Process Optimization for Siderophore Production and ...

Process Optimization for Siderophore Production and Evaluation of Bioefficacy and Root Colonizing Potential of Alcaligenes sp. P.R. Patel1, S.S. Shaikh2 and R.Z. Sayyed3 1,2,3

Department of Microbiology, Arts, Science & Commerce College, Shahada–425409, India E-mail: [email protected]

ABSTRACT We report production of siderophores by Alcaligenes sp. and further improvement in yield after optimization of condition at a shake flask level. The highest siderophores concentration was obtained in SAM during 30 hrs of incubation with 1% inoculum level and 6.0g/L of K2HPO4, pH 7, urea, mannitol, tyrosine. The optimization of cultural and growth condition resulted in further increase in siderophore productivity. Ferric iron supported the growth, yield at 100 µm concentration, further increase in Fe++ Concentration affected siderophore productivity. Bacterization of siderophore rich broth of Alcaligenes sp. enhanced seed germination, shoot height, root length, number of leaves, chlorophyll content of wheat (Triticum aestivum) and peanut (Arachis hypogaea) and showed good root colonizing potential. Keywords: Siderophore, Optimization, Alcaligenes sp.

INTRODUCTION Rhizosphere is a site of complex interactions between plant roots and diverse group of root associated microorganisms (Mishra & Kumar 2009). The grandness of this root associated population is critical for maintenance of overall plant health, nutrient uptake and tolerance of environmental stress (Cook 2002). This group of microbes is called Plant growth promoting rhizobacteria (PGPR) directly enhance plant growth by a variety of mechanisms, namely fixation of atmospheric nitrogen, production of siderophores for providing iron to the plant root, solubilization of minerals such as phosphorus and synthesis of phytohormones (Roesti et al. 2006; Khurana and Sharma 2006; Shaikh et al. 2004, Shaikh and Sayyed 2015). Siderophores are low molecular weight iron chelating agents produced by most bacteria and fungi under iron limiting conditions (Saharan & Nehra 2011). Production of siderophore is a device of antagonism siderophore producing microorganism competes for Fe (III) with pathogenic microorganism and inhibit their growth (Leong 1986; Loper & Buyer 1991).

140 Advances in Bio and Medico Sciences

This has been regarded as an eco-friendly approach to environmentally hazardous chemical pesticide. Many different environmental factors affect the synthesis of siderophores, notably the chemical nature of the organic carbon and energy source (Sayyed et al. 2010), metals (Sayyed & Chincholkar 2010), Fe3+ (Sreedevi et al. 2014) amino acids (Sayyed et al. 2010, 2011) and organic nitrogen sources (Sayyed et al. 2005, 2010). Any factor influencing siderophore production influences the performance of PGPR in plant growth promotion and phytopathogens suppression (Sharma & Kaur 2010). Therefore present study was aimed to regulate the cultural parameter for improved siderophore production and to evaluate the potential of plant growth promotion, efficient adsorption of heavy metal ions and effective root colonization by Alcaligenes sp.

MATERIALS AND METHODS SOURCE AND MAINTENANCE OF CULTURE Alcaligenes sp. was used in this study was previously isolated and identified (Sayyed and Patel 2011) on the basis of biochemical characteristics and 16s rRNA sequencing and submitted to gene bank under accession number NCBI accession N. HQ443704.1.

SIDEROPHORE PRODUCTION, DETECTION AND ESTIMATION For growth and siderophore production Alcaligenes sp. (6 X 106 cells ml-1) was grown independently in 500 ml flask containing 100 ml succinic acid medium (SAM) (Meyer and Abdallah 1978) at 28 ± 2ºC at 120 rpm for 24– 48 h. After the incubation, cell density was measured at 620 nm by using double beam UV–Visible spectrophotometer [1240, Shimadzu, Japan]. The detection and estimation of siderophores were performed following the centrifugation at 15 min 5,000 rpm at 4ºC and cell free supernatant was assayed for qualitative estimation of siderophore by using Chrome Azurol Sulphonate (CAS) test (Schwyn and Neilands, 1987) and quantitative estimation by the CAS shuttle assay (Payne 1994).

INOCULUM DEVELOPMENT A loopful of culture of Alcaligenes sp. from nutrient agar slant was separately grown in each 100 mL of iron deficient SAM (Schwyn and Neilands 1987) at 29ºC for 24-30 h with constant shaking at 120 rpm.

PROCESS OPTIMIZATION FOR ENHANCED YIELD SIDEROPHORE Optimization of Physico-chemical Parameter The effect of various media like SAM, Cas-amino acid medium, Barbhaiyya and Rao (BR) and nutrient broth on growth and siderophore production

Process Optimization for Siderophore Production and Evaluation of Bioefficacy 141

was evaluated. In order to evaluate the influence of water source siderophore production and growth was carried out in tap water based SAM and was compared with that of distilled water based SAM. To study the influence of sugars each 100ml of SAM was externally supplemented separately with 1.0 gL-1 each glucose, dextrose, sucrose, mannitol, starch, maltose, lactose and fructose. To study the influence of nitrogen source ammonium sulfate in SAM was replaced individually with urea, yeast extract, meat extract, peptone, ammonium ferric citrate, ammonium ferrous sulfate, ammonium chloride, ammonium acetate, ammonium oxalate, ammonium dihydrogen phosphate, ammonium per sulfate, ammonium solution, ammonium molybdate, casein and beef extract. To study the influence of organic acid Succinic acid in SAM was replaced individually with malic acid, lactic acid, formic acid, acetic acid, citric acid, propionic acid and oxalic acid. In order to observe the effect of different amino acids SAM was differently fortified with 1.0 gL-1 each of serine, lysine, alanine, threonine, cysteine, arginine, tyrosine and methionine. To observe the influence of cell volume, growth and siderophore production was carried put with varying levels (1-5 mg%) of cell mass of of Alcaligenes sp. To observe the variation of phosphate source, K2HPO4 was taken in the range of 0-6 g L-1 and KH2PO4 was taken in the range of 0-3 g L-1 of SAM.

Optimization of Cultural Conditions To observe the effect of incubation period Alcaligenes sp. was separately grown in SAM at 29ƕC for 48 h, Samples were withdrawn after every 6 h intervals and were subjected for growth measurement and siderophore estimation. To observe the effect of pH, Alcaligenes sp. was grow in SAM prepared with different pH from 3-10.

Influence of Metal Ions For detecting the influence of different heavy metals on growth and siderophore production, Alcaligenes sp. was separately grown in SAM, separately supplemented with 100-ʹͲͲͲ Ɋ ǡ MnCl2.4H2O, NiCl.6H2O, ZnSO4.7HO, ZnCl2, FeSO4, CuSO4, CuCl2, HgCl2, FeCl3.6H2O, AgNO3 and COCl2.

BIOEFFICACY TEST The bioefficacy test was carried out to check the performance of siderophore producing isolate for efficient plant growth promotion. Seeds like wheat (Triticum aestivum) and groundnut (Arachis hypogaea) were the first surface sterilized with 0.1% HgCl2, repeatedly washed 2-3 times with distilled water and pasteurized with a siderophore rich broth of Alcaligenes sp. (112 CFU/ml) broth and were then dried in shadow. These seeds (5 wheat seeds/pot and 3 groundnut seeds/pot) were sown in pots containing ͳͲͲ Ɋ


independently added in various pots. Soil moisture was maintained at 60% level throughout the experiment and was observed up to 30 and 45 days respectively. After incubation period bioefficacy of siderophore coated seeds was calculated as the percent increase in seed germination, increase in shoot length, increase in root length, an increase in the number of leaves and chlorophyll content.

ROOT COLONIZATION Only those bacteria which colonize plant root can exert their plant growth promotion effect. For this purpose, the population densities of isolates on the rhizoplane of wheat and peanut were determined by taking the plate count of soil (10-5 dilution was used) and the number of colonies (CFU/gm soil) was taken as a measure of root and seed colonization.

STATISTICAL ANALYSIS Data generated from bioefficacy test were analyzed statistically using Students ‘t’ test. The significance of the differences between control and test groups was determined by the Students ‘t’ test and values of P ζͲǤͲͷ taken to imply statistical significance (Parker 1979).

RESULTS AND DISCUSSION SIDEROPHORE PRODUCTION, DETECTION AND ESTIMATION In shake flask studies, change in the color of SAM from colorless to fluorescent green after 24 h incubation indicated siderophore production, it was further confirmed by CAS test, where the addition of CAS to cell free supernatant changed the blue color of CAS to orange while no color change occurred in the control (un-inoculated SAM). Change in the color of CAS reagent was due to the fact that siderophore present in the supernatant chelates the iron from CAS reagent and results in a color change from blue to orange red (Schwyn and Neilands 1987). The amount of siderophore estimated by CAS shuttle assay was 92.61% units.

PROCESS OPTIMIZATION FOR ENHANCED YIELD SIDEROPHORE Optimization of Physico-chemical Parameter Alcaligenes sp. produced maximum siderophore (92.61% units) in SAM as compared to Cas-amino acid and Barbhaiyya and Rao medium (Table 1). This indicated that SAM is most suitable for siderophore production by Alcaligenes sp. Distilled water based SAM gave better siderophore yields (92.61%) in contrast to tap water based SAM (88.07%).Optimum siderophore yield (88.98%) obtained in SAM supplemented with mannitol (Table 1), other sugars shows variable effect. An optimum siderophore yield of 94.39% units was obtained in SAM supplemented with urea. Urea was proved to be a best utilizable nitrogen source it gave maximum siderophore

Process Optimization for Siderophore Production and Evaluation of Bioefficacy 143

units, while ammonium solution and meat extract stimulate the growth of Alcaligenes sp. (Table 1). Among organic acids, succinic acid was found to be suitable for siderophoregenesis as it gave 92.61% units of siderophore (Table 1). All amino acids under test supported siderophore production. However, tyrosine resulted in the production of maximum siderophore units, i.e. 90.39% (Table 1). The amount of cell mass determines the growth and production of siderophore. From various levels of cell mass, it is clear that 1.0 mg% cell mass was optimum, i.e. 93.81% unit for maximum siderophore production (Table 1). Among the various combinations of the K2HPO4 and KH2PO4 6 gL-1 and 3 gL-1 was found to give the maximum (90.07%) siderophore yield as compared to other combinations (Table 1).

Optimization of Cultural Condition In growth and siderophore production, a lag phase of 6 h was observed, siderophore production started after 12 h of incubation, increased up to 30 h and declined thereafter. 30 h of incubation, resulted in the maximum (88.23%) siderophore production (Table 1). Neutral pH (7.0), gave maximum (93.71%) siderophore units, increasing pH towards alkalinity affected siderophore production (Table 1). Table 1: Influence of Various Parameters on Siderophore Genesis by Alcaligenes sp. NS (4 g L-1) AM AFC AFS AC AA AO ADHP APS ASL AS Urea YE ME Pep Cas BE

% SU 71.10 Nd Nd 91.10 88.98 92.51 88.10 83.34 34.88 92.61 94.39 68.81 Nd 36.65 35.72 65.11

pH 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10 Nd Nd

% OAC SU (4 g L-1) Nd MA Nd LA Nd FA 29.13 AA 29.42 CA 65.11 PA 89.33 OA 93.71 SA 90.21 Nd 91.01 Nd 88.10 Nd 36.65 Nd 35.77 Nd Nd Nd Nd Nd Nd Nd

AA % SU (1 g L-1) 57.90 Ser 77.07 Lys Ala 60.79 Thr 51.01 Cys Arg 76.97 Tyr 92.61 Met Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd

PS % INCU % INOC % MED % % Sugars % SU (h) SU (mg%) SU SU SU (1 g L-1) SU K2HP KH2P 87.65 Glu 78.88 0 0 6 1 93.81 SAM 92.61 87.22 Dex 88.54 1.0 0.5 60.61 12 36.12 2 83.43 CAA 59.33 85.99 Suc 2.0 1.0 63.06 18 59.32 3 75.21 BR 38.63 89.33 Man 88.98 3.0 1.5 68.81 24 79.12 4 67.74 NB Nd 85.72 Sta 83.70 4.0 2.0 88.98 30 88.23 5 58.32 Nd Nd 63.79 Mal 76.65 5.0 2.5 89.03 36 61.02 Nd Nd Nd Nd 90.39 Lac 73.74 6.0 3.0 9 90.07 Nd Nd Nd Nd Nd Nd 60.79 Fru 76.38 Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd Nd

NS, Nirogen source; OAC, Organic acid; AA, Amino acid; PS, Phosphate source; K2HP, K2HPO4; KH2P, KH2PO4; INC, Incubation; INOC, Inoculum; MED, Media; SU, Siderophore units; -, No siderophore units; Glu, Glucose; Dex, Dextrose; Suc, Sucrose; Man, Mannose; Sta, Starch; Mal, Maltose; Lac, Lactose; Fru, Fructose; AM, Ammonium molybdate; AFC, Ammonium ferric citrate; AFS, Ammonium ferrous sulphate; AC, Ammonium chloride; AA, Ammonium acetate; AO, Ammonium oxalate; ADHP, Ammonium dihydrogen phosphate; APS, Ammonium per sulphate; ASL, Ammonium solution; AS, Ammonium sulphate; YE, Yeast extract; ME, Meat extract; Pep, Peptone; Cas, Casein; BE, Beef extract; MA, Malate; LA, Lactate; FA, Formate; AA, Acetate; CA, Citrate; PA, Propionate; OA, Oxalate; SA, Succinate; Ser, Serine;Lys, Lysine; Ala, Alanine; Thr, Threonine; Cys, Cystein; Arg, Arginine; Tyr, Tyrosine; Met, Methionine.

Influence of Metal Ions Maximum growth and siderophore yield of Alcaligenes sp. was observed at 100 PM concentration of MnCl2.4H2O, NiCl2.6H2O, ZnSO4.7H2O, ZnCl2, FeSO4, FeCl3 and CoCl2. Further increase in metal ion concentration above 100


(M affected siderophore production (Fig. 1). This may be due to repression of siderophore synthesizing genes. Sayyed and Chincholkar (2010) reported ͳͲ Ɋ 2 and CoCl2, ʹͲ Ɋ 2, MgSO4, ZnCl2 and ZnSO4 severely affected siderophores production. 80 70 MnCl2

% Siderophore unit

60

NiCl2

50

ZnSO4 40

ZnCl2

30

FeSO4 FeCl3

20

CuSO4

10

CUCl2 CoCl2

0 100

200

400

600

800

1000

1200

1400

Concentration of metal ion (ɊM)

Fig. 1: Siderophore Production in SAM Metals in Presence of Varing Concentrations of Heavy Metals by Alcaligenes sp.

BIOEFFICACY TEST In case of bacterized seeds significant increase in plant growth was observed in individual heavy metal spiked soil as compared to the control seeds. Bacterized wheat and peanut seeds resulted good effect on seed germination, shoot height, root length, number of leaves and chlorophyll content. In presence of Alcaligenes sp. coated seeds grown in MnCl2 spiked soil, a 40% increase in germination of wheat and 33.3% rise in germination of peanut seeds grown in MnCl2, NiCl2, ZnSO4, ZnCl2, FeSO4, FeCl3, CuSO4 and CuCl2 spiked soil was recorded. Percent increase in shoot length of wheat plant was recorded at 42.28% in the presence of CoCl2 and for peanut it was 25.80% with NiCl2, ZnSO4, FeSO4, CuSO4 and CuCl2. Increase in root length of wheat was noted as 31.57% in the presence of FeCl3 and CuCl2, whereas 45.0% increase in peanut root length was observed with MnCl2, NiCl2 and ZnSO4. Number of wheat leaves increased by 25.0% in the presence of MnCl2, ZnSO4, ZnCl2 and FeSO4 while the leaves of peanut increased by 50.0% in the presence of MnCl2. Chlorophyll content of wheat and peanut plant in the presence of CuSO4 raised by 36.84% and 35.29% respectively (Table 2).

Process Optimization for Siderophore Production and Evaluation of Bioefficacy 145 Table 2: Influence of Alcaligenes sp. on Wheat and Peanut Seeds in Heavy Metal (100 PM) Spiked Soil MnCl2 NiCl2 Wheat 40.0 25.0 (0.0021)* (0.024)* Peanut 56.6 29.9 (0.0074)* (0.047)* 28.57 (0.0025)* Peanut 23.33 (0.0033)*

ZnSO4 25.0 (0.024)* 29.9 (0.047)*

Wheat

24.24 (0.021)* 25.80 (0.0031)*

Wheat

18.75 13.33 18.75 (0.0060)* (0.042)* (0.0060)* 45.0 45.0 45.0 (0.00038)* (0.00038)* (0.00038)*

Peanut

24.24 (0.021)* 25.80 (0.0031)*

Nil (01.0) NS 33.39 (0.121)

25.0 (0.158) NS 40.0 (0.036)*

Wheat

ಪ (0.565) NS 15.38 (0.287) NS

25.58 (0.0056)* Nil (01.0) NS

Wheat

36.96 35.10 72.19 (0.00015)* (0.00020) * (0.00013) * 68.51 70.68 72.19 (0.00081)* (0.0033)* (0.00013)*

Wheat Peanut

25.0 (0.158) NS 50.0 (0.0096)*

21.87 (0.013)* Peanut 08.30 (0.629) NS

Peanut

% Increase in Germination of Seeds ZnCl2 FeSO4 FeCl3 CuSO4 CuCl2 25.0 25.0 25.0 25.0 25.0 (0.024)* (0.024)* (0.024)* (0.024)* (0.024)* 29.9 29.9 29.9 29.9 29.9 (0.047)* (0.047)* (0.047)* (0.047)* (0.047)* % Increase in Shoot Length of Plant 16.66 24.24 21.87 7.40 21.87 (0.030)* (0.021)* (0.011)* (0.35) NS (0.011)* 23.33 25.80 23.33 25.80 25.80 (0.0031)* (0.0033)* (0.0033)* (0.0033)* (0.0033)* % Increase in Root Length of Plant 23.52 27.70 31.57 27.70 31.57 (0.0041)* (0.0018)* (0.00080)* (0.0018)* (0.00080) * 42.10 38.88 42.10 38.88 42.10 (0.00060)* (0.0010)* (0.00060)* (0.0010)* (0.00060)* % Increase in Number of Leaves of Plant 25.0 25.0 Nil Nil Nil (0.158) NS (0.158) NS (01.0) NS (01.0) NS ((01.0) NS 40.0 40.0 40.0 40.0 40.0 (0.036)* (0.036)* (0.036)* (0.036)* (0.036)* % Increase in Chlorophyll Content of Leaves 04.0 ಪ ಪ 36.84 18.51 (0.48) NS (0.13) NS (01.0) NS (0.00041)* (0.0808)* 15.38 35.29 15.38 26.66 ಪ (0.287) NS (0.024)* (0.047)* (0.042)* (0.118) NS % Increase in CFU 31.24 71.87 54.25 69.12 22.65 (0.0012) * (0.0036) * (0.0038) * (0.00026) * (0.0108) * 36.64 71.87 67.72 22.72 29.16 (0.0008)* (0.00036)* (0.0073)* (0.0048)* (0.032)*

CoCl2 25.0 (0.024)* Nil (01.0) NS

AgNO3 25.0 (0.024)* Nil (01.0) NS

HgCl2 Nil (0.373)NS Nil (01.0) NS

28.57 (0.0025)* 20.68 (0.166)NS

24.24 13.79 (0.021)* (0.057)* 23.33 20.68 (0.0033)* (0.166)NS

23.52 (0.0041)* 20.68 (0.0010)*

23.52 23.52 (0.0041)* (0.0041)* 20.68 35.29 (0.0010)* (0.0022)*

Nil (01.0) NS 33.33 (0.121)

Nil (01.0) NS 25.0 (0.158)

Nil (01.0) NS 25.0 (0.158)

ಪ (0.800) NS 18.51 (0.242) NS

ಪ (0.329) NS ಪ (0.589) NS

29.41 (01.0)NS ಪ (0.196)

34.43 21.42 02.94 (0.00059) * (0.0108) * (0.338) NS 34.0 38.50 54.46 (0.026)* (0.00075)* (0.222) NS

The results are expressed% increase in the regarding parameter (values are the mean of 3 replicates). Values in parenthesis are the values generated by comparing control with test by the Students ‘t’ test. *Values weretaken to be statistically significant at P ζ ͲǤͲͷǢ different at P ζ ͲǤͲͷǤ ǣ Ψ regarding parameter. ಪ:% decrease as compared to control treatment in regarding parameter.

ROOT COLONIZATION The population densities from rhizosphere soil also showed the abundance of Alcaligenes sp.

CONCLUSION Alcaligenes sp. yielded 94.39% siderophore units. The experimental results showed that the organism is resisted heavy metals and produce siderophore. Maximum siderop ͳͲͲ Ɋ various metal ions. Thus, such isolate can be soundly influenced the heavy metal contaminated sites in nature for the bioremediation. The potential of Alcaligenes sp. to promote growth of wheat and peanut seeds under natural soil conditions and as well as in heavy metal spiked reflected the potential of the organism as promising bioinoculant with an added advantage of bioremediation of heavy metal contaminated soil.

ACKNOWLEDGEMENT Author RZS is thankful to University Grants Commission, New Delhi for providing financial support in the form of Major Research Project to carry out this work.


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