effect of siderophore on plant growth promotion. · effect of siderophore on plant growth...

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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

International Journal of Applied and Pure Science and Agriculture (IJAPSA)

Volume 02, Issue 03, [March - 2016] e-ISSN: 2394-5532, p-ISSN: 2394-823X


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




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)




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).




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.



@IJAPSA-2016, All rights Reserved 5

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




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




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.

Bibliography

[1] Aditi Bhattacharya (2010) Siderophore mediated metal uptake by Pseudomonas fluorescens and its comparison to

iron chelation, Cey.J.Sci (bio sci), 39(2):147-155.

[2] Alok Sharma, B. N. Johri (2003), Growth promoting influence of siderophore-producing

[3] Pseudomonas strains GRP3A and PRS9 in maize (Zea mays L.) under iron limiting conditions, Microbiol. Res, 158:

243–248.




[4] Bholay A. D., Jadhav Priyanka.U., Borkhataria B. V.,Mayuri, V. Dhalkari (2012), Fluorescent Pseudomonads as

Plant Growth Promoting Rhizobacteria and their Siderophoregenesis, IOSR Journal of Pharmacy and Biological

Sciences, 3(1): 27-32.

[5] Eli Bar-Ness, Yitzhak Hadar, Yona Chen, AbrahanShanzer and Jacqueline Libman (1992), Iron Uptake by Plants

from Microbial Siderophores, Plant physiol, 99:1329-1335.

[6] Alemu (2013), Isolation of Pseudomonas fluorescensspecies from rhizospheric soil of faba bean and assessment of

their siderophores production, International Journal of Advanced Research, 1(8): 203-210.

[7] Govind Gupta, Shailendra Singh Parihar, Narendra Kumar Ahirwar, Sunil Kumar Snehi and Vinod Singh (2015),

Plant Growth Promoting Rhizobacteria (PGPR): Current and Future Prospects for Development of Sustainable

Agriculture, J Microb Biochem Technol, 7(2): 096-102.

[8] Girija Ganeshan, Manoj Kumar (2005), Pseudomonas fluorescens, a potential bacterial antagonistic to control plant

diseases, Journal of plant intraction, 1(3): 123-134.

[9] H.Manjunatha and M.K.Naik (2013).Biological and molecular characteristics of fluorescent Pseudomonas isolate

from crop rhizosphere soil, In.J.Sci.Res and Tech, 1(1): 18-22.

[10] K. S. Prathibha and K.G. Siddalingeshwara (2013), Effect of plant growth promoting Bacillus subtilis and

Pseudomona fluorescens as Rhizobacteria on seed quality of sorghum, International Journal of current microbiology

and applied sciences, 2(3): 11-18.

[11] Mansoureh Sadat SharifiNoori and HalimiMohd Saud (2012), Potential plant growth promoting activity of

Pseudomonas species isolated from paddy soil in Malaysia as biocontrol agent, Journal of Plant Pathology and

Microbiology, 3(2): 1-4.

[12] MehriInes,Khessairi Amel,Turki Yousra,SaidiNiela, DalyImen, Meyer Jean Marie and Hassen Abdennasseur (2012),

Effect of dose response of zinc and manganese on siderophores induction. American Journal of Environmental

Science, 8(2):143-055

[13] PreethiRavindran, Tara Menon (2015), Role of siderophoreProducing Rhizobacteria in plant growth promotion, Life

science international research journal, 2 spl Issue: 37-43.

[14] R.Z Sayyed, N.S Gangurde, P.R Patel, S.A Joshi and S.B Chincholkar, “Siderophore production by

Alcaligenesfaecalis and its application for growth promotion in Archis hypogea”, Indian Journal of Biotechnol, 9:

302-307.

[15] Saharan. K, Sarma. M.V.R.K, Prakash. A, Bisaria. V. S and Sahai.V (2009), Application of Fluorescent

Pseudomonads Inoculant Formulations on Vigna mungo through Field Trial, World Academy of Science,

Engineering and Technology, 3: 700-704.

[16] Deshwal VK, Singh SB, Chubey A and Kumar (2013), Isolation and characterization of Pseudomonas strains from

Potatoes Rhizosphere at Dehradun Valley, India, International Journal of Basic and Applied Sciences, 2(2): 53-55.

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