Indian Journal of Experimental Biology Vol. 39, May 2001, pp. 464-468

Siderophore production by fluorescent pseudomonads colonizing roots of certain crop plants

R D Yeole, B P Dave & H C Dube*

Department of Life Sciences, Bhavnagar University, Bhavnagar 364 002, India

Received 8 Seprember 2000: revised 23 .January 2001

Twelve fluorescent Pseudomonas isolates colonizing roots of four crop plants, chill i, cotton, groundnut and soybean , were examined for extracellular siderophore production in different media under iron deficient conditions. While all the or­ganisms produced siderophores, they varied in the quantity of siderophores produced and in their preference to the medium. The siderophores were invariably hydroxamates (pyoverdine) of trihydroxamate type which formed bidentate ligands with Fe Ill ions.

Siderophores (sid=iron, phores=bearers) are low mo­lecular weight ( < 1000 D) iron chelating compounds produced by microorganisms under iron stress condi­tions 1.2. No system analogous to siderophores has been found for any other metal ion thus, making iron unique in requiring such specific ligands. A large number of bacteria and fungi are known to produce siderophores under iron limiting conditions in the soil. The root coloniz~ng bacteria, called rhizobacteria, produce siderophores that determine their role as deleterious rhizobacteria (ORB) or as plant growth promoting rhizobacteria (PGPR). PGPR produce sid­erophores having greater binding potentials which deprive the ORB of their iron nutrition3

. Based on this mechanism, the siderophore-producing organisms (mostly fluorescent Pseudomonas) have been used in biological control of plant pathogens as well as for obtaining higher yield from crop plants4

.

In the present study, we have examined sidero­phore production by 12 rhizobacterial isolates (fluo­rescent Pseudomonas) obtained from chilli, cotton, groundnut, soybean, characterized their chemical na­ture and iron binding properties.

Materials and Methods Organisms-Twelve fluorescent Pseudomonas

isolates used in this study were obtained from the rhizoplanes of 4 crops and grown on King's B me­diums. The method of their isolation and identification have been described earlier6

. The organisms were designated as CHRBl, CHRB2, CHRB3 (from chilli);

*Correspondent author: Email: hcdube@hotmail.com

CORB 1, CORB2, CORB3, CORB4 (from cotton); GNRB 1, GNRB2, GNRB3 (from groundnut); and SBRBI, SBRB2 (from soybean).

Siderophore production-Four media used were Sand's succinate medium7

, Mayer & Abdallah me­dium8, Philson & Llinas medium9 and Scher & Baker medium 10

• The glasswares were washed with HCI (6 M) to remove traces of iron, while the media were rendered iron free by treating them with 8 hydroxy­quinoline dissolved in chloroform as suggested by Messenger and Ratledge 11

• Aliquots (50 mL) of the media were dispensed in conical fla ks (100 mL) and autoclaved at !Sib psi. These were inoculated with 106 cells/mL of fluorescent pseudomonads obtained from 18 hr old cultures grown on King's B mediums at 28°-30°C. Forty-eight hr old cul:ures were centri­fuged at 10,000 rpm, and the cell-free supernatants were examined for extracelluLar siderophore produc­tion by standard assays, as given in following para­graphs.

FeC/3 test 12-To 0.5 mL of cell free culture super­natant was added to 0.5 mL of 2% of aqueous FeCI3

solution. Appearance of orange or red-brown colour indicated the presence of siderophore.

Chrome azurol sulphonate {CAS) assa/3 --To 0.5 mL of CAS assay solution wa added 0.5 mL of cell free culture supernatant. The change in colour of the blue dye to orange indicated the presence of sidero­phores. CAS assay solution was prepared as-A 6 mL of hexadecyl trimethylammoniumbromide (10 mM; HDTMA) solution was placed in a volumetric flask (100 mL) and diluted with water. A mixture of 1.5 mL iron (III) solution (1 mM FeCb.6H20 in 10 mM

YEOLE eta/.: SIDEROPHORE PROUDCTION BY FLUORESCENT PSEUDOMONADS COLONIZING ROOTS 465

HCI) and 7.5 mL of 2 rnM aqueous CAS dye solution was slowly added under stirring. Anhydrous pipera­zine (4.307 g) was dissolved in water and 6.25 mL of HCI (12 M) was added to get a buffer solution (pH 5.6). It was added to above volumetric flask made to 100 mL with distilled water to make CAS assay solu­tion.

CAS agar plate test13 -Pseudomonas isolates were streaked on CAS agar plates for 48 hr at 30°C. For­mation of orange halos around the colonies indicated the presence of siderophores. CAS agar plates were prepared as-CAS (60.5 mg) was dissolved in dis­tilled water (50 mL) and mixed with 10 mL of iron (III) solution (1 mM FeCI3.6H20 in 10 rnM HCI) with stirring. This solution was slowly added to 72.9 mg of HDTMA dissolved in 40 mL of water. The resultant dark blue liquid was autoclaved at 1 S lb psi. Also, a mixture of 750 mL, H20; 100 mL, (10xMM9 salts); 15 g, agar; 30.2 g, and pipes, (12.0 g of SO% wlw NaOH solution to raise the pH of pipes to 6.8) were also autoclaved. After cooling to 50°C, 30 mL of ca­samino acid (10%), the carbon source and supple­ments like vitamjns and antibiotics (0.2%) after mak­ing a sterile solution were added. The dye solution was finally added as sterile solution, along the glass wall, with enough agitation to achieve mixing without generation of foam. Each plate contained 30 mL of blue agar.

Spectrophotometric assa/-Cell-free culture su­pernatants were examined for their absorption maxi­mum in Shimadzu UV-Vis 160 A spectrophotometer. A peak at or near 405 nm indicated the presence of siderophore.

Chemical nature of siderophores

Hydroxamate, catecholate, and carboxylate nature of the siderophores was examined by following tests.

Hydroxamate nature

Neilands' spectrophotometric assay 2-To l mL of cell-free supernatant, was added 1-5 mL of freshly prepared 2% aqueous FeCI3 solution, and absorbance between 400-600 nm was noted. A peak between 420-450 nm, indicated the hydroxamate nature of the sid­erophores.

Tetrazolium salt test14- To a pinch of tetrazolium

salt, was added 1-2 drops of 2 N NaOH and 0.1 mL of the test culture supernatant. Instant appearance of a red to deep-red colour indicated the presence of hy­droxamate siderophores.

Catecholate nature Neilands' spectrophotometric ass a/ -Formation

of wine-coloured complex by addition of 1-5 mL of freshly prepared 2% aqueous FeCI3 to 1 mL of the test sample, that absorbed maximally at 495 nm, indicated catecholate nature of siderophores.

Arnow's test15-To 1 mL of cell-free culture super­natant, was added 1 mL of 0.5 N HCI, 1 mL of nitrite molybdate reagent and 1 mL of 1 N NaOH, followed by distilled water to make the volume to S mL. Ab­sorbance was read at 500 nm using 2, 3 dihydroxy­benzoic acid as standard.

Carboxylate nature Vogel's chemical test16-To 3 drops of 2 N NaOH,

was added 1 drop of phenolphthalein and then water was added until light pink colour developed. Disa­pearance of pink colour on addition of test sample, indicated carboxylate nature of siderophores.

Spectrophotometric assay 17-To 1 mL of cell free culture supernatant was added 1 mL of 250 11M CuS04 and 2 mL of acetate buffer (pH 4). Copper complex was observed for absorption maximum be­tween 190-280 nm. There is no specific wavelength for absorption of copper complex and, thus, the entire wavelength (190-280 nm) was scanned to observe the peak of absorption of siderophore.

Measurement of siderophore concentration18-

Standard curves were prepared for absorbance (630 nm) of desferroxamine mesylate (DFOM; standard for hydroxamate siderophores) divided by the absorbance (630 nm) of the reference solution as a function of siderophore concentration (20-100 ~.that yielded a linear relationship. Reference solution contained all the ingredients except the siderophore. Cell-free cul­ture filtrate (0.5 mL) wa~ added to 0.5 mL of CAS assay solution and NArer was measured at 630 nm. Siderophore concentration was calculated from the standard curve.

Determination of mono-, di-, and trihydroxamate nature of siderophores

Spectrophotometric method12 -pH dependent ab­sorption maxima of ferrate hydroxamate siderophores have been used to distinguish ferric complexes of mono-, di- and trihydroxamates. Ferric complexes were examined spectrophotometrically for a shift in Arnax (nm) at different pH. Little (3-8 nm) or no shift when pH of the growth medium varied from 4 to 7 indicated trihydroxamate nature, while a wide shift (up to 80 nm) indicated dihydroxamate nature. Mono

466 INDIAN J EXP BIOL, MAY 200 I

hydroxamate siderophores showed a shift (500-520 nm) when pH dropped to 4.

Electrophoretic method12 -Paper electrophoresis was performed with a flat-bed device, with 4% formic acid at pH 2. Cell free supernatants of 48 hr old cul­tures, grown at 30°C, were spotted on Whatman No. 3 paper. The electrophoresis was run at approximately 30 Ycm· 1 for 1-2 hr. The paper was dried to remove traces of formic acid and sprayed on both sides with 2% aqueous FeCJ3 solution. Ferric monohydroxamate and dihydroxamate complexes formed deep-purple and pink-purple colours, respectively, while ferric trihydroxamates gave red colour reaction.

Binding properties of hydroxamate siderophores19

-Binding property i.e., the number of bonds the li­gand formed with metal ions was detected by sta­ble/unstable nature of colour of ferrate siderophores at different pH. It is stable (red) over a wide pH range for hexadentates. Tetra-, and bidentate siderophores

are unstable and show a change in colour from red to pink-purple or deep-purple colour respectively.

Results and Discussion Results of siderophore productio by twelve fluo­

rescent Pseudomonas isolates on 4 media (Table 1) suggested that succinate medium supported sidero­phore production by all the test isolates as evidenced by positive FeCI3 test, CAS assay, CAS agar plate test and spectrophotometric assay. Only 3 organisms (CORB 1, GNRB2 and SBRB2), produced sideropho­res on all the media, while other 3 (CHRB 1, GNRB I and GNRB3) produced siderophores on 3 media (ex­cept Scher and Baker medium). These six organisms (CHRB 1, CORB 1, GNRB 1, GNRB2, GNRB3 and SBRB2) were examined for detailed studies related to siderophore production on succinate medium which emerged as the most preferred medium and supported siderophore production by all the isolates.

Table 1-Siderophore production by 12 Pseudomonas isolates in four media after 48 hr of growth

Pseudomonas Media FeC13 CAS isolates test assay

CHRBI

CHRB2

CHRB3

COREl

CORB2

CORB3

I 2 3 4

I 2 3 4

I 2 3 4

I 2 3 4

I 2 3 4

I 2 3 4

+ + +

+

+

+ + + +

+

+

+

+ + +

+

+

+ + + +

+

+

+

CAS Spectra- Pseudomonas Media FeC13 CAS agar Photo- isolates test assay plate metric assay test Amax (nm)

+ + +

+

+

+ + + +

+

+

+

408 406 404

406

407

406 406 407 405

405

+

405

CORB4

GNRBI

GNRB2

GNRB3

SBRBI

SBRB2

I 2 3 4

I 2 3 4

I 2 3 4

I 2 3 4

I 2 3 4

I 2 3 4

+

+ + +

+ + + +

+ + +

+

+ + + +

+

+ + +

+ + + +

+ + +

+

+ + + +

CAS Spectra-agar Photo-plate metric assay test Amax (nm)

+

+ + +

+ + + +

+ + +

+

+ + + +

403

404 404 403

402 405 406 +

402 405 406

404

407 404 405 407

Media taken were-(1}-Succinate medium; (2}-Mayer & Abdallah; (3}-Philson& Llinas; and (4}-Scher & Bake_r _____ .

YEOLE eta/.: SIDEROPHORE PROUDCTION BY FLUORESCENT PSEUDOMONADS COLONIZING ROOTS 467

Results of the tests for the chemical nature of sid­erophores (Table 2) suggested that the siderophores produced by all the six Pseudomonas isolates were of hydroxamate type, as indicated by positive Neilands' spectrophotometric assay and tetrazolium salt test. Pyoverdine nature of hydroxamate nature was evident by their absorption maximum between 407-412 nm (Table 1). These results corroborate the findings of Messenger and Ratledge'' who have reported hydrox­amate nature for pyoverdine siderophores. Mayer and Abdallah8 have reported production of mixed type of siderophores (hydroxamate and catecholate) by fluo­rescent Pseudomonas isolates. Among the 6 isolates, CHRB I was the highest siderophore producer (21.7xl04 g/mL), and SBRB2 the least (16.8xl0-4

g/mL). According to the amount of siderophores pro-

duced, the organisms could be arranged in a sequence as-CHRBl > CORBl > GNRBI > GNRB2 = GNRB3 > SBRB2.

Difference in the quantity in siderophore produced is a logical happening, and several reports have indi­cated such variations20

, which changes with time, space and environment in which the organism oper­ates.

Results of detection of mono-, di- and trihydrox­amate nature of siderophores produced by three potent isolates CHRBl, COREl and GNRBI and (Table 3), indicated that the siderophores were trihydroxamates as they showed a shift in Amax (3-7 nm) at different pH. Dihydroxamate with a broad shift in Amax (6-80 nm) and monohydroxamate (500-520 nm at pH 4) could not be detected. Trihydroxamate nature is

Table 2-Detection of chemical nature (hydroxamate, catechol ate, carboxylate) of siderophores of 6 fluorescent Pseudomonas isolates

Pseudomonas Hydroxamate Neilands' spectro- Tetrazolium

isolates photometric salt test assay

CHRBl 420 + CORSI 425 + GNRBI 430 + GNRB2 440 + GNRB3 428 + SBRB2 445 +

Catechol ate Neilands' spectro- Arnow's

photometric test assay

Carboxylate Vogel's Spectra-

test photometric assay

Siderophore concentration xJ0-4 g/mL

80.7 21.7 19.2 18.2 18.2 16.8

Table 3-Determination of mono-, di-, and trihydroxamate nature of siderophores by spectrophotometric and electrophoretic methods and their binding properties

Pseudomonas "-max (nm) of ferrate Amax (nm) Colour of ferrate Inference Colour of ferrate Binding isolates sidero~hores shift siderophores in hydroxamates properities

pH "-max (nm) electrophoresis

CHRBI 4 420 7 Red Trihydroxamate Red 5 420 Red 6 421 Red Bidentate 7 426 Deep blue 8 427 Deep blue 9 420 Deep blue

CORBI 4 421 5 Red Trihydroxamate Red 5 421 Red 6 425 Red Bidentate 7 425 Deep blue 8 426 Deep blue 9 426 Deep blue

GNRBI 4 420 7 Reddish brown Trihydroxamate Red 5 421 Red 6 422 Red Bidentate 7 425 Deep blue 8 427 Deep blue 9 427 Dee blue

468 INDIAN 1 EXP BIOL, MAY 2001

known to be more common, which by virtue of pres­ence of 2 atoms of 0 2 in each hydroxamic acid allows formation of hexadentate ligand, as each Oz forms a bidentate ligand21

• This is a common property among the hydroxamates. Results of electrophoretic mobil­ities of hydroxamate siderophores with 4% formic acid at pH 2, also suggested the trihydroxamate nature as evidenced by formation of reddish-brown colour, on spraying with 2% aqueous FeCh solution.

In tests for ligand properties i.e., stable/unstable nature of coloured ferrate siderophores at different pH (Table 3), the siderophores of 3 Pseudomonas isolates CHRB 1, CORB I and GNRB 1 formed red to deep blue colour at pH range 4-9, indicating their bidentate nature. If the number of hydroxamates and ligands produced was correlated, it was found all trihydrox­amates formed hexadentate ligands, but the present siderophores, which were trihydroxamates showed bidentate ligands. This was an important observation which was difficult to interpret, as the available lit­erature does not provide any explanation.

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