Production of auxins, gibberellins and cytokinins by Azotobacter vinelandii ATCC 12837 in chemically-defined media and dialysed soil media

Soil Biol. Biochem. Vol. 18, No. I, pp. 119-120, 1986 Printed in Great Britain. All rights reserved

0038-0717/86 $3.00+0.00 Copyright © 1986 Pergamon Press Ltd

SHORT COMMUNICATION PRODUCTION OF AUXINS, GIBBERELLINS A N D CYTOKININS BY AZOTOBACTER VINELANDH ATCC 12837 IN CHEMICALLY-DEFINED MEDIA A N D DIALYSED SOIL MEDIA J. GONZALEZ-LOPEZ, V. SALMERON, M. V. MARTINEZ-TOLEDO, f . BALLESTEROS and A. RAMOS-CORMENZANA Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain

(Accepted 15 July 1985)

Azotobacter is one of the microorganisms which are able to synthesize large amounts of biologically-active substances. It has been reported that. A. chroococcurn (Brown and Burlingham, 1968; Brown and Walker, 1970), A. vinelandii (Lee et al., 1970; Azcon and Barea, 1975) and A. paspali (Barea and Brown, 1974) produce plant growth regulators in chemically-defined media, but their liberation in natural habitats is not known. Gonzalez-Lopez and Vela (1981) described a medium for Azotobacter culture (dialysed soil medium) which is similar to the natural environment of this organism. We have studied the liberation in culture supernatants of auxins, gibberellins and cytokinins by A. vinelandii ATCC 12837 to understand the influence of nutrient conditions on the synthesis of hormones by this microorganism. Liberation of auxins, gibberellins and cytokinins was studied in Burk's medium (Wilson and Knight, 1952) Burk's medium amended with 0.3% of ammonium nitrate, dialysed soil medium (Gonzalez-Lopez and Vela, 1981), and dialysed soil medium plus 0.5% glucose. The growth curves of A. vinelandii in chemically-defined media and dialysed soil media were determined by plate counts (Miles and Misra, 1938). Bacterial cultures (200 ml) incubated at 26-28°C on a shaker with gentle agitation, were collected after 1, 3, 7 and 15 days and centrifuged at 5000 rev min -~ for 15 min. The supernatant was reduced to 50 ml by evaporation under vaccuum and extracted sequentially with ethyl acetate and n-butanol by the procedure described by Tien et al. (1979). The extracts were filtered through 0.45#m membrane filters, and chromatographed by descending paper chromatography using as the solvent system freshly mixed isopropanol: ammonia : water ( 10: 1: 1) by volume. After development, chromatograms were examined for fluorescence under u.v. radiation (wavelength, 350 nm) before and after spraying with chromogenic reagent (5% conc. H2SO4 in methanol). R/values of authentic standards ranged from 0.3 to 0.4 for IAA (indolyl-3-acetic acid; Merck), 0.5-0.7 for GA 3 (Sigma) and 0.7-0.8 for kinetin (Sigma). Chromatogram portions not treated with chromogenic reagent were dried for at least 7 days to remove solvents and 10 equal strips representing the sequence of R/values 0.1-1.0 were eluted and used in bioassays. Auxins were assayed by measuring length increase of oat coleoptile segments (Nitsch and Nitsch, 1956). Gibberellins were assayed by extension of lettuce hypocotyls (Frankland and Wareing, 1960). Cytokinins were assayed using the radish cotyledon expansion test (Lethan, 1971). To minimize interferences from gibberellins and auxins in cytokinins bioassays the chromatograph

(used for these assays) were heated at 115°C for 20 rain; this destroyed the gibberellins and auxins, but not the cytokinins. The amounts of growth regulators were calculated from dose response curves obtained with authentic substances and presented as #g equivalents ml -t of culture supernatant. Uninoculated media were also extracted, chromatographed and assayed as controls. When A. vinelandii was cultured in dialysed soil media, it lysed rapidly after 96h (Fig. 1). In the oat coleoptile, substances with R/0.3-0.5 eluted from chromatograms of extracts from cultures possessed auxin activity equivalent to 0.2-2/~g IAA ml -~. In the lettuce hypocotyl bioassay, significant increases were produced with R/values between 0.2-0.8, giving a peak of activity at R/ 0.6. This was equivalent to 0.8-3.1/ag GA 3 ml-~ culture supernatant fluid. Azotobacter cultured in dialysed soil media showed cytokinin activity at R:O-O. I, 0.2-0.5, and 0.6--0.8 equivalent to 1.8--4.4#g ml -~ culture. When A. vinelandii was cultured in dialysed soil medium with added glucose in the oat coleoptile bioassay, substances with R/0.3-0.5 eluted from chromatograms possessed auxin activity equivalent to 0.4-0.9/~g IAA ml -~ (Fig. 1). In the lettuce hypocotyl bioassay, significant increases were pro-

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Fig. 1. Growth curves ( 0 ) and time-course of the production of auxins (A), gibberellins ( . ) and cytokinins (C)) by Azotobacter vinelandii ATCC 12837 in dialysed soil medium ( ) and dialysed soil medium with the addition of 0.5% glucose ( - - - ) . Five replicates were prepared from each culture medium.

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Fig. 2. Growth curves (O) and time-course of the production of auxins (A), gibberellins (El) and cytokinins (O) by Azotobacter Hnelandii ATCC 12837 in Burk's nitrogen free medium ( ) and Burk's medium with the addition of 0.3% NH4NO~ ( ). Five replicates were prepared from each culture medium.

duced with R/ values between 0.2 to 0.7, giving a peak of activity at B I 0.6. This was equivalent to 0.1-2.2 ug ml culture. Azotobacter cultures showed cytokinin activity at R t 0.3-0.5. 0.643.7. and 0.8-0.9. This was equivalent to 0.5-3.5,ug ml '. The production of phytohormones and the growth curve of A. t,h~elandii cultured in N-free medium are shown in Fig. 2. In the oat coleoptile bioassay, substances with Rr 0.3-0.5 eluted from chromatograms of extracts from cultures possessed auxin activity equivalent to 0.9 2.61tgml '. In the lettuce hypocotyl bioassay, significant increases were produced with R/ values between 0.1~).7 giving a peak of activity at R t 0.5-0.6. This was equivalent to 0.8,ug ml t. Heated chromatograms from extracts of A. z,inelandii cultures showed activity at R t 0 O.1 and 0.6-0.8. This was equivalent to 0.9 1.05,ugml-'. When A. Hnelandii was cultured in Burk's medium with the addition of 0.3% ammonium nitrate, in the oat coleoptile bioassay, substances with R t 0.3-0.5 eluted from chromatograms possessed auxin activity equivalent to 0.8,ug IAA (Fig. 2). In the lettuce hypocotyl bioassay, significant increases were produced with R r values between 0 to 0.7, giving a peak of activity at RI 0.5-0.6 equivalent to 1.9-5.9 ,ug m l - ' culture supernatant fluid. Heated chromatograms from extracts of Azotobacter cultures showed cytokinin activity at RI 0-0.1, 0.2-0.4 and 0.6-0.8. This was equivalent to 1 1.2,ugml ~ (Fig. 2). The production of auxins, gibberellins and cytokinins were influenced by the growth and incubation time. Auxins produced by Azotobacter were released continuously, especially when the bacteria were grown in dialysed soil medium (Fig. I) and N-free medium (Fig. 2). Auxin production in dialysed soil medium was about twice that in dialysed soil medium with glucose. Auxin production in N-free medium was about three times that in medium containing ammonium nitrate. Lee et al. (1970) reported that the IAA production of A. rinelandii was reduced in the presence of combined nitrogen, but Tien et al. (1979) reported that combined nitrogen had little effect on the production of IAA by Azospirillum brasilense. Our data suggest that the presence of combined nitrogen reduced auxin production, since the bacterial populations were very similiar in both media. The increase in auxins produced when A. vh~elandii was cultured in dialysed soil medium without glucose compared with dialysed soil medium containing glucose were very significant, because in dialysed soil medium, bacterial populations, after 15 days were about I07

cells ml -~ (Fig. I) and in dialysed soil medium with glucose about 109 cells ml ' Culture supernatants of A. t,hlelandii contained at least three gibberellin-like substances. Gibberellins were released continuously when Azotobacter were cultured in N-free medium (Fig. 2) and dialysed soil medium (Fig. 1). Gibberellin production increased in dialysed soil medium compared with dialysed soil medium with glucose, and especially, compared with the bacterial populations in both media. Gibberellins produced in Burk's medium containing ammonium nitrate was about six times that in N-free medium. The results show that combined nitrogen increases gibberellin production. Culture supernatants contained at least three substances possessing cytokinin activity. Cytokinins were released continuously only when Acotobacter was cultured in dialysed soil medium. In chemically-defined media the cytokinins were produced in similar quantities. However, when A. t,inelandii was cultured in dialysed soil media (with or without glucose) the production of cytokinins was about three times that in defined media, after 15 days. The results suggest that culture supernatants of A. t~inelandii ATCC 12837 contain at least three gibberellin-like substances, auxin and at least three substances possessing cytokinin activity. When A. t~inelandii was cultured in dialysed soil medium, an increase in phytohormone synthesis was detected (especially per cell unit), and this suggests that Azotobacter have the potential to stimulate plant growth through the production of phytohormones. REFERENCES

Azcon R. and Barea J. M. (1975) Synthesis of auxins, gibberellins and cytokinins by Azotobacter vinelandii and Azotobacter beijerinckii related to effect produced on tomato plants. Plant and Soil 43, 609-619. Barea J. M. and Brown M. E. (1974) Effect of plant growth produced by Acotobacter paspali related to synthesis of plant growth regulating substances. Journal o f Applied Bacteriology 37, 583-593. Brown M. E. and Burlingham S. K. (1968) Production of plant growth substances by Azotobacter chroococcum. Journal o f General Microbiology 53, 135-144. Brown M. E. and Walker N. (1970) Indolyl-3-acetic acid formation by Azotobacter chroococcum. Plant and Soil 32, 250-253. Frankland B. and Wareing P. F. (1960) Effect of gibberellic acid on hypocotyl growth of lettuce seedling. Nature 185, 255-256. Gonzalez-Lopez J. and Vela G. R. (1981) True morphology of the Azotobacteraceae-filterable bacteria. Nature 289, 588-590. Lee M., Breckenridge C. and Knowles R. (1970) Effect of some culture conditions on the production of indole-3acetic acid and gibberellin-like substances by Azotobacter vinelandii. Canadian Journal o f Microbiology 16, 1325-1380. Lethan D. S. (1971) Regulators of cell division in plant tissues XII. A cytokinin bioassay using excised cotyledon. Physiologia Plantarum 25, 391-396. Miles A. A. and Misra S. A. (1938) The estimation of the bactericidal power of blood. Journal o f Hygiene, Cambridge 38, 732-736. Nitsch J. P. and Nitsch C. (1956) Studies on the growth of coleoptile and first internode sections. A new, sensitive, straight-growth test for auxins. Plant Physiology 31, 94-111. Tien T. M., Gakins M. H. and Hubbell D. H. (1979) Plant growth substances produced by Azospirillum brasilense and their effect on the growth of pearl millet (Pennisetum americanurn L.). Applied and Environmental Microbiology 37, 1016-1024. Wilson P. W. and Knight S. C. (1952) Experiments in Bacterial Physiology, p. 49. Burgess, Minneapolis.