EFFECT OF SIDEROPHORE ON PLANT GROWTH PROMOTION. HENA.Y.PARMAR1 AND HEMLATTA CHAKRABORTY2 1,2 Department 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 Page 60
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 Page 61
fermentation (Glucose, Lactose), Oxidize test, Nitrate reduction test, Gelatin hydrolysis, Citrate utilization and growth at 4oc and 41oc 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) Page 62
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). Page 63
Absorbance maxima of siderophore 0.45 0.4 Absorbance. 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 380 390 400 410 420 430 440 450 Wavelength (nm) 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 undiluted 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. Page 64
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 Page 65
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 Page 66
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. Page 67
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