinema sp. (99.0% similarity) and Leptolyngbya sp. (95.0% similarity).
IAA production was maximum (51.06 µg/ml) when strain Leptolyngbya sp. was cultivated in BG11 media at pH 9.0, whereas
Geitlerinema sp. produced a maximum of 67.87 µg/ml in ASN III media at pH 9.0 in 14 days of incubation. This is the first report
of IAA production by Geitlerinema sp. and Leptolyngbya sp.

Keywords: Geitlerinema sp., Indole-3-acetic acid and Leptolyngbya sp.

Introduction (Trp 3 IAM 3 IAA); and iii) indole-3-pyruvic acid (IPA)

Petrochemical based-fertilizers and toxic pesticides,
which have caused detrimental effects to soil, water
supplies, foods & animal health, have engendered
considerable interest in evaluation of renewable
biofertilizers1. Moreover, they are very expensive and
their continuous and extensive use leads to a loss of
organic humus, ends up with deterioration of fertility of
top soil and a decrease in soil porosity. These problems
can be reduced by application of plant growth regulators
or phytohormones that play important roles in plant
responses to growth and development, resistance to biotic
and abiotic stresses2 . Cyanobacteria are prokaryotic
photosynthetic microorganisms that produce a wide array
of biologically active substances like proteins, vitamins,
carbohydrates, amino acids, polysaccharides and plant-
growth regulators3 . Among phytohormones, indole-3-
acetic acid (IAA) is a natural auxin that directs many
aspects of plant growth and development including
induction and regulation in cell division, root extension,
vascularization, apical dominance and tropisms 4,5 .
Pathways for microbial synthesis of IAA are as follows:
i) indole-3-acetonitrile (IAN) pathway (Trp 3 indole- 3-
acetaldoxime 3 IAN 3 IAA); ii) IAM pathway
*Author for correspondence
E-mail: vara5277@gmail.com
pathway (Trp 3 IPA 3 indole-3-acetaldehyde [IAAld] 3
IAA). IAM and IPA pathways are utilized by bacteria
more commonly than IAN pathway and these pathways
are all Trp dependent6. Presence of phytohormones in
cyanobacterial extract may induce rice callus in micro-
propagation and also influence germination of seeds like
paddy, ladies finger and sunflower7,8. Several bacterial
and cyanobacterial strains require L- tryptophan as a
substrate for IAA production, which functions as a
precursor in production of IAA9. This study demonstrates
efficiency of cyanobacteria isolated from fresh water
and estuarine for production of IAA in BG11/ASN media
with L- tryptophan.
Experimental Section
Sample Collection, Isolation and Purification
Cyanobacterial samples were collected from fresh
water as well as estuarine water source in different
regions of Tamil Nadu, India. Samples collected from
fresh water bodies were inoculated in BG-11 medium
and estuarine samples were inoculated in ASN-III
medium. Pure cultures of cyanobacterial isolates were
done from processed samples maintained in poly-bag by
serial dilution techniques. These plates were incubated
at room temperature (RT) for 8 to 14 days. After
incubation, a single colony or array was transferred into
the fresh appropriate medium and kept for incubation.
582 J SCI IND RES VOL 72 SEPT - OCT 2013
IAA Production and Preparation of IAA Standard by using
Salkowski’s Reagent
Cyanobacteria isolated from estuarine as well as
fresh water environments were screened for production
of phytohormone IAA/indole derivatives by Salkowski’s
colorimetric method6. Colorimetric Salkowski assay was
performed at initial screening for the presence of IAA
and further confirmed by FTIR studies. IAA producing
cyanobacteria are identified by 16S rRNA sequencing
and sequences submitted to NCBI Genbank. IAA
production by Geitlerinema sp. and Leptolyngbya sp.
is the first time reporting that these species of
cyanobacteria are efficiently producing IAA in BG11/
ASN media.
For standard solution, 1.0 mg of IAA (Hi-media) was
dissolved in 1.0 ml of methanolic water and used as stock.
From stock solution, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 µl
were taken, made-up into 1000 µl with appropriate
volume of distilled water and 1000 ml of Salkowski’s
reagent was added and incubated in dark for 30 min.
After incubation, absorbance was read at 535 nm in
spectrophotometer.
Detection of IAA in Cyanobacteria
Pure cultures of cyanobacterial isolates (100 mg)
from fresh water samples were inoculated in duplicates
into BG-11 medium at pH 9.0, whereas cyanobacterial
isolates from estuarine water samples were inoculated
into ASN-III medium at pH 9.0 and kept for incubation
at RT for 14 days. Both media were amended with 4
mg/ml of tryptophan (filter sterilized 0.22 mM and 30
mm diam millipore membrane) in an aseptic conditions
and maintained in 16h: 8h (Light: Dark) in first week and
changed into 8h: 16h (Light: Dark) in second week
onwards. After 14 days of incubation, culture filtrates
were harvested by centrifugation at 10,000 RPM for 10
min and filtrates were subjected to Salkowski’s
colorimetric assay and absorbance was read at 535 nm.
Strain Identification by 16S rRNA Analysis
Two cyanobacterial isolates that showed maximum
IAA production were microscopically identified initially
and further confirmed by 16S rRNA gene sequencing.
For 16S rRNA analysis, genomic DNA was isolated and
PCR was performed using genomic DNA as template
with universal primers (8F and 1462R). PCR program
was run on BIO-RAD Thermal cycler, USA, with
following settings: 5 min initial denaturation at 94° C, 32
cycles of 30 s at 94° C, 1 min at 54° C, 2 min at 72° C
and amplicons were sequenced. Sequences of 16S rRNA
gene were compared with NCBI sequence database
(GenBank) through BLAST (www.ncbi.nlm.nih.gov/
BLAST).
Results and Discussion
Cyanobacterial IAA Biosynthesis
Cyanobacterial pure cultures (15) were isolated from
fresh and estuarine water sources and screened for their
ability to produce IAA. Among 15 isolates, two strains
(BGA-44 and PK-12) showed maximum IAA production
extr acellularly in respective BG11/ASN media
supplemented with (4 mg/ml) tryptophan in 14 days of
incubation. Total amount of IAA was quantified by
Salkowski colorimetric method and that were compared
with authentic IAA. Authenticity and reliability of this
standard graph were confirmed since R2 value is 0.995.
Maximum production of IAA (51.06 µg/ml) was by BGA-
44 when strain was cultivated in BG11 media with
tryptophan (4 mg/ml) at pH 9.0, whereas there was no
detectable IAA production in BG11 media without
tryptophan. Similarly, PK-12 produced a maximum of
67.87 µg/ml in ASN III media with tryptophan (4 mg/ml)
at pH 9.0, while no IAA production was detected in
ASNIII media, which is devoid of tryptophan. These
results clearly demonstrated tryptophan dependency on
IAA production by both isolates that obtained from
different environments. IAA production with tryptophan
induction by free living and associated cyanobacteria is
well documented9-12. Many reports13-15 state that number
of bacteria produce IAA with and without tryptophan
induction. Presence of auxin like growth promoting
substances has been shown in cyanobacteria including
Oscillatoria annae8, A. platensis9 and Nostoc10 .
Identification of Strains by 16S rRNA Sequencing
Maximum IAA producing strains (BGA-44 and PK-12)
were identified under light microscope. Morphological
characteristics of BGA-44 were similar to Leptolyngbya
sp. whereas PK-12 showed morphological characteristics
similar to Geitlerinema sp.16. However, molecular-based
tools like 16S rRNA gene sequencing are extensively
used for bacterial identification17,18. Hence, in present
study, 16S rRNA gene sequence analysis was carried
out using genomic DNA that was isolated from IAA
producing cyanobacterial strains (Fig. 1) as template with
16S rRNA specific universal primers (8F and 1462R)
for PCR amplification (Fig. 2). Sequences of 16S rRNA
gene were compared with NCBI sequence database
(GenBank) through BLAST. Strains were confirmed by
blasting resulting sequence to NCBI sequence database.
 VENKATESH BABU et al: INDOLE-3-ACETIC ACID FROM FILAMENTOUS CYANOBACTERIA 583

1 2 3 4 5 6

10 kb

1 kb 115
.5kbKb
11 Kb
500 bp
005 Kb

Fig. 2— 16S rRNA amplification for strains Leptolyngbya and

Geitlerinema sp. [Lane 1: Leptolyngbya, Lane 2: Geitlerinema,
Fig. 1— Genomic DNA isolation from Leptolynbya sp. and
Lane 3 & 4: Positive control (bacteria), Lane 5: Negative control,
Geitlerinema sp. (Lane 1: 1kb ladder, Lane 2: Leptolyngbya sp.
Lane 6: 1Kb Ladder]
and Lane 3: Geitlerinema sp.)

a)

b)
Fig. 3— Phylogenetic tree constructed by MEGA 5.10 for: a) Leptolyngbya sp.; and b) Geitlerinema sp.

Strain BGA-44 was most closely related to Leptolyngbya accession numbers JX317745 for Leptolyngbya sp. and
sp. with 95% similarity, while PK-12 showed 99% JX317744 for Geitlerinema sp. Phylogenetic tree
similarity with Geitlerinema sp. 16S rRNA gene (Fig. 3) was obtained by applying neighbour-joining
sequences were submitted to NCBI GenBank under method. This is the first report about IAA production by
584 J SCI IND RES VOL 72 SEPT - OCT 2013
both Leptolyngbya sp. and Geitlerinema sp. and about
Leptolyngbya sp. from fresh water and Geitlerinema
sp. from estuarine that had the ability to produce IAA.
Conclusions
This study is the first on production of IAA/
derivatives of indole (auxin-like compounds) by
Leptolyngbya sp. and Geitlerinema sp. and shows its
dependency for tryptophan on IAA production. This study
also emphasizes for analysis of genes corresponding IAA
production so that pathways involved in IAA can be
elucidated for commercial production of IAA for
sustainable agriculture.
Acknowledgement
Authors thank University Grants Commission
(UGC), New Delhi, for financial support.
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