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Prodigiosin and the inhibition of Aspergillus niger by Serratia marcescens in soil
Soil Biol. Biochent Vol. 12., pp. 295 to 296 {) Pergamon Press Ltd 1980. Printed in Great Britain
0038-0717/80/0501-0295502.00/0
SHORT C O M M U N I C A T I O N Prodigiosin and the inhibition of AspergUlus niger by Serratia marcescens in soil WILLIAM D. ROSENZWEIG* a n d G. STOTZKY Laboratory of Microbial Ecology, Department of Biology, New York University, New York, NY 10003, U.S.A. (Accepted 25 October 1979)
Pigments produced by some chromogenic bacteria possess antimicrobial activity (Abraham and Florey, 1949; Baron, 1950). Prodigiosin produced by Serratia marcescens (Abraham and Florey, 1949; Barron, 1950), prodigiosin-like pigments (e.g., prodiginine) produced by various actinomycetes (Gerber, 1971; Gerber and Lechevalier, 1976), pyocyanine produced by Pseudomonas aeruginosa (Caltrider, 1967), iodinin produced by Chromobacterium iodinum (Abraham and Florey, 1949; Baron, 1950), and violacein produced by C. violaceum (Abraham and Florey, 1949; Baron, 1950) have been shown to exhibit antimicrobial activity. Previous studies indicate that S. marcescens inhibits Aspergillus niger in soil. The inhibition was maintained when the soil was amended with 6% (v/v) montmorillonite, whereas the addition of 6% kaolinite eliminated the antagonism (Rosenzweig and Stotzky, 1979). We have attempted to determine whether prodigiosin, which is a potent inhibitor of fungi in laboratory media, was involved in this inhibition in soil. Nutrient-cyclohexamide agar (nutrient agar [NA], 23.0 g; cycloheximide, 100 nag; and water, 1 1, pH 6.8) was used for replica plating of bacteria from soil, and bacterial cultures were maintained and grown on this medium without cycloheximide. Sabouraud-dextrose-rose bengal-streptomycin agar (Sabouraud-dextrose agar I'SDA], 65.0g; rose bengal, 33.3 rag; streptomycin sulfate [Sigma Chem. Co.], 80 mg; and water, I l, pH 5.6) was used for the replica plating of fungi from soil. Fungal cultures were maintained and grown and the antifungal activity of prodigiosin was investigated on this medium without rose bengal and streptomycin sulfate. Nutrient broth (nutrient broth [NB], 8.0 g; and water, 1 l, pH 6.8) was used to grow S. marcescens for extraction of prodigiosin. Soil (K) was amended with either montmorillonite (Volclay, Panther Creek-Aberdeen, American Colloid Co.; K6M) or kaolinite (Continental, R.T. Vanderbilt Co.; K6K) to yield 6% clay (v/v). The pH of the K, K6K, or K6M soil was 5.1, 4.8 and 5.5, respectively. Other chemical and physical characteristics of these soil-clay mixtures have been reported by Babich and Stotzky (1977). A. niger was grown for 3-5 days on slants of S D A , and S. marcescens was grown for 2-3 days on slants of NA, both at 25 _+ 2°C, and the slants were then flooded with 0.85% sterile saline and agitated on a Vortex-Genie. A 0.1 ml inoculum of A. niger was added to the center of a soil dish containing 40 g of sterile soil, adjusted to 30Pa (½ bar) tension water content, and a 0.1 ml inoculum of S.
* Present address: Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ 08903, U.S.A.
marcescens was placed near the periphery, approximately 2.5 cm from the center of the soil dish. Control plates were centrally inoculated with the fungus only. After inoculation, the soil dishes were placed in a humidifier-incubator, which was maintained at 25 + 2°C. The soil dishes were replicated at various times after inoculation to appropriate selective media (Stotzky, 1965, 1973). The replicated agar dishes were incubated at 25 + 2°C, and radial growth of A. niger was measured after 2-3 days of incubation. Three replicate soil dishes were used for each variable, and experiments were performed at least twice. A non-pigmented mutant of S. marcescens was prepared by exposing NA plates containing a lawn of the bacterium to u.v. radiation for 30 s. After exposure, the plates were wrapped in aluminum foil, placed in the dark to prevent photoreactivation, and non-pigmented mutants were subcultured after 48 h on NA. Gram-staining and various biochemical tests were performed to verify that the stable mutants were S. marcescens. Prodigiosin was extracted according to the procedure of Williams et al. (1971): 100 ml of a 72-h culture of S. marcescens in NB was centrifuged at 10,000O for 10min; the supernatant was discarded; the cells were resuspended in 50 ml 1 N NaOH and digested by boiling for 1 h in a water bath; prodigiosin was extracted f r o m t h e digest with absolute ethanol and then from the ethanolic solution with petroleum ether; the petroleum ether extract was dried in a boiling water bath; and the residue was dissolved in 50°/, ethanol. A. niger was inoculated into the center of the soil dish, and 0.1 ml of extracted prodigiosin (in 50% ethanol) or 0.1 ml 50% ethanol was added near the periphery, 2.5 cm from t h e center. The soil dishes were incubated at 25 ___2°C and replicated at various times. SDA plates were prepared with the following amendments: unamended or uniformly amended with either 1% extracted prodigiosin (1 vol prodigiosin in 50% ethanol/99 vol SDA) or 1% (v/v) 50% ethanol. Three sets of plates with each amendment were prepared. The pH of each set of plates was adjusted with HCI or NaOH to 4.0, 5.0, or 6.0. A. niger was inoculated onto the center of each plate and radial measurements of growth were made daily, for 3 days, in four directions. When A. niger and S. marcescens were inoculated into separate sites in the soils, a non-pigmented strain of S. marcescens was as effective as a pigmented strain in inhibiting the growth of A. niger in the K and K6M soils. There was no inhibition by either strain in the K6K soil (Tablel). When placed into K, K6K, and K6M. soils," neither extracted prodigiosin in 50% ethanol nor 50% ethanol alone influenced the growth of A. niger. The mean radial extension of A. niger after 12 days was 35.0 ___0 mm in all treatments. The pH ot' the soils (4.8-5.5) had no apparent influence on the activity of the prodigiosin.
295
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Short communications
Table 1. Inhibition of Aspergillus niger by pigmented (P) and non-pigmented (NP) strains of Serratia marcescens in soil amended with kaolinite or montmorillonite ~ Clay added None 6 ~ kaolinite 6 ~ montmorillonite Control 4
Strain P NP P NP P NP
Growth 2 (mm + SEM) 26.7 25.9 35.0 35.0 22.8 25.6 35.0
+ 2.76 ± 2.42 +_ 0 _+ 0 + 2.51 + 2.12 + 0
p3 >0.500 >0.500 >0.200
Fungus inoculated into the center of the soil dish and the bacterium near the periphery. 2 Mean linear radial extension. 3 Probability, two-tailed t-test comparing growth in presence of pigmented strain to that of non-pigmented strain. 4 Dishes containing only A. niger. Growth of A. nioer was inhibited on SDA amended with 1~,,, prodigiosin in 50~o ethanol. The degree of inhibition was influenced by the pH of the medium and increased as the pH decreased; the mean linear radical extension (in mm) after 3 days was 21.0 __. 0.21 at pH 4.0, 25.5 + 0.10 at pH 5.0, and 32.5 _ 0.38 at pH 6.0. Conversely, the mean linear radial extension of A. niger after 3 days on plates containing only SDA (control) or SDA amended with 1~o of a 50% ethanol solution (control for the prodigiosin solvent) was 42.5 + 0 at all pH values. The difference between the control values in the soil (35.0 ± 0) and on the agar (42.5 ± 0) plates was due to the different method of measurement: in soil they were made with a soil replicator having a radius of 35 mm, whereas on agar they were made directly on the Petri plate which had a radius of 42.5 mm. Extracted prodigiosin inhibited the growth of A. niger in pure culture, and the antifungal activity of the extracted pigment increased as the pH of the medium decreased. However, extracted prodigiosin failed to inhibit the growth of A, niger in soil, regardless of the pH, The ability of prodigiosin to inhibit the growth of A. niger on agar but not in soil suggested that either the cell-free pigment was inactivated rapidly in soil but not in agar; that the pigment was adsorbed to soil particles, such as clay minerals; or that the low solubility of prodigiosin in water, as compared to its high solubility in lipoid solvents (Abraham and Florey, 1949; Baron, 1950; Williams et al., 1971). prevented
the entrance of inhibitory amounts of prodigiosin into the aqueous soil phase where it could interact with A. niger. Furthermore, a non-pigmented mutant of S. marcescens was as effective as a pigmented strain in inhibiting the growth of A. niger in soil, again indicating that prodigiosin was not involved in the inhibition of the fungus by the bacterium in soil. Inasmuch as the inhibition of A. niger by S. marcescens and Agrobacterium radiobacter (not a producer of prodigiosin) was eliminated by the addition of glucose to the soils (Rosenzweig and Stotzky, 1979), factors other than the antimicrobial activity of prodigiosin (e.g., competition for energy and carbon sources) were responsible for the inhibition of the fungus by S. marcescens in soil. REFERENCES
ABRAHAME. P. and FLOREY H. W. (1949) Antibiotics from chromogenic bacteria. In Antibiotics (H. W. FIorey, Ed.) Vol. l, pp. 537-565. Oxford University Press, London. BARICH H. and STOTZKV G. (1977) Effect of cadmium on fungi and on interactions between fungi and bacteria in soil: Influence of clay minerals and pH. Applied and En~'ironmental Microbiology 33, 1059-1066. BARON A. L. (1950) Handbook of Antibiotics. Reinhold. New York. CALTRIDER P. G. (1967) Pyocyanine. In Antibiotics (D. Gottlieb and P. D. Shaw, Ed.) Vol. 1, pp. 117-121. Springer-Verlag, New York. GERBER N. N. (1971) Prodigiosin-like pigments from Actinomadura (Nocardia) pelletieri Journal of Antibiotics 24, 636-640. GERBER N. N. and LECHEVALIER M. P. (1976) Prodiginine (prodigiosin-like) pigments from Streptomyces and other aerobic actinomycetes. Canadian Journal of Microbiology 22, 658-667. ROSENZWEIG W. D, and STOTZKY G. (1979) Influence of environmental factors on antagonism of fungi by bacteria in soil: Clay minerals and pH. Applied and Environmental Microbiology 38, 1120-1126. STOTZKY G. (1965) Replica plating technique for studying microbial interactions in soil. Canadian Journal of Microbiology 1 I, 629-636. STOTZKY G. (1973) Techniques to study interactions between microorganisms and clay minerals in vivo and in ~,itro. In Modern Methods in the Study of Microbial Ecology (T. H. Rosswall, Ed.), Bulletins from the Ecological Research Committee (Stockholm) 17, 17-28. WILLIAMS R. P., GOTT C. L., QADRI S. M. H. and SCOTT R. H. (1971) Influence of temperature of incubation and type of growth medium on pigmentation in Serratia marcescens. Journal of Bacteriology 106, 438-443.
0038-0717/80/0501-0295502.00/0
SHORT C O M M U N I C A T I O N Prodigiosin and the inhibition of AspergUlus niger by Serratia marcescens in soil WILLIAM D. ROSENZWEIG* a n d G. STOTZKY Laboratory of Microbial Ecology, Department of Biology, New York University, New York, NY 10003, U.S.A. (Accepted 25 October 1979)
Pigments produced by some chromogenic bacteria possess antimicrobial activity (Abraham and Florey, 1949; Baron, 1950). Prodigiosin produced by Serratia marcescens (Abraham and Florey, 1949; Barron, 1950), prodigiosin-like pigments (e.g., prodiginine) produced by various actinomycetes (Gerber, 1971; Gerber and Lechevalier, 1976), pyocyanine produced by Pseudomonas aeruginosa (Caltrider, 1967), iodinin produced by Chromobacterium iodinum (Abraham and Florey, 1949; Baron, 1950), and violacein produced by C. violaceum (Abraham and Florey, 1949; Baron, 1950) have been shown to exhibit antimicrobial activity. Previous studies indicate that S. marcescens inhibits Aspergillus niger in soil. The inhibition was maintained when the soil was amended with 6% (v/v) montmorillonite, whereas the addition of 6% kaolinite eliminated the antagonism (Rosenzweig and Stotzky, 1979). We have attempted to determine whether prodigiosin, which is a potent inhibitor of fungi in laboratory media, was involved in this inhibition in soil. Nutrient-cyclohexamide agar (nutrient agar [NA], 23.0 g; cycloheximide, 100 nag; and water, 1 1, pH 6.8) was used for replica plating of bacteria from soil, and bacterial cultures were maintained and grown on this medium without cycloheximide. Sabouraud-dextrose-rose bengal-streptomycin agar (Sabouraud-dextrose agar I'SDA], 65.0g; rose bengal, 33.3 rag; streptomycin sulfate [Sigma Chem. Co.], 80 mg; and water, I l, pH 5.6) was used for the replica plating of fungi from soil. Fungal cultures were maintained and grown and the antifungal activity of prodigiosin was investigated on this medium without rose bengal and streptomycin sulfate. Nutrient broth (nutrient broth [NB], 8.0 g; and water, 1 l, pH 6.8) was used to grow S. marcescens for extraction of prodigiosin. Soil (K) was amended with either montmorillonite (Volclay, Panther Creek-Aberdeen, American Colloid Co.; K6M) or kaolinite (Continental, R.T. Vanderbilt Co.; K6K) to yield 6% clay (v/v). The pH of the K, K6K, or K6M soil was 5.1, 4.8 and 5.5, respectively. Other chemical and physical characteristics of these soil-clay mixtures have been reported by Babich and Stotzky (1977). A. niger was grown for 3-5 days on slants of S D A , and S. marcescens was grown for 2-3 days on slants of NA, both at 25 _+ 2°C, and the slants were then flooded with 0.85% sterile saline and agitated on a Vortex-Genie. A 0.1 ml inoculum of A. niger was added to the center of a soil dish containing 40 g of sterile soil, adjusted to 30Pa (½ bar) tension water content, and a 0.1 ml inoculum of S.
* Present address: Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ 08903, U.S.A.
marcescens was placed near the periphery, approximately 2.5 cm from the center of the soil dish. Control plates were centrally inoculated with the fungus only. After inoculation, the soil dishes were placed in a humidifier-incubator, which was maintained at 25 + 2°C. The soil dishes were replicated at various times after inoculation to appropriate selective media (Stotzky, 1965, 1973). The replicated agar dishes were incubated at 25 + 2°C, and radial growth of A. niger was measured after 2-3 days of incubation. Three replicate soil dishes were used for each variable, and experiments were performed at least twice. A non-pigmented mutant of S. marcescens was prepared by exposing NA plates containing a lawn of the bacterium to u.v. radiation for 30 s. After exposure, the plates were wrapped in aluminum foil, placed in the dark to prevent photoreactivation, and non-pigmented mutants were subcultured after 48 h on NA. Gram-staining and various biochemical tests were performed to verify that the stable mutants were S. marcescens. Prodigiosin was extracted according to the procedure of Williams et al. (1971): 100 ml of a 72-h culture of S. marcescens in NB was centrifuged at 10,000O for 10min; the supernatant was discarded; the cells were resuspended in 50 ml 1 N NaOH and digested by boiling for 1 h in a water bath; prodigiosin was extracted f r o m t h e digest with absolute ethanol and then from the ethanolic solution with petroleum ether; the petroleum ether extract was dried in a boiling water bath; and the residue was dissolved in 50°/, ethanol. A. niger was inoculated into the center of the soil dish, and 0.1 ml of extracted prodigiosin (in 50% ethanol) or 0.1 ml 50% ethanol was added near the periphery, 2.5 cm from t h e center. The soil dishes were incubated at 25 ___2°C and replicated at various times. SDA plates were prepared with the following amendments: unamended or uniformly amended with either 1% extracted prodigiosin (1 vol prodigiosin in 50% ethanol/99 vol SDA) or 1% (v/v) 50% ethanol. Three sets of plates with each amendment were prepared. The pH of each set of plates was adjusted with HCI or NaOH to 4.0, 5.0, or 6.0. A. niger was inoculated onto the center of each plate and radial measurements of growth were made daily, for 3 days, in four directions. When A. niger and S. marcescens were inoculated into separate sites in the soils, a non-pigmented strain of S. marcescens was as effective as a pigmented strain in inhibiting the growth of A. niger in the K and K6M soils. There was no inhibition by either strain in the K6K soil (Tablel). When placed into K, K6K, and K6M. soils," neither extracted prodigiosin in 50% ethanol nor 50% ethanol alone influenced the growth of A. niger. The mean radial extension of A. niger after 12 days was 35.0 ___0 mm in all treatments. The pH ot' the soils (4.8-5.5) had no apparent influence on the activity of the prodigiosin.
295
296
Short communications
Table 1. Inhibition of Aspergillus niger by pigmented (P) and non-pigmented (NP) strains of Serratia marcescens in soil amended with kaolinite or montmorillonite ~ Clay added None 6 ~ kaolinite 6 ~ montmorillonite Control 4
Strain P NP P NP P NP
Growth 2 (mm + SEM) 26.7 25.9 35.0 35.0 22.8 25.6 35.0
+ 2.76 ± 2.42 +_ 0 _+ 0 + 2.51 + 2.12 + 0
p3 >0.500 >0.500 >0.200
Fungus inoculated into the center of the soil dish and the bacterium near the periphery. 2 Mean linear radial extension. 3 Probability, two-tailed t-test comparing growth in presence of pigmented strain to that of non-pigmented strain. 4 Dishes containing only A. niger. Growth of A. nioer was inhibited on SDA amended with 1~,,, prodigiosin in 50~o ethanol. The degree of inhibition was influenced by the pH of the medium and increased as the pH decreased; the mean linear radical extension (in mm) after 3 days was 21.0 __. 0.21 at pH 4.0, 25.5 + 0.10 at pH 5.0, and 32.5 _ 0.38 at pH 6.0. Conversely, the mean linear radial extension of A. niger after 3 days on plates containing only SDA (control) or SDA amended with 1~o of a 50% ethanol solution (control for the prodigiosin solvent) was 42.5 + 0 at all pH values. The difference between the control values in the soil (35.0 ± 0) and on the agar (42.5 ± 0) plates was due to the different method of measurement: in soil they were made with a soil replicator having a radius of 35 mm, whereas on agar they were made directly on the Petri plate which had a radius of 42.5 mm. Extracted prodigiosin inhibited the growth of A. niger in pure culture, and the antifungal activity of the extracted pigment increased as the pH of the medium decreased. However, extracted prodigiosin failed to inhibit the growth of A, niger in soil, regardless of the pH, The ability of prodigiosin to inhibit the growth of A. niger on agar but not in soil suggested that either the cell-free pigment was inactivated rapidly in soil but not in agar; that the pigment was adsorbed to soil particles, such as clay minerals; or that the low solubility of prodigiosin in water, as compared to its high solubility in lipoid solvents (Abraham and Florey, 1949; Baron, 1950; Williams et al., 1971). prevented
the entrance of inhibitory amounts of prodigiosin into the aqueous soil phase where it could interact with A. niger. Furthermore, a non-pigmented mutant of S. marcescens was as effective as a pigmented strain in inhibiting the growth of A. niger in soil, again indicating that prodigiosin was not involved in the inhibition of the fungus by the bacterium in soil. Inasmuch as the inhibition of A. niger by S. marcescens and Agrobacterium radiobacter (not a producer of prodigiosin) was eliminated by the addition of glucose to the soils (Rosenzweig and Stotzky, 1979), factors other than the antimicrobial activity of prodigiosin (e.g., competition for energy and carbon sources) were responsible for the inhibition of the fungus by S. marcescens in soil. REFERENCES
ABRAHAME. P. and FLOREY H. W. (1949) Antibiotics from chromogenic bacteria. In Antibiotics (H. W. FIorey, Ed.) Vol. l, pp. 537-565. Oxford University Press, London. BARICH H. and STOTZKV G. (1977) Effect of cadmium on fungi and on interactions between fungi and bacteria in soil: Influence of clay minerals and pH. Applied and En~'ironmental Microbiology 33, 1059-1066. BARON A. L. (1950) Handbook of Antibiotics. Reinhold. New York. CALTRIDER P. G. (1967) Pyocyanine. In Antibiotics (D. Gottlieb and P. D. Shaw, Ed.) Vol. 1, pp. 117-121. Springer-Verlag, New York. GERBER N. N. (1971) Prodigiosin-like pigments from Actinomadura (Nocardia) pelletieri Journal of Antibiotics 24, 636-640. GERBER N. N. and LECHEVALIER M. P. (1976) Prodiginine (prodigiosin-like) pigments from Streptomyces and other aerobic actinomycetes. Canadian Journal of Microbiology 22, 658-667. ROSENZWEIG W. D, and STOTZKY G. (1979) Influence of environmental factors on antagonism of fungi by bacteria in soil: Clay minerals and pH. Applied and Environmental Microbiology 38, 1120-1126. STOTZKY G. (1965) Replica plating technique for studying microbial interactions in soil. Canadian Journal of Microbiology 1 I, 629-636. STOTZKY G. (1973) Techniques to study interactions between microorganisms and clay minerals in vivo and in ~,itro. In Modern Methods in the Study of Microbial Ecology (T. H. Rosswall, Ed.), Bulletins from the Ecological Research Committee (Stockholm) 17, 17-28. WILLIAMS R. P., GOTT C. L., QADRI S. M. H. and SCOTT R. H. (1971) Influence of temperature of incubation and type of growth medium on pigmentation in Serratia marcescens. Journal of Bacteriology 106, 438-443.