High production of pyoluteorin and 2,4-diacetylphloroglucinol by Pseudomonas fluorescens S272 grown on ethanol as a sole carbon source

JOURNALOF FERMENTATION AND Vol. 86, No. 6, 559-563. 1998

BIOENGINEERING

High Production of Pyoluteorin and 2,4-Diacetylphloroglucinol by Pseudomonas fluorescens S272 Grown on Ethanol as a Sole Carbon Source ZHAN

YUAN,’ SONG CANG,’

MOTOKO

MATSUPUJI,2 KUNIHO NAKATA,2 YOSHIMOTO’*

YASUNORI

NAGAMATSU,’

AND AKIHIRO

Faculty of Applied Biological Science, Hiroshima University, l-4-4 Kagamiyama, Higashi-Hiroshima 739-O&W and Central Research Laboratories, Mercian Corporation, 4-9-l Johnan, Fujisawa 251-0057,2 Japan Received 21 May 1998/Accepted 3 September 1998

Pseudomonas fruorescens S272 newly isolated from a soil sample produced a considerable amount of pyoluteorin and 2,4-diacetylphloroglucinol when grown on ethanol as a single carbon source. The copraduction of approximately 150 &ml of pyoluteorin and approximately 500 ,ug/mf of 2,4_diacyl-phforogluciuol was achieved by tlask cultutivation in a medium containing approximately 2% ethanol. A high C/N ratio and inorganic phosphate limitation in the medium were also important factors to be considered for optimization of production of the antibiotics. [Key words:

ethanol substrate,

pyoluteorin,

2,4-diacetylphloroglucinol,

Pseudomonas fluorescens is well documented as a biocontrol agent against fungi causing plant root disease (1). It produces several antifungal substances such as pyoluteorin (PLT) (2, 3), 2,4-diacetylphloroglucinol (DAPG) (4, 5), phenazine-1-carboxylic acid (6), and hydrogen cyanide (7) which are considered to contribute to the suppression of the plant diseases. Genes involved in the biosynthesis of PLT (8) and DAPG (9) were isolated and characterized. Of the aforementioned antibiotics, PTL was the most toxic to Pythium ultimum (3) although DAPG also suppressed mycelial growth of the fungus (5, 10). It was reported that P. fluorescens usually produced 0.1-20 pg/ml of these antibiotics extracellularly when grown in nutritional liquid media. Genetic approaches were attempted for their production. Sigma factor S deficiency (RpoS- mutant) led to overproduction of PLT and DAPG (11, 12), and amplification of the housekeeping sigma factor enhanced the production of these antibiotics (13). Pyrroloquinoline quinone (PQQ)-negative mutant, defective in glucose dehydrogenase activity and alcohol utilization, also gave a several times more improved yield of PLT, but not DAPG (14). In regard to PLT, it was found that a high amount (about lOO/~g/ml) was produced when hydrocarbon-assimilating P. aeruginosa was cultivated in 10% n-paraffin medium (15). The chemical synthesis and derivatization of PLT have been reported (16, 17). In this paper, we describe a fluorescent pseudomonad newly isolated from soil that produced a considerable amount of PLT and DAPG (Pig. 1) when it grown in media containing ethanol as a sole carbon source. Other fermentation conditions that favored the antibiotic production are also described. MATERIALS Microbial strain study was isolated

Pseudomonasfluorescens]

Toyosaka, Hiroshima and maintained on a YPG agar slant (0.5% yeast extract, 0.5% Polypepton, 1.0% glucose and 1.5% agar, pH 6.8). The other P. Jluorescens strains were obtained from the Institute for Fermentation, Osaka (IFO). Media and cultivation Basal medium contained (per liter of tap water): 15 ml of ethanol, 3 g of NH4N03, 2 g of yeast extract, 1 g of KH2P0.,, 2 g of NaCl, 0.5 g of MgS04.7Hz0, and 1 ml of mineral solution (0.3 g each of CuS04. 7Hr0, ZnSO.,. 7Hz0, CoS047Hz0, FeS04. 7H20 and MnS04.4H20 in 100 ml of distilled water, pH 2.3), pH 6.8. In tests for antibiotic production, ethanol as a carbon source and NHaOj as a nitrogen source were replaced by other sources at various concentrations. Cultivation was carried out in test tubes (16 mm x 180 mm) each containing 6 ml of the medium or 5OO-ml Sakaguchi flasks each containing 50 ml of the medium on a reciprocal shaker (150rpm) at 28°C. Seed culture was prepared by incubating the strain in a test tube containing 6 ml of YPG medium for 2 d at 28°C and inoculated 2.0% into the culture medium. Time-course experiments were performed using a Sakaguchi flask (50 ml medium) and 5 ml of the culture broth was taken dialy during the cultivation and stored at -85°C until used for assays. Thin-layer chromatography (TLC) The culture broth was centrifuged and 1 ml of the supernatant was extracted with 1 ml of CHC&. The CHC& extract was

AND METHODS

ii

P. fluorescens S272 used in this from a soil sample collected in

i)H

Pyoluteorin FIG. 1. Antibiotics pyoluteorin produced by P. fluorescence S272.

* Corresponding author. 559

COCHs 2,4-Diacetylphloroglucinol and 2,4-diacetylphloroglucinol

560

J. FERMENT.BIOENG.,

YUAN ET AL.

evaporated to dryness, dissolved in a small volume of CHC& and spotted on a silica gel plate 7OFz,, (Wako, Hiroshima). The plate was developed with the solvent, CHC&-MeOH-HZ0 (80 : 15 : 1). Products were visualized by spraying the plate with p-anisaldehyde reagent (0.5 ml p-anisaldehyde and 5 ml H2S04 in 95 ml of 95% ethanol) followed by heating for 3 min at 110°C. Both PLT and DAPG produced showed both orange spots with Rf values of 0.45 and 0.75, respectively. High performance liquid chromatographic (HPLC) The CHC& extract from 1 ml of the supernatant assay was obtained as described above, dissolved in 100 /rl of the mobile phase and applied to HPLC (Toso CCPM) equipped with a reverse phase YMC-Pack A312 S-5 12OA (ODS) (6 mm x 150 mm) (Yamamura Chemical Co., Kyoto). The mobile phase was 50% CH&N (pH adjusted to 2.0 with H3P04), run at 1 ml/min and monitored at 310 nm. Ten ~1 of the sample was injected. PLT and DAPG were eluted with retention times of 4.10 and 10.05 min, respectively, and quantified from each standard curve based on the peak area between 0.1 /Ig and 1Opg. The assay was carried out in duplicate and the mean values are shown. Other assays Bacterial growth was measured by the optical density (OD) at 660nm after 5- or lo-fold dilution of the culture broth with 0.85% NaCl. Bioautography using Bacillus subtilis M45T was carried out by a previously described method (18). Antibiotic isolation and identification Strain S272 was cultivated in twenty 500-ml Sakaguchi flasks each containing 50ml of the basal medium (for PTL production) or the modified medium in which 0.3% NH4N03 was replaced by 0.5% Polypepton (for DAPG production). After 3-d cultivation, both cultures were centrifuged and the supernatants were pooled, solid NaCl was added to a final concentration of 3% and the sample was extracted twice with 300ml of CHC13. The extracts, containing 85 mg of PLT and 546 mg of DAPG, were evaporated to dryness in vacua. Purification was performed by column chromatography on a silica gel C200 (Wako, Hiroshima) (22mmx2OOmm). The crude extract was dissolved in 20 ml of CHC& and applied to the column. After washing with 50 ml of CHC&, the column was eluted with 300 ml of CHCls-MeGH (30 : 1). Five-ml fractions were collected and examined for the presence of antibiotics by thin-layer chromatography (TLC). The active fractions were pooled, evaporated and purified by preparative thin layer chromatography on a silica gel 60PFZs4 (Merk) with CHC&-MeOH-HZ0 (80 : 15 : 1). Bands of PLT (Rf 0.45) and DAPG (Rf 0.78) detected by UV light were removed and eluted with CHCls-MeOH (5 : 1). One hundred each of CHC& and 10% NaCl aqueous solution were added to the eluates and vigorously mixed. The CHC& layer was separated and evaporated to dryness. Pure PTL and DAPG were obtained as white or yellowish-white powder. Their yields from 2 I of the cultured broth were approximately 80mg and 440 mg, respectively. Products were identified by ‘H and 13CNMRs and FAB-MS spectrum as follows. For PLT (CllH7C12N03, mw 272), ‘H NMR (400Mz, acetone-d6) 69.65 (lH, s), 7.84 (lH, s), 7.26 (lH, t, J=8.0Hz), 7.12 (lH, s), 6.50 (lH, d, J=8.0Hz); 13C NMR (lOOMHz, MeOH-d4) 6185.59, 157.77, 132.62, 132.13, 121.62, 119.18, 115.31, 111.67, 108.01; FAB-MS m/z 272 (M+); UVlggH(E]L) 205 (423), 220 (312), 255 (254), 310 (907).

TABLE 1.

Effect of carbon sources on antibiotic production by P. fruorescens S272

Carbon source Ethanol Glucose Glycerol Fructose Galactose Sucrose Maltose Lactose

PLT*

Growth @D-W)

(pg/ml)

1.70 1.66 3.52 3.90 1.92 0.80 2.10 0.76

56 0 28 0 0 0 0 0

(pg/GDsso) 40.0 0 8.0 0 0 0 0 0

DAPGb (Irg/ml)

Medium: Basal medium containing 1.2% (w/v) of the indicated carbon source (6 ml/test tube). Cultivation time: 3 d. a Pyoluteorin (PLT). b 2,4-Diacetylphloroglucinol (DAPG).

For DAPG (CloHroOs, mw 210), ‘H NMR (4OOMz, acetone-d6) 616.30 (3H, br, s), 5.85 (lH, s), 2.60 (lH, s); 13CNMR (100 MHz, acetone-b) 6204.56, 172.69, 170.06, 104.67, 95.62, 32.76; FAB-MS m/z 211 (M+H)+; UVlggH(E&) 210 (579), 270 (2060). RESULTS Effect of carbon source on antibiotic production

Strain 5272 was cultivated in media containing 1.2% each of a variety of carbon sources and 0.3% NH4N03 as a nitrogen source, and the antibiotic production was measured. The results are shown in Table 1. This strain grew well on glycerol and fructose, less efficiently on glucose, galactose, maltose and ethanol, and poorly on sucrose and lactose. Production of PLT was observed only when it was grown on ethanol or glycerol. The production titer was about 2 times higher in ethanol medium (56 pg/ml) than in glycerol medium (26 pg/ml), but it was 5 times greater in ethanol medium when expressed as the yield per cell mass (ODsao). The another antibiotic, DAPG, was not produced in these media in which NH4N03 was used as a sole nitrogen source. Table 2 shows the results of time-course assays in ethanol and glycerol media. Cell growth reached a stationary phase 1 d after cultivation and antibiotic production reached a plateau 2 d after cultivation in both types of media. Effect of nitrogen source on antibiotic production

The efficiency of antibiotic production was examined using various nitrogen sources added to ethanol or glycerol medium (Table 3). The nitrogen sources tested were NHfi03, Polypepton and soybean meal. The organic nitrogen sources were better for the antibiotic producTABLE 2.

Antibiotic production by P. fruorescens S272 in ethanol or glycerol medium

Carbon source Ethanol

Glycerol

Cultivation (d) 1 2 3 4 1 2 3 4

PLT Growth (GD6so) @g/ml) (pg/ODW) 1.85 1.75 1.75 1.73 3.48 3.10 3.05 2.95

18 30 36 35 1 16 18 20

9.7 17.1 20.2 20.2 0.3 4.6 5.9 6.8

DAPG @g/ml) 0 0 0 0 0 0 0 0

Medium: Basal medium containing 1.2% (v/v) of ethanol or glycerol as a carbon source (50 ml/Sakaguchi flask).

ANTIBIOTIC PRODUCTION

VOL. 86, 1998 TABLE 3. Effect of nitrogen source on antibiotic production by P. Jluorescens S272 in ethanol or glycerol medium

C source

Medium (w/v %) N source

Ethanol (1.2)

Glycerol (1.2)

NHfi03 (0.3) Polypepton (0.5) Soybean meal (0.5) NH4NOI (0.3) Polypepton (0.5) Soybean meal (0.5)

Growth (GDsso) 2.00 2.79 3.10 3.50 3.88 3.95

31 124 88 21 38 16

Medium Ethanol N source no. (v/v %) (w/v%) Al A2 A3 A4 Bl B2 B3 B4 B5 B6 Bl B8 B9 BlO

31 822 546 21 38 632

Medium: Basal medium containing ethanol or glycerol as a carbon source and the indicated nitrogen source (50 ml/Sakaguchi flask). Cultivation time: 3 d.

tion than the inorganic nitrogen source, NHdNOl. It was determined that the organic nitrogen sources gave a considerably high yield of DAPG which was never produced in media containing NH4N0s as the nitrogen source. The effect of the organic nitrogen sources of the enhancement of the antibiotic production was also more significant in ethanol medium than in glycerol medium. The yields of PLT and DAGP were highest in ethanolPolypepton medium (124 ,ug/ml and 698 pg/ml, respectively) while those in glycerol-Polypepton medium were low (38 /*g/ml and 0 pg/ml, respectively). When soybean meal was used, a high yield of both DAPG and PLT was obtained in ethanol and glycerol media. Furthermore, the antibiotic production was also several times higher in ethanol medium than in glycerol medium. The effect of ethanol and nitrogen source concentrations on antibiotic production was examined using the ethanol-Polypepton media containing ethanol concentrations ranging from 1.5% to 4.0% and Polypepton at the concentration of 0.3%, 0.5% or 0.7%. The antibiotics were assayed daily during 4-d cultivation and the results at 3 d when the optimum production was obtained are shown in Table 4. The production of both antibiotics was significantly high in 0.5% Polypepton media. However, no DAPG production was observed in 0.3% or 0.7% Polypepton medium while PLT yield decreased to one-third in 0.3% Polypepton medium and one-sixth in 0.7% Polypepton medium compared to 0.5% Polypepton medium. The yield reduction was not overcome by decreasing or increasing the amount of ethanol. When NH4NOs was used as the nitrogen source, its optimum TABLE 5.

1.5 1.5 1.5 3.0 1.5 1.5 1.5 2.0 2.0 2.0 3.0 3.0 3.0 4.0

Growth (ODsso)

PLT

2.4 2.3 2.4 1.8 4.0 3.9 3.8 4.1 3.1 4.9 4.0 5.2 5.3 0.1

30 15 11 1 43 130 26 52 160 17 38 61 5 0

0.3 0.4 0.5 0.5 0.3 0.5 0.7 0.3 0.5 0.7 0.3 0.5 0.7 0.5

DAGP PLT+DAPG

(&ml) .,0 0 0 0 0 558 0 0 527 0 0 0 0 0

Od

Al A2 A3 A4 Bl B2 Cl c2 c3 c4 Dl D2 D3 D4

1.5 2.0 2.5 3.0 1.5 1.5 1.5 1.5 2.0 2.0 1.5 1.5 2.0 2.0

Ethanol added (w/v %) 2d Id 0 0 0 0 0.5 0 0 0.5 0.5 0 1.5 1.0 1.0 0

0 0 0 0 0 0.5 1.0 0.5 0 0.5 0 0.5 0 1.0

30 15 11 1 43 688 26 52 687 17 38 61 5 0

Medium: Basal medium containing the indicated % of ethanol and either NHdNOs (A) or Polypepton (B) as nitrogen source (50ml/ Sakaguchi flask). Cultivation time: 4 d.

concentration was 0.3% in 1.5% (v/v) ethanol media. NH4N03 addition more than 0.5% resulted in little or no antibiotic production. These results show that the optimum C/N ratio for antibiotic production in ethanol media was around 4 or more, i.e., a nitrogen-limited condition. Effect of intermittent feeding of ethanol on antibiotic production Since strain S272 was not tolerant to more than 3% (w/v) of ethanol, intermittent feeding of ethanol was tested for improvement of antibiotic production using Polypepton as a nitrogen source. Ethanol was fed at an initial concentration of 1.5% in the medium at various intervals for 2d and finally fixed at 3% or less. The assays were carried out 4 d after cultivation. The results are shown in Table 5. Strain 272 grew to almost similar extents between 3% and 1.5% ethanol under non-fed conditions. PLT yield increased gradually with the addition of increased amounts of ethanol from 1.5% to 3.0% (from 118 /*g/ml to 169/*g/ml) while DAPG production was not correlated either with initial ethanol concentration or ethanol feeding. When more than 2.0% ethanol was initially added in the medium. Intermittent feeding of ethanol was found to improve the PLT

Effect of intermittent feeding ethanol on antibiotic production by P. jluorescens S272 in ethanol-Polypepton

Medium no.

561

TABLE 4. Effect of concentrations of ethanol and nitrogen sources on antibiotic production by P. fluorescens S272 in ethanol medium

PTL DAGP Total @g/ml) 0 698 458 0 0 616

FROM ETHANOL SUBSTRATE

medium

Growth (GD,)

PLT

DAGP

Total

total 1.5 2.0 2.5 3.0 2.0 2.0 2.5 2.5 2.5 2.5 3.0 3.0 3.0 3.0

2.8 2.7 3.0 3.0 3.0 2.8 2.4 2.6 2.8 2.6 2.3 2.1 3.0 2.9

118 146 151 169 124 131 149 147 194 200 165 144 173 182

@g/ml) 845 420 0 0 14 534 364 184 0 210 0 0 0 90

963 566 151 169 198 665 513 331 194 410 165 144 173 272

Medium: Basal medium containing 0.5% Polypepton as a nitrogen source and intermittently fed ethanol as indicated (50 ml/Sakaguchi flask). Cultivation time: 4 d.

562

J. FERMENT.BIOENG.,

YUAN ET AL. TABLE

6.

P. fluorescens strain s212

IF03081

1.0

01 0

I

I

0.05

0.1

KH,PO,

I 0.15

I

I

0.2

0.25

0.3

concentration (%)

FIG. 2. Effect of KH2P04 concentration on PLT production. Media used were basal medium containing 1.5% (v/v) ethanol and 0.5% Polypepton with various concentrations of KH2P04 (50ml/ Sakaguchi flask). Cultivation was carried out for 3 d. Symbols: 0, PLT; m, growth.

IF03507

IF03925

IF01 3922

Pyoluteorin productivity by four other P. fluorescens strains Carbon source

pH

Growth (ODWJ)

PLT @g/ml)

ethanol glycerol glucose ethanol glycerol glucose ethanol glycerol glucose ethanol glycerol glucose ethanol glycerol glucose

5.5 6.1 3.1 8.1 1.3 6.6 4.9 7.3 6.7 6.2

5.2 6.8 2.8 1.2 6.2 4.2 3.2 6.2 5.6 3.0 4.9 3.9 4.8 6.4 5.3

44

7.1

6.8 5.9 1.3 5.2

36 0 0 0 0 0 0 0 0 0 0 0 0 0

Medium: Basal medium containing 1.2% (w/v) of the indicated carbon source and 0.5% of Polypepton. Cultivation time: 3 d.

specific for strain S272. production with a yield increase from 10% to 30%. Thus, the highest yield of PLT (200 pg/ml) was achieved in a medium with an initial ethanol concentration of 2.0% and feed of 0.5% ethanol. However, DAPG production was maximal in medium with 1.5% ethanol added under non-fed conditions (845 pg/ml). The PLT yields similarly improved with ethanol feeding when NH4N03 was used as a nitrogen source (unpublished data). Effect of inorganic phosphate on antibiotic production When the effect of medium composition on antibiotic production was tested by omission of a component from the medium, it was found that K2HP04 affected greatly the antibiotic production although little or no influence was observed from the other constituents, such as NaCl, minerals or MgS04. Figure 2 shows the results when K2HP04 was added in the range of zero to 0.3% into ethanol-Polypepton medium. K2HP04 reduced PLT and cultivation in a medium without production, K2HP04 resulted in the highest PLT production. Inhibition by K2HP04 was observed with additions of more than 0.02%. When shown as percent of the control (no K2HP04), PLT production was about 60% in 0.02%, 45% in 0.04% and a constant level of 35% in the range of 0.1% to 0.3% K2HP04. Bacterial growth was decreased by K2HP04 limitation, but PLT was produced in inverse proportion to K2HP04 concentration. Antibiotic production by some other P. jhorescens Four additional four P. fluorescens strains strains were tested for antibiotic production in ethanol medium. The experiment was carried out using the media containing ethanol, glycerol or glucose as a carbon source and Polypepton as a nitrogen source. The results are shown in Table 6. None of the four strains tested produced the antibiotics in any of the three carbon-source media, while strain S272 produced PLT in both ethanol and glycerol media. All the test strains grew well on glycerol and also comparatively well on glucose, but to a lesser extent on ethanol. However, strain S272 was different from the test strains in that it grew better on ethanol than on glucose. The results indicate that neither the efficiency of antibiotic production nor its dependence on ethanol is inherent to any P. jfuorescens strains, but is

DISCUSSION

P. fiuorescens is known to produce

several antifungal substancess (pyrrolnitrin, phenazine-1-carboxylate, DAPG, PTL and HCN). Keen et al. (19) showed that DAPG-producing P. jfuorescens strains collected worldwide were divided into two phenotypically distinct groups in terms of this antibiotic production. One group is capable of producing DAPG, PTL and HCN, and the other group produced DAPG and HCN, but not PTL. Our newly isolated strain belonged to the former group although HCN production was not examined. Detailed bioautographic tests with CHC& extracts of broth supernatants showed that strain S272 did not produce bioactive substances other than PLT and DAPG. It has been reported that P. fluorescens produced PTL or DAPG extracellularly at a level of 0.1-20 ,ug/ml in nutrient broths (4, 9, 11, 13, 14, 20). Their production was influenced by the carbon source in the medium. Glucose was used for optimization of PLT production by strain HV37a (20), while it did not improve for PLT production by strains F113 (4) and Pf-5 (9), which gave good yields of PTL (20 pg/ml) and DAPG (15 pg/ml) when the bacteria were grown on mannitol or sucrose and glycerol, respectively. However, strain S272 produced antibiotics only when grown on ethanol or glycerol. The antibiotic production was about 2 times more in ethanol medium than in glycerol medium. The nitrogen source also affected the antibiotic production. Both PTL and DAPG were produced in Polypepton or soybean meal medium, but only PTL was produced in NH4N03 medium. Since concentrations of more than 0.3% for NH4N03 and more than 0.5% for Polypepton caused a severe reduction in antibiotic production, it seems likely that it is necessary to restrict the nitrogen source concentration in the medium to C/N ratio greater than 4 for optimum antibiotic production. We also found that inorganic phosphate limitation was required for the increased antibiotic production by strain S272. Consequently, the best yield of about 150 /*g/ml of PLT and 500,~g/ml of DAPG, were obtained in about 2% ethanol medium. Their yields were about lo-fold greater than those of other

VOL. 86, 1998

ANTIBIOTIC PRODUCTION

antibiotic producing strains grown on their most suitable carbon sources (4, 9, 20). Current genetic improvements, such as the RpoS mutation (11, 12), amplification of the house-keeping sigma factor gene (13) and PQQ negative mutation (14), were reported to lead to overproduction of PLT or DAPG, or both. However, their yields did not exceed 50 pg/ml. In addition to this investigation, we also found that ethanol was the most effective carbon source for production of the biosurfactant rhamnolipid by P. aeruginosa IF0 3924 (21). Osman et al. (22) reported similar results, that Pseudomonas BOP 100 gave high yields of rhamnolipid and phenazine dye when grown in ethanol medium. These results suggest that ethanol could be an important and preferred carbon source for the production of secondary metabolites by certain bacteria. Recently, it has been evidenced that the rhamnolipid production in P. aeruginosa is controlled by a cell-density dependent signal factor N-(3-oxodecanoyl)-1-homoserine lactone (23). In this respect, we found that production of PLT and DAPG by P. fluorescens S272 was considerably enhanced by exogeneously added N-(3-oxodecanoyl)-lhomoserine lacton (unpublished data), suggesting that the antibiotic production in this strain is regulated under signal-factor mediated control, and that significantly increased production of signal factor may occur when the bacteria are cultured in ethanol medium.

8.

9.

10.

11.

12.

13.

14.

ACKNOWLEDGMENT We would like to thank Mr. Takashi Nakajima of Central Research Laboratories, Mercian Corporation for measuring rHand r3C-NMR and FAB-MS spectra. REFERENCES 1. O’Sulivan, D. J. and O’Gara, F.: Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogens. Microbial. Rev., 56, 662-676 (1992). 2. Howell, C. G. and Stipanovic, R. C.: Suppression of Pythium Itium-induced damping-off of cotton seedlings by Pseudomonas fluorescens and its antibiotic, pyoluteorin. Phytopathology, 70, 712-715 (1980). 3. Maurhofer, M., Keel, C., Schnider, U., Voisard, C., Hass, D., and Defago, D.: Influence of enhanced antibiotic production in Pseudomonas fluorescens strain CHAO on its disease suppressive capacity. Phytopathology, 82, 190-195 (1992). 4. Shanahan, P., O’Sullivan, D. J., Simpson, P., Glennon, J. D., and O’Gara, F.: Isolation of 2,4-diacetylphloroglucinol from a pseudomonad and investigation of physiological parameters influencing its production. Appl. Environ. Microbial., 58, 353358 (1992). 5. KeU, C., Schneider, U., Maurhofer, M., Voisard, C., Laville, J., Burger, U., Wirthner, P., Haas, D. F., and Defago, G.: Suppression of root diseases by Pseudomonas fluorescens CHAO: Importance of the bacterial secondary metabolite 2,4diacetylphloro-glucinol. Mol. Plant-Microbe Interact., 5, 4-13 (1992). 6. Thomashow, L. S. and Weller, D. M.: Role of phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. J. Bacterial., 170, 3499-3508 (1988). I. Voisard, C., Keel, C., Haas, D., and Defago, G.: Cyanide production by Pseudomonas jfuorescens helps suppress black

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