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EFFECT OF SIDEROPHORE ON PLANT GROWTH PROMOTION.
HENA.Y.PARMAR1 AND HEMLATTA CHAKRABORTY
2
1,2Department of Microbiology, K.J. Somaiya College of Science and Commerce, Vidyavihar, Mumbai
Abstract
At present fertilizer has become essential to modern agriculture to feed the growing population.
Though chemical fertilizers increase crop production; their overuse has hardened the soil,
decreased fertility, strengthened pesticides, polluted air and water, and released greenhouse gases,
thereby bringing hazards to human health and environment as well. Due to the adverse effect of
chemical fertilizers and the presence of plant pathogens, apart from using chemically based
methods present study provides a biological platform to increase plant productivity. It has been
reported that many siderophore producing bacterial and fungal strains have their potentials in
plant growth promotions because although iron is abundant in the soil it is unavailable to plants
because of its low solubility. Thus siderophore produce will chelate iron and make it available to
the plant. Pseudomonas fluorescens was able to produce extracellular water soluble yellow green
siderophore of pyoverdine type in succinate medium. Siderophore produced was also proved to be
useful for plant growth promotion due to increase in root length, shoot length and number of leaves
of leguminous plants like Lens Culinaris and Phaseolus lunatus when grown under iron limiting
conditions with siderophore supplements. Thus siderophore can be used in combination with other
biofertilizers to increase crop productivity.
Key words: chemical fertilizers, decreased fertility, biological platform, siderophore, Pseudomonas
fluorescens, plant growth promotion, biofertilizers.
I. Introduction
In modern cultivation process indiscriminate use of fertilizers, particularly the nitrogenous and
phosphorus, has led to substantial pollution of soil, air and water. Excessive use of these chemicals
exerts deleterious effects on soil microorganism, affects the fertility status of soil and also pollutes
environment. The application of these fertilizers on a long term basis often leads to reduction in pH
and exchangeable bases thus making them unavailable to crops and the productivity of crop declines
[6]. Thus due to such adverse effects of chemical fertilizers at present there is an urgent need of a
biological agent which can be used in place of such chemical fertilizers.
It has been observed that Plant growth promoting rhizobacteria (PGPR) can stimulate growth by
one or more different mechanisms. The direct mechanisms include production of growth hormones
like IAA, phosphate-solubilization and uptake of iron, whereas indirect mechanisms include check on
phytopathogens by the release of HCN, antibiotics and siderophores. Among PGPR, fluorescent
pseudomonads (FLPs) possess several properties best suited for survival and colonization in the
rhizosphere environment [2]. Pseudomonas species have also been known for their siderophore
production for many years and therefore many reports on the isolation and characterization of their
siderophores have been published. The importance of siderophore extends their applications in
agriculture, biotechnology and medicine. Siderophores are low molecular weight high affinity iron
chelators produce by microorganisms under iron limited conditions. Siderophores are multidentate,
organic, oxygen donor ligands that usually have Hydroxamate, Catecholate or Carboxylate moieties.
Siderophores facilitate the solubilization and transport of iron into the cell by cognate transport system
[3]. Siderophores efficiently deplete iron from the environment making it less available to certain
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competing microorganisms including plant pathogens. Therefore this property of the siderophore
produced by many rizobacteria may be of use to enhance their survival in the rhizosphere [12].
On the basis of this, present study was carried out to provide a biological platform by investigating the
involvement of siderophore in plant growth promotion. This was done by studying iron nutrition of
the leguminous plant. For this study, intense siderophore producers were isolated from spinach roots
surface. The effectiveness of Siderophore from the obtained strain was then analysed for plant growth
promotion by monitoring growth of the plants with increase in root length, shoot length and number of
leaves with colour.
II. Materials and Methods
2.1. Collection of sample
Spinach roots with soil attached on it is taken as a sample (Kalidas, Mulund west). Healthy plants
were obtained by uprooting the plant along with its root [8].
2.2. Isolation of siderophore producers
Isolation was carried out as given by H. Manjunatha et al (2013)[8] with slight modification. 1gram of
roots with its surface soil was cut from the plant and transferred into 250ml of conical flask containing
100ml of sterile distilled water and incubated on shaker at RT for 6-7 hrs. 1ml was taken from this
medium and was serially diluted up to 10-4,
10-5
, 10-6
and about 0.1ml of this was surface spread on
King’s B medium to isolate the colonies. The plates were incubated at 28oC for 48 hr. The colonies
showing yellow pigmentation on King’s B medium were taken for further studies. The obtained
isolates were then grown as pure culture on sterile King’s B medium and isolates was maintained by
weekly transfer on sterile King’s B and on Nutrient agar slants.
2.3. Isolation of intense siderophore producers in liquid media.
Siderophore production was studied using succinate medium (SM) consisting of following
components: 1L of distilled water contains K2HPO4 (4 g), KH2PO 4(6 g), Succinic acid (3 g),
(NH4)2SO 4 (1 g), MgSO4 (0.2 g), pH-7. Method was carried out as given by R.Z Sayyed et al (2005)
[13] with slight modification. In this present study, 25ml succinate medium was inoculated with 0.1ml
of inoculum and incubated on shaker for 48 h at 28oC.Among the isolates obtained the organism
responsible for intense siderophore production in SM medium is selected for further studies.
2.4. Confirmation of extracted siderophore.
Siderophore produced by the selected isolate is extracted by centrifugation of SM media at 15000rpm
for 20-25 min. The supernatant is collected and the cell pellet is discarded. Supernatant is
spectrophotometrically scanned from 380-450 nm for maximum absorbance and look for
Fluorescence under UV light [11].
2.5. Identification of detected siderophore producing organism.
A Nutrient broth suspension of the isolate was made from 24hrs old culture of the isolate grown on
King’s B agar slant. The identification was done by using following characteristics:
2.5.1. Morphological characteristics of the isolate involved the microscopic determination of gram
nature, shape, arrangement and motility of organism in nutrient broth suspension.
2.5.2. Cultural characteristics involved determination of size, shape, color, elevation, opacity,
consistency of the colony on Nutrient agar.
2.5.3. Biochemical characteristics involved inoculation of saline suspension of isolate in various
biochemical media. The following biochemical test was performed which includes Carbohydrate
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fermentation (Glucose, Lactose), Oxidize test, Nitrate reduction test, Gelatin hydrolysis, Citrate
utilization and growth at 4oc and 41
oc was observed. For identification and confirmation of isolate
Berge’s manual was referred.
2.6 Plant Study
Plant study was carried according to Preethi Ravindran et al (2015) and R.Z Sayyed et al (2005) [12]
[13] with modification. The seeds of Lens Culinaris (Masoor dal i.e. red lentils) and Phaseoluslunatus
(large white Lima Beans) were surface sterilized with Distilled water to remove any surface bacteria.
These seeds were then allowed to germinate on sterile moistened cotton cloth. This was followed by
regular moistening with sterile distilled water. After germination these seeds were collected and
immersed in media containing essential nutrients in the form of salts supplemented with iron. This can
serve as a control. On the other hand some seeds of Lens Culinaris and Phaseoluslunatus were
immersed in same media but under iron limiting conditions. This was followed by incubation on
shaker at RT for 24hrs. Soil was collected and autoclaved to reduce the microbial load. 8-9 germinated
seeds of Lens Culinaris along with 3-4 germinated seeds of Phaseoluslunatus from the flask with iron
limiting conditions were immersed for 10-15 min in siderophore released by the isolate under study.
These treated seeds i.e. form the control medium, iron deprived media and iron deprived along with
Siderophore supplemented conditions were then transferred to the soil and allow to grow for
minimum 7-8 days cycle under pot culture conditions. Growth of the plants was monitored as shoot
length, root length and number of leaves with its colour
III. Results and Discussion
Siderophore producing bacteria were isolated from washings of spinach root surface. Three
bacterial colonies were obtained which were able to produce diffusible yellow green florescent
pigment around them on Kings B (KB) Agar. Three isolates which obtained above was allowed to
produce siderophore in liquid medium i.e. succinate medium (Figure 1). The result showed that all the
three isolate were able to produce diffusible yellow green pigment but out of these three, one isolate
were visually producing intense yellow green diffusible pigment. This was also compare with the
control where there was pyocynin production by Pseudomonas aeruginosa. Thus Siderophore
produced by selected isolate was of pyoverdine type (Figure 2). This isolate obtained was selected for
further study.
Figure 1. Siderophore in succinate media(right), control (left)
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Figure 2. No blue pigment on KA (left), control (right)
The yellow green diffusible pigment obtained after separation of cells of selected isolate was
confirmed as siderophore from the result obtained by addition of FeCl3. The production of reddish
brown precipitate indicates the presence of siderophore. This is because of iron acquisition by
siderophore molecule (Figure 3).
Figure 3. Siderophore iron complex (right tube), control (left tube)
In the research study adopted by Bholay A. D et al (2012) [3] they determine the effect of iron
concentration on the siderophore production where the Succinate medium was supplemented with iron
(FeCl3) at conc. 1 to 50 µM in different sets, for both the Pseudomonas species separately. Following
the inoculation and incubation at 28oC for 24 hours at 120 rpm, the siderophore was confirmed due to
appearance of reddish –brown colour in all the tubes containing different concentration of FeCl3.
The presence of siderophore was even further confirmed by UV Spectrophotometric scan from 380
nm to 450nm of yellow green diffusible pigment after its separation from the cells. Thus from the
graph shown in the figure 8, it was observed that the supernatant showed maximum absorbance at 410
nm which is a typical characteristics of siderophore obtained from previous research studies. This
supernatant was also showing fluorescence when it was observed under UV light (Figure 4 and 5).
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Figure 4. Graph of UV Spectrophotometric scan of siderophore
Figure 5. Fluorescence of Siderophore under UV light.
In the research reported by Mehri Ines et al (2012) [11] Spectrophotometric analysis of the un-
diluted bacteria supernatant showed an absorption area between 350 and 450 nm with a sharp peak at
about 400 nm, characterizing the siderophore of pyoverdine type. The maximum absorbance obtained
for the strain Pseudomonas S29 was at 400 nm. The other strains saved the maximum absorbance
between 405 and 410 nm. The maximum absorbance also varied from 400 to 410 nm which may
indicate the diversity of compounds produced by their obtained strains. They reported that this
multiplicity may be due to the nature and the number of the aminoacyl residues in the peptide moiety.
Aditi Bhattacharya (2010) reported the presence of siderophore, a polar substance with bands of
absorption at different wavelengths such as 260 nm and 402 nm, 448nm, absorbance maxima of 365
and 410 nm for pyoverdins and its ferric chelate, respectively, 350 – 600 nm in absence and presence
of iron. A shift on the longer wavelength side after iron chelation has also been observed in this study.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
380 390 400 410 420 430 440 450
Wavelength (nm)
Absorbance maxima of siderophore
Ab
sorb
an
ce.
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Peak shift on the longer wavelength side has also been observed with respect to calcium, cadmium,
lead, chromium, Nickel, copper, Manganese, Magnesium.
Thus siderophore producing bacteria was identified on the basis of Morphogical, Cultural and
Biochemical Characteristics. Morphologically they were Gram negative motile rods which have the
ability to grow even at 4oc. Biochemical characteristics revealed the presence of oxidase, gelatinase
and nitratase enzyme along with citrate utilization. Thus by comparing the obtained characteristics
with standards for Pseudomonades in Bergey’s manuals, obtained organism was found to be
Pseudomonas fluorescens.
3.2. Results for plant growth promotion
Siderophore producing plant growth promoting rhizobacteria have shown to play a vital role in
Iron nutrition of the plant and therefore in plant growth promotion leading to healthy plants [13]. It
was therefore checked in the present study whether the plant is capable of utilizing microbial
siderophores for iron nutrition. For this leguminous plants like Lens Culinaris (Masoor dal i.e red
lentils) and Phaseoluslunatus (large white Lima Beans) were grown under iron limiting conditions
and under iron limiting conditions with siderophore supplements. Plants growth under Iron
supplemented conditions was used as control (Table 1 and 2). Better growth in terms of increase in
root length, shoot length and number of leaves was observed in plant grown under iron limiting
conditions with siderophore supplements as when compare to the plants grown under iron limiting
conditions (Figure 6 and 7).
Mansoureh Sadat et al (2012) [10] also reported that Pseudomonas fluorescens forms a major
constituent of Rhizobacteria that encourage the plant growth through their diverse mechanisms. In this
investigation, 20 strains of Pseudomonads isolated from the rhizosphere soils of paddy areas in
Malaysia and were screened for their plant growth promoting activity. All the 20 tested isolates of
Pseudomonads were positive for the production of siderophores and HCN, while of the 20 antagonist
bacteria strains, 15 strains (75%) showed positive for the production of plant growth-promoting
hormone, IAA. All the twenty bacterial isolates (except DL21) inhibited the pathogen in the dual
culture assay. Following API 20NE biochemical identification kit, of the 20 isolates, 15 strains were
identified as Pseudomonas fluorescens, 3 isolates belong to the species of P.luteola, one isolates to the
P.aeruginosa.
Table 1. Effect of siderophore produce by Pseudomonas fluorescens on the growth of Lens Culinaris (Masoor dal i.e
red lentils)
Shoot length
(cm)
Root length
(cm)
Number of
leaves
Control
7
1.5
13
Iron deprived conditions
5
3
10
Iron deprived and siderophore supplemented
conditions.
9
5
19
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Figure 6. Effect of siderophore on the growth of Lens Culinaris (A- Iron deprived, B- control, C- Iron deprived and
Siderophore treated)
Table 2: Effect of siderophore produce by Pseudomonas fluorescens on the growth of Phaseoluslunatus (large white
Lima Beans)
Shoot length (cm)
Root length
(cm)
Number of
leaves
Control
15
4.5
9
Iron deprived conditions
10
3
6
Iron deprived and siderophore
supplemented conditions.
17.5
6.5
13
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Figure 7. Effect of siderophore on the growth of Phaseoluslunatus, control (right), Iron deprived (middle), Iron
deprived plus Siderophore treated (left).
IV. Conclusion
Thus in the present study Pseudomonas fluorescens was able to overcome the major problem
related to the adverse effects of chemical fertilizers on plant growth and productivity. Thus a
biological platform was built to combat this problem. Pseudomonas fluorescens produce extracellular
water soluble yellow green Siderophore which was proved to be useful for plant growth promotion
due to increase in root length, shoot length and number of leaves of leguminous plants like Lens
Culinaris and Phaseoluslunatus when grown under iron limiting conditions with siderophore
supplements. Thus siderophore can be used in combination with other biofertilizers to increase crop
productivity.
V. Acknowledgements We Thank K.J. Somaiya college of Science and Commerce, Vidyavihar, Mumbai for providing
us facilities during the course of our research work.
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