Indian J. Agric. Res.

, 42 (2) : 153 -156, 2008

SIDEROPHORE PRODUCTION BY PLANT GROWTH

PROMOTING RHIZOBACTERIA
Alka Gupta and Murali Gopal
Department of Microbiology,
Central Plantation Crops Research Institute,
Kudlu P.O., Kasaragod - 671 124, Kerala, India
ABSTRACT
Under iron-limiting conditions, many bacteria secrete ferric iron-specific ligands, generically
termed siderophores, which aid in sequestering and transport of iron. We report here the production
of siderophores by selected plant growth promoting rhizobacteria. Production of siderophores by plant
growth promoting rhizobacteria was detected via the chrome azurol S assay, a general test for
siderophores, which is independent of siderophore structure.

Siderophores are low molecular weight
compounds that are produced under iron-limiting
conditions, chelate the ferric ion (Fe 3+) with a
high specific activity, and serve as vehicles for
the transport of Fe (III) into a microbial cell. In
rhizosphere, the availability of iron for microbial
assimilation is extremely limiting (Buyer and
Sikora, 1990; Loper and Buyer, 1991). To survive
in such environment, organisms secrete iron-
binding ligands called siderophores which can
bind the ferric ion and make it available to the
host organisms.
Recent research as have shown the
involvement of these compounds in plant growth
promotion (Bakker et al., 1986; 1987; Dileep
Kumar and Dube, 1993; Gupta, 1995). The root
colonizing bacteria, which directly or indirectly
stimulate plant growth are referred as plant
growth promoting rhizobacteria (PGPR). They
promote growth of several annual crops by
increased uptake of nitrogen (Bakker et al., 1991)
and phosphorus (Linderman, 1992), synthesis
of plant growth promoting substances
(Frankenberger and Arshad, 1995) and uptake
of iron through siderophores (Sindhu et al.,
1997). PGPR produce siderophores having
greater binding potentials (Schippers et al.,
1988).
In the present study, we have screened
ten PGPR isolates for siderophore production. The
details of the PGPR isolates are given in Table 1.
King’s B agar medium (King et al.,
1954) was used for growth and maintenance of
Pseudomonas sp., Pseudomonas fluorescens and
Pseudomonas striata, Sabouraud’s agar medium
was used for Bacillus coagulans, Brevibacillus
brevis and Enterobacter sp. whereas Yeast
Extract Glucose agar medium was used for
growth and maintenance of Bacillus sp. The
PGPR isolates were raised in their respective broth
media by inoculating @ 107 cells/50 ml of the
culture medium and incubated under shake
culture conditions (150 rpm) at 28±20C. The
cultures were incubated for 24-48 h. These broth
cultures were used for spotting onto the chrome
azurol S (CAS) agar medium plates and
incubated at 28±20C. Chrome azurol S agar
medium was prepared according to the protocol
of Schwyn and Neilands (1987) with slight
modifications, wherein PIPES buffer was
replaced with HEPES and the MM9 salts
consisted of K2HPO4 (2%), KH2PO4 (2 %),
MgSO4 (25 %), CaCl2 (12.5 %) and NH4Cl (10
mM). After 72 hours of incubation, plates were
observed for orange halo formation around
the bacterial growth. The size of the orange
halo around the colony of each PGPR
isolate was measured. The broth cultures
were also used to inoculate iron free SM
medium (Meyer and Abdallah, 1978)
consisting of K 2HPO4 (6.0 g/l), KH 2PO4 (3.0
g/l), MgSO4 .7H2 O (0.2 g/l), (NH 4) 2 SO 4 (1.0
154 INDIAN JOURNAL OF AGRICULTURAL RESEARCH

Table 1. Details of PGPR isolates

Organism
Bacillus coagulans
Bacillus sp.
Bacillus polymyxa
Brevibacillus brevis
Enterobacter sp.
Pseudomonas sp.
Pseudomonas fluorescens
Pseudomonas striata
Azospirillum brasilense
Enterobacter sp.
Isolate No. Plant growth promoting attributes
BbN Production of phytohormones
BAC Mineral phosphate soubilization
BbP Mineral phosphate soubilization
BbC Production of phytohormones
EB-RS-1 Production of phytohormones and antibiotics
Z2 Production of phytohormones and antibiotics
PFII Production of antibiotics
PS1 Mineral phosphate solubilization
SP7 Atmospheric nitrogen fixation and production of phytohormones
EG-ER-2 Production of plant growth promoting hormones

Table 2. Siderophore production by PGPR isolates on CAS agar plates

PGPR isolates Isolate no. Siderophores
Production Diameter of orange
halo (mm)
Bacillus coagulans BbN - -
Bacillus sp. BAC - -
Bacillus polymyxa BbP - -
Brevibacillus brevis BbC + 6.7
Enterobacter sp. EB-RS-1 + 11.2
Pseudomonas sp. Z2 + 15.8
Pseudomonas fluorescens PFII + 26.0
Pseudomonas striata PS1 - -
Azospirillum brasilense SP7 + 7.2
Enterobacter sp. EG-ER-2 + 18.4
+ = Siderophore production;
- = No siderophore production.

g/l) and succinic acid (4.0 g/l) at the rate of 1%
(v/v) inoculum. The PGPR cultures were
incubated at 28±20C for 24-72 h with constant
shaking at 120 rpm. After the incubation, the
culture broths were centrifuged in a refrigerated
centrifuge at 10,000 rpm for 10 min. The cell
free supernatants were subjected to quantitative
estimation of siderophores by CAS-shuttle assay
(Payne, 1994). In this assay, 0.5 ml of culture
supernatant was mixed with 0.5 ml of CAS
reagent and absorbance was measured at 630
nm against a reference consisting of 0.5 ml of
uninoculated broth and 0.5 ml of CAS reagent.
Siderophore content in the aliquot was calculated
by using the following formula:
Ar - As
% Siderophore units = x 100
Ar
Where,
Ar = absorbance of reference at 630 nm (CAS
reagent) and As = absorbance of sample at 630
nm.
Out of ten PGPR isolates tested, only
six were found to produce siderophores (Table
2). The other four PGPR isolates did show
some growth on CAS agar medium but no
orange halo formation (Fig. 1). Pseudomonas
fluorescens produced maximum amount of
siderophores (as shown by the size of the
orange halo around the bacterial colony and
the % siderophore units), followed by
Enterobacter sp., Pseudomonas sp.,
Enterobacter sp., Azospirillum brasilense and
Brevibacillus brevis. Same trend was observed
in both qualitative and quantitative detection
of siderophores produced by various PGPR
isolates (Tables 2 and 3). Siderophore producing
 Vol. 42, No. 2, 2008 155
Table 3. Siderophoregenesis by PGPR isolates
PGPR isolate Isolate No. Incubation time (h) % siderophore units*
Bacillus coagulans BbN 48 h -
Bacillus sp. BAC 32 h -
Bacillus polymyxa BbP 48 h -
Brevibacillus brevis BbC 52 h 21
Enterobacter sp. EB-RS-1 52 h 38
Pseudomonas sp. Z2 24 h 51
Pseudomonas fluorescens PFII 24 h 76
Pseudomonas striata PS1 32 h -
Azospirillum brasilense SP7 72 h 24
Enterobacter sp. EG-ER-2 48 h 64
*as determined by CAS-shuttle assay.

Fig. 1. Siderophores produced by PGPRs on CAS-agar medium

PGPRs, particularly Pseudomonas spp., play and Albouvette, 1993) by depriving the pathogen
vital role in stimulating plant growth and also in from iron nutrition, thus resulting in increased
controlling several plant diseases (Lemanceau crop yield (O’Sullivan and O’Gara, 1992).
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