In Vivo and in Vitro Interactions between Pseudomonas fluorescens and Pythium ultimum in the Suppression of Damping-off in Tomato Seedlings

Biological Control 19, 1– 8 (2000) doi:10.1006/bcon.2000.0840, available online at http://www.idealibrary.com on

In Vivo and in Vitro Interactions between Pseudomonas fluorescens and Pythium ultimum in the Suppression of Damping-off in Tomato Seedlings Malin Hultberg, 1 Beatrix Alsanius, and Peter Sundin* Department of Horticulture, Swedish University of Agricultural Sciences, P.O. Box 55, SE-230 53 Alnarp, Sweden; and *Department of Environmental Assessment, Swedish University of Agricultural Sciences, P.O. Box 7050, SE-750 07 Uppsala, Sweden Received March 1, 1999; accepted March 31, 2000

INTRODUCTION Pseudomonas fluorescens 5.014 and its mutant 5-2/4, which is capable of 2,4-diacetylphloroglucinol production, were evaluated for biological control of Pythium ultimum on tomato seedlings. Bacterization of tomato seeds with these strains reduced the development of P. ultimum-induced damping-off expressed as plant weight, disease index, and fungal colonization. The protection against Pythium-induced damping-off did not differ significantly between the strains in comparisons of fresh weight of tomato seedlings. However, strain 5-2/4 protected the seedlings to a higher extent in comparisons of disease index. The time of introduction of the pathogen and the amount of fungal inoculum were important for disease development. Strain 5.014 inhibited P. ultimum to a larger extent than the mutant 5-2/4 on King’s medium B, a medium that promotes siderophore production. HPLC analysis showed that strain 5-2/4 produced a significantly lower amount of siderophores. The mutant was also observed to produce a lower amount of extracellular protease in vitro compared to strain 5.014. On yeast–malt medium, mutant strain 5-2/4 inhibited the growth of P. ultimum to a larger extent than 5.014 and produced a high amount of the antibiotic 2,4-diacetylphloroglucinol. P. ultimum exposed to 2,4-diacetylphloroglucinol in vitro formed disrupted mycelium. A significant increase of glutathione, a reducing tripeptide important for cellular protection, was noted in P. ultimum after exposure to the antibiotic compound (10 ␮g/ml). © 2000

Damping-off caused by Pythium spp. is a serious problem for plant producers (Moulin et al., 1994) and has caused severe economic losses in commercial greenhouse vegetable production. In hydroponic systems, low amounts of competing microbes and a facilitated dissemination of the pathogen increase the potential for disease development (Stanghellini and Rasmussen, 1994). Several different methods of reducing disease incidence have been assessed. The use of fungicides is questionable because of environmental concerns and the potential for undesired residues in the fruit (Vanachter et al., 1983). Use of fungicides is also prohibited in Sweden in plant cultures with continuous harvest. The use of detergent to lyse zoospores has shown promise to reduce the infection (Stanghellini et al., 1996). However, not all Pythium spp. produce zoospores as the infective propagule. Oospores, sporangia, and the mycelia have also been implicated in infection of the plant (Endo and Colt, 1974). Compared to other microorganisms inhabiting the rhizosphere, both beneficial and deleterious, Pythium spp. have a poor competitive ability and often act only as primary colonizers (Tedla and Stanghellini, 1992). The low competitive ability and early pathogenesis offer possibilities to develop efficient biocontrol systems. Fluorescent pseudomonads have been widely tested for biological control of Pythium spp. because of their rapid growth rate and an ability to colonize the rhizosphere to a large extent (Fukui et al., 1994). In addition to competition for limited carbon sources in the rhizosphere, antagonism can be ascribed mainly to the production of secondary metabolites, in particular antibiotics, siderophores, and cyanide (O’Sullivan and O’Gara, 1992). Pseudomonas fluorescens Migula 5.014 and its mutant 5-2/4, the strains used in the present study, have been previously shown to be efficient colonizers of tomato seedlings (Hultberg and Waechter-Kristensen,

Academic Press

Key Words: biological control; damping-off; 2,4-diacetylphloroglucinol; protease; Pseudomonas fluorescens; Pythium ultimum; siderophores; glutathione.

1 To whom correspondence should be addressed. Fax: ⫹46 40 460441. E-mail: [email protected].

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1049-9644/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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1998). The aim of this investigation was to further evaluate these strains as biocontrol agents against damping-off induced by Pythium ultimum Trow var. ultimum. A seedling assay was used to investigate the effect of seed bacterization with regard to plant weight, disease index, and fungal and bacterial colonization on P. ultimum-infected seedlings. In addition, the inhibition of P. ultimum in vitro on different media was determined and the production of antifungal substances by the bacterial strains was examined. Also, the effect of the antibiotic 2,4-diacetylphloroglucinol (phl) on the content of glutathione (GSH) in P. ultimum was investigated. GSH protects cells against oxidative damage, free-radical damage, and other types of toxicity (Meister, 1988; Chesney et al., 1996). It is therefore of interest to examine whether phl affects GSH concentration. To further investigate the interactions between the beneficial bacteria, the seedling, and the pathogenic fungus, attempts were made to detect siderophore and antibiotic production by the bacteria in the rhizosphere. MATERIAL AND METHODS

Bacterial Isolates and Production of Antifungal Substances The bacterial strains, Pseudomonas fluorescens strain 5.014 and its mutant 5-2/4, have been previously described by Hultberg and Waechter-Kristensen (1998). In short, the wild type, which showed growth promotion on tomato and antagonism against P. ultimum in initial studies, was transformed with the gene fragment required for production of phl by P. fluorescens Q2-87. This gene fragment has previously been cloned and described by Bangera and Thomashow (1996). The transformation was done at Washington State University, Pullman, by triparental mating as described by Kim (1995). The obtained mutants were screened in a bioassay and compared to the parental strain, and strain 5-2/4 was chosen for further experiments (Alsanius et al., 1998). The production of phl in yeast–malt medium (YM) (Bangera and Thomashow, 1996) was determined by HPLC analysis as described by Bonsall et al. (1997), using a Nucleosil 100-5C 18 column. Extraction of phl from the rhizosphere was done according to Bonsall et al. (1997) using 4 g of root tissue (fresh weight). For the detection of extracellular protease activity, a medium developed by Maurhofer et al. (1994) was used. Also, dual inoculation of the fungus and bacteria was performed on this medium. For detection of cellulase production, cellulose-containing plates were used (1 and 0.5% cellulose), with yeast extract and blood-base added as carbon sources. Cyanide production was investigated according to Castric (1975) and the cyanogenic strain P. fluorescens Q2-87 (Vincent et al., 1991) was included as a positive control.

The production of fluorescent siderophores was verified by fluorescence under UV light (Blak-Ray Lamp, 366 nm) on King’s medium B (KB) (King et al., 1954). An HPLC method, developed by Nowak-Thompson and Gould (1994) for detection of pyoverdine/pseudobactintype siderophores, was used to investigate the amount of siderophores produced by the strains in pure culture in KB broth. A Nucleosil 100-5C 18 column was used for analysis. The production of siderophores in the rhizosphere was investigated by the use of chrome azurol S (CAS) assay solution (Schwyn and Neilands, 1987). Four-day-old seedlings nonbacterized or bacterized with 5.014 or 5-2/4 were put in wells (microtiter plate) containing the CAS solution, and color change from blue to orange was monitored at a wavelength of 620 nm. The seedlings used in this experiment were grown under the condition described above for 4 days and washed in a 100 ␮M EDTA solution before the start of the experiment. Fungal Isolate and Cultivation The Pythium isolate used for the present study was kindly provided by Dr. W. Wohanka, Forschungs-anstalt Geisenheim, Germany and defined by reverse dotblot hybridization (Le´vesque et al., 1998) as belonging to the main molecular group of P. ultimum (Dr. A. C. Le´vesque, PARC AgriCanada, Canada, personal communication). The fungus was kept on corn-meal agar (CMA; Difco) and potato– carrot agar during laboratory storage and repeatedly reisolated from tomato seedlings to avoid loss of pathogenicity. A kanamycin-resistant mutant was obtained by sublethal mutation (Martin and Semer, 1997). The mutant was used to follow the colonization of the introduced P. ultimum only. Fungal inoculum was prepared by growing the fungus for 3 weeks in 20 ml of Schmitthenner’s medium (Adams, 1971). After this time, the P. ultimum isolate had developed abundant hyphal swellings. The mycelium was removed from the liquid medium and briefly homogenized in a blender. The density of the fungal inoculum was determined using a counting chamber and viability was determined. The inoculum was added to the filter paper in a 1-ml volume. For viable counts, the fungus was reisolated on CMA containing 200 ␮l/ml kanamycin. To suppress the growth of general root microflora, rifampicin was also added (50 ␮l/ml). Preliminary experiments in our laboratory showed that this did not affect the growth of the fungus. The same result, no growth inhibition of P. ultimum by rifampicin at this concentration, was also observed by Howie and Suslow (1991). Biocontrol Experiment Tomato seeds (Lycopersicon esculentum Mill., cultivar Gitana (Svalo¨f-Weibull Tra¨dgård AB, Hammenho¨g, Sweden; product ID 290 639; F1)) were used for

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SUPPRESSION OF Pythium BY P. fluorescens

the experiment. The seeds were agitated (200 rpm) in a bacterial suspension in Ringer solution: Na 2S 2O 3 䡠 5H 2O, 0.5; KCl, 0.075; NaCl, 2.15; CaCl 2, 0.12; g/liter, pH 6.6). The optical density of the suspension was 0.96 at 620 nm, and the seeds were agitated in the suspension for 8 h. The control seeds were agitated in sterile Ringer solution. The seeds were then transferred to petri dishes with filter papers (Munktell No. 0B 7 cm) soaked with 5 ml of nutrient solution, NO 3-N, 12.5; P, 1.2; K, 4.7; Mg, 1.1; S, 0.6; Ca, 4 mM; Mn, 10; B, 25; Cu, 0.75; Fe, 18.3; Zn, 5 ␮M; pH 5.6 (KNO 3, Ca(NO 3) 2 䡠 4H 2O, MgSO 4, KH 2PO 4, Mg(NO 3) 2, NH 4NO 3, FeEDTA, MnSO 4 䡠 2H 2O, ZnSO 4 䡠 7H 2O, H 3BO 3, CuCl 2 䡠 2H 2O, NaMoO 4.2H 2O). Each petri dish contained 10 seeds. The fungal inoculum was added to the seeds at two inoculum densities, either 2.6 ⫻ 10 3 or 6.5 ⫻ 10 2 cfu per petri dish. The inoculum was added either directly after bacterization or with an 18-h delay. The different treatments are summarized in Table 1. The seeds were kept in the dark for 4 days at 23°C. At day 5, they were transferred to light (110 ␮mol/m 2/s, 16 h photoperiod) and 21°C, and 2 ml more of the nutrient solution was added. At day 7, the experiment was terminated. The disease was measured with a modified version of Rafin and Tirilly’s (1995) disease index, sorting the seedlings into five symptom classes: 0 ⫽ healthy seedling, 1 ⫽ necrotic root tip, 2 ⫽ necrotic area above the root tip, 3 ⫽ partly necrotic root and less development of cotyledons, and 4 ⫽ severe necrosis, root length ⬍2 cm and no cotyledons. The seedlings were homogenized with 3 ml of detergent (0.1% peptone and 0.2% sodium hexametaphosphate in water) and vortexed twice for 30 s. The sample was diluted and viable counts were recorded. Growth on CMA amended with kanamycin (200 ␮l/ml) and rifampicin (50 ␮l/ml) (selective for P. ultimum) was determined after 20 h at 26°C. Growth on KB amended with rifampicin and cycloheximide (100 ␮l/ml of each) (selective for 5.014 and 5-2/4) was determined after 48 h at 26°C. All antibiotics were obtained from Sigma Chemical Co. (St. Louis, MO). Media and equipment were autoclaved before use, and the antibiotics were added after cooling.

sterilized samples, double-strength media were used to avoid dilution when the agar was added. The media and the bacterial suspensions were poured into 9-cm petri dishes. Also, KB amended with 10 ␮M FeCl 3 was included in the in vitro assay. Agar blocks (5 mm in diameter) of a 1-week-old fungal culture (CMA) were transferred to the solidified media and the solidified bacterial suspensions. The fungal growth was determined after 24 h by measuring the colony radius, which was compared to the control (fungal growth on solidified media and Ringer solution in which bacteria had not been cultivated). YM broth was used to examine the effect of phl on P. ultimum. The bacteria (5.014 and 5-2/4) were grown in YM broth for 60 h and the broth was then filter-sterilized. Mycelium was added to the 3-ml broth and incubated for 5 days at 26°C. As controls, YM broth with the addition of a known amount of phl (10 and 50 ␮g/ml) and without phl, was included. The content of GSH in P. ultimum and medium was determined according to Andersson et al. (1993). The cellular contents of GHS were expressed as amount (nmol)/mg of cell protein. Cell protein was determined according to Lowry et al. (1951) with bovine serum albumin as the standard. Statistics All experiments were performed in four replicates and repeated once, in addition to the HPLC analysis of siderophores, which was performed in three replicates and repeated twice. The results of viable counts are expressed as mean ⫾ SE after log transformation (Angle et al., 1996). All data, except the fresh weight from the biocontrol experiment, were analyzed using analysis of variance (ANOVA) and Fisher’s LSD with P ⬍ 0.05 considered significant. The fresh-weight data were analyzed with ANOVA and proc REGWQ (SAS, 1989). RESULTS

Biocontrol Experiment In Vitro Experiments For the assay of in vitro antagonism of the bacteria against Pythium, four media were used: YM, KB, 10% tryptic soy broth (0.1⫻ TSB) (Difco), and TSB (full strength). The inoculated media were incubated at 22°C at 200 rpm for 24 and 60 h. A bacterial suspension, consisting of Ringer solution containing approximately 10 8 cfu/ml of strain 5.014 or 5-2/4 (optical density at 620 nm, 0.96), was also included in the in vitro assay. After incubation, the media and the bacterial suspension were either autoclaved with agar (BactoAgar 1.5%) (Difco) or filter-sterilized and autoclaved agar solution was added to the filtrate. For the filter-

Significantly less disease developed on the seedlings exposed to the high inoculum density and bacterized with strain 5-2/4 (Table 1). All bacterized treatments tended toward lower disease index compared to the nonbacterized treatments. The nonbacterized treatment exposed to a high inoculum dose of the pathogen at the start of the experiment had the largest degree of fungal colonization as determined by viable count. Bacterization significantly reduced the fungal colonization in both bacterial treatments receiving the high inoculum density. Strain 5-2/4 also decreased the fungal colonization in the seedlings receiving the low amount of fungal inoculum with delayed exposure. When seeds

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HULTBERG, ALSANIUS, AND SUNDIN

TABLE 1 The Effect of Bacterization with Pseudomonas fluorescens Strains 5.014 and 5-2/4 on Damping-off Caused by Pythium ultimum, Measured as Fresh Weight (FW) of 7-Day-Old Tomato Seedlings, Disease Index (DI), and Colonization by the Pathogen and P. fluorescens a Treatment

Colonization

P. fluorescens

P. ultimum

FW (g)

DI

P. ultimum

P. fluorescens

1, – b 2, – 3, 5.014 4, 5-2/4 5, – 6, 5.014 7, 5-2/4 8, – 9, 5.014 10, 5-2/4 11, – 12, 5.014 13, 5-2/4

– HI c, S d HI, S HI, S HI, D e HI, D HI, D LI f, S LI, S LI, S LI, D LI, D LI, D

0.63 ⫾ 0.02 0.39 ⫾ 0.08 0.42 ⫾ 0.07 0.55 ⫾ 0.05 0.48 ⫾ 0.01 0.52 ⫾ 0.03 0.54 ⫾ 0.03 0.47 ⫾ 0.03 0.55 ⫾ 0.2 0.51 ⫾ 0.01 0.54 ⫾ 0.01 0.61 ⫾ 0.02 0.58 ⫾ 0.03

0 2.1 ⫾ 0.3 1.2 ⫾ 0.9 0.9 ⫾ 0.5* e 0.8 ⫾ 0.1 0.6 ⫾ 0.2 0.2 ⫾ 0.1* 1.1 ⫾ 0.2 0.6 ⫾ 0.2 0.7 ⫾ 0.1 0.7 ⫾ 0.2 0.5 ⫾ 0.1 0.3 ⫾ 0.03

– 3.3 ⫾ 0.3 2.5 ⫾ 0.1* 1.9 ⫾ 0.2* 2.6 ⫾ 0.1 1.9 ⫾ 0.2* 1.9 ⫾ 0.1* 2.2 ⫾ 0.4 2.5 ⫾ 0.1 2.3 ⫾ 0.2 2.7 ⫾ 0.1 2.4 ⫾ 0.1 1.7 ⫾ 0.1*

– – 6.0 ⫾ 0.3 7.5 ⫾ 0.1 – 6.3 ⫾ 0.1 6.4 ⫾ 0.1 – 7.3 ⫾ 0.1 7.9 ⫾ 0.1 – 6.3 ⫾ 0.3 6.5 ⫾ 0.1

a The bacterial and fungal colonization is presented as log cfu/g FW of root. Statistical analyses (ANOVA and REGWQ) of fresh weight showed significant differences between the three factors: time of inoculation of the fungus, the density of the fungal inoculum, and bacterization versus no bacterization. b –, Not added. c HI, High inoculum density (2.6 ⫻ 10 3 cfu/dish). d S, Inoculation at the start of the experiment. e D, Inoculation with an 18-h delay. f LI, Low inoculum density (6.5 ⫻ 10 2 cfu/dish). * The value differs from the control (unbacterized roots exposed to the same fungal treatment) at P ⬍ 0.05.

were exposed to the pathogen immediately after bacterization, disease development was more extensive than in treatments exposed to the pathogen 18 h later. No difference was noticed between the strains in comparisons of fresh weight of the seedlings. In Vitro Inhibition of P. ultimum and Production of Antifungal Substances Fungal growth on solidified Ringer solution with or without bacteria did not differ significantly between any of the treatments (Table 2). On 10% TSA and full-strength TSA, growth of the fungus was inhibited by the bacterial strains compared to the control. When the fungus was inoculated on full-strength TSA in which the bacteria had grown for 60 h, the fungus did not grow. Both the wild type 5.014 and the mutant 5-2/4 inhibited growth of P. ultimum on 10% TSA and full-strength TSA media. On autoclaved YM medium, growth inhibition of the fungus by the wild type was less after both 24 and 60 h. However, strain 5-2/4 caused more inhibition, which increased with time. For the filter-sterilized samples, growth inhibition was less but a similar pattern was seen. On KB, the wild strain 5.014 showed a significantly higher level of growth inhibition of the fungus than the mutant 5-2/4, suggesting a decreased production of siderophores by the mutant. After the addition of Fe 3⫹, the growth inhibi-

tion of P. ultimum was reversed for strain 5.014 and partly reversed for strain 5-2/4, considering only filtersterilized samples after 60 h of incubation (Table 2). HPLC analysis for detection of siderophores of pseudobactin/pyoverdine-type showed a decreased production of siderophores by the mutant. The strains produced three major peaks of which two showed spectra very similar to the published spectra of pyoverdine (Abdallah, 1991) and the third absorbed in the same region but to a lower degree. Comparing the area of the two siderophores, as defined by the spectra, the mutant produced significantly, 47 and 33%, respectively, compared to the wild type. However, no differences were observed by comparing the bacterial media under UV light. HPLC analysis indicated that a small amount of phl was produced in KB broth by the mutant. Also, no differences could be found between the treatments in the production of siderophores in the rhizosphere as measured by CAS assay (data not shown). Production of the antibiotic substance phl in the range of approximately 50 ␮g/ml of YM broth by strain 5-2/4 was indicated by HPLC analysis. The effect on P. ultimum, when grown in sterile-filtered YM broth in which 5-2/4 had been cultured, was compared to the fungal growth in YM broth with added phl standards. The result of this experiment further suggested that the concentration of phl in the bacterial broth was

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SUPPRESSION OF Pythium BY P. fluorescens

TABLE 2 The in Vitro Inhibition of Pythium ultimum on Different Media in Which Pseudomonas fluorescens Strains 5.014 and 5-2/4 Had Been Cultured for 24 and 60 h a Autoclaved samples growth (%)

Filter-sterilized samples growth (%)

Treatment

24 h

60 h

24 h

60 h

Ringer solution ⫹5.014 ⫹5-2/4 10% TSA ⫹5.014 ⫹5-2/4 TSA ⫹5.014 ⫹5-2/4 YM ⫹5.014 ⫹5-2/4 KB ⫹5.014 ⫹5-2/4 KB ⫹ Fe 3⫹ ⫹5.014 ⫹5-2/4

100a b 92a 102a 100a 79b 80b 100a 56b 57b 100a 89b 76b 100a 33b 65c ND c ND ND

100a 91a 106a 100a 37b 46b 100a 0b 0b 100a 85b 0c 100a 8b 36c ND ND ND

100a 102a 100a 100a 78b 78b 100a 60b 49c 100a 96a 98a 100a 78b 77b ND ND ND

100a 100a 94a 100a 44b 41b 100a 0b 0b 100a 95a 36b 100a 44b 67c 100a 93a 85b

a The bacterial growth medium, autoclaved or filter-sterilized, was solidified and P. ultimum was inoculated. The fungus was grown for 24 h on the medium after which the colony radius was measured. Statistical analysis was performed on the original data and standard deviations were within 10%. Average growth compared to the control (%) is shown. b Values followed by different letters differ significantly, P ⬍ 0.05, n ⫽ 4. Values were compared within each treatment and time. c ND, Not determined.

between 10 and 50 ␮g/ml medium. Phl could not be isolated from the rhizosphere. However, the extraction efficiency of phl from the roots was less than 20%, and it is possible that phl was present in concentrations below the detection limit. When P. ultimum was grown in YM broth in which strain 5-2/4 had been cultured for 60 h, an effect on colony development was observed, as disrupted mycelium formation. Cellular GSH concentration of P. ultimum was significantly affected by exposure to phl (Table 3). When the fungus was exposed to a small amount of phl (10 ␮l/ml), GSH level increased and cysteine content decreased compared to the YM treatment. Similar findings were observed when P. ultimum was grown in filter-sterilized bacterial broth from strain 5-2/4. Likewise, the same growth pattern, disrupted mycelium formation, was observed with addition of 10 ␮l/ml phl to the medium. A decrease in GSH, cysteine, and total cell protein levels was noted after increasing the phl concentration to 50 ␮l/ ml. A markedly lowered growth was also observed visually. Increasing the phl concentration resulted in further inhibition of P. ultimum. The mean concentra-

tion of GSH in noninoculated YM medium was 29.4 nmol/ml; after 5 days of fungal growth, the GSH concentration was significantly increased to 41.0 nmol/ml. However, no increase was observed after addition of phl (10 and 50 ␮l/ml) or after cultivation in 5-2/4 filtersterilized bacterial broth. Extracellular protease activity was detected for strain 5.014 after a 24-h incubation. The mutant showed a much weaker expression of protease and only a small halo was observed after 144 h. A high production of protease was seen at 26°C, but also when incubated at 4°C strain 5.014 had detectable protease activity after 72 h. A concomitant inoculation of the bacteria and the fungi on skim-milk plates did not result in any inhibition of fungal growth when the fungus reached the clarified halos. No cellulase production could be detected for the strains. Cyanide production by 5.014 or 5-2/4 could not be detected in any of the media used (data not shown). DISCUSSION

A possible interpretation of the suppression of colonization by the fungus (Table 1) is exclusion by competition or nutrient depletion, indicating the importance of high establishment in the rhizosphere of the biocontrol organism. The experimental system used in the present study is highly artificial and, compared to a natural infection, the fungal inoculum was present in high concentrations. It is therefore not possible to make any conclusion concerning the amount of beneficial rhizosphere bacteria needed for suppression of an infection under natural conditions. Since the mutant 5-2/4 tended to be more protective than the wild type

TABLE 3 Cellular Glutathione (GSH) and Cysteine Levels, Expressed as nmol/mg Cell Protein, and Total Cell Protein in mg in Pythium ultimum after Exposure to Filter-Sterilized Bacterial Culture Broth and to 2,4-Diacetylphloroglucinol (Phl) a Internal content Treatment YM YM YM YM YM a

b

(5.014) (5-2/4) (phl 10 ␮l ml ⫺1) (phl 50 ␮l ml ⫺1)

Cysteine 42.2 ⫾ 0.3a 38.8 ⫾ 0.1b 25.1 ⫾ 0.6c 29.6 ⫾ 0.4d 20.1 ⫾ 0.3e

c

GSH

Protein

40.6 ⫾ 3.1a 50.2 ⫾ 4.6a 69.7 ⫾ 3.8b 73.6 ⫾ 2.1b 38.1 ⫾ 4.2a

2.5 ⫾ 0.1a 2.3 ⫾ 0.2a 2.7 ⫾ 0.1a 2.3 ⫾ 0.1a 1.5 ⫾ 0.3b

The fungus was grown for 5 days in filter-sterilized yeast malt broth, in which Pseudomonas fluorescens 5.014 and 5-2/4 had been cultured for 60 h. Pythium ultimum was exposed to phl at concentrations of 10 and 50 ␮g/ml. b Yeast malt broth. c Values followed by different letters within a column differ significantly, P ⬍ 0.05. Mean ⫾ SE shown, n ⫽ 4.

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5.014, production of phl in the rhizosphere could be involved. However, as phl could not be isolated from the rhizosphere, this is only a speculation. The degree of bacterial colonization of tomato seeds is in agreement with previous results (Hultberg and WaechterKristensen, 1998). When the bacteria were suspended in Ringer solution, and the suspensions were autoclaved or filtersterilized, there was no effect on fungal growth (Table 2). This suggests that the growth inhibition observed in nutrient-rich media was caused by exudation of antimicrobial metabolites. The concentration of nutrients in the rhizosphere, and in the medium, should affect the production of secondary metabolites since adequate carbon levels are required to sustain production (Elad and Baker, 1985). The difference in inhibition of P. ultimum by both strains on full-strength TSA and 10% TSA, where greater inhibition could be observed on the full-strength medium, supports this. Fluctuating carbon levels in the rhizosphere may be one reason for the difference between in vivo and in vitro effects of the same bacterial isolates. In the rhizosphere, nutrients become available in a pulsing manner (Lynch, 1990). The bacteria are starved for shorter or longer periods and might therefore not produce the antifungal substances that have been identified in the nutrient-rich artificial medium. Production of antibiotics has been shown to be affected not only by the quantity but also by the quality of the available carbon source (James and Gutterson, 1986), and Keel et al. (1992) found that malt agar was a suitable substrate for phl production. Several different antibiotics have been shown to be effective against P. ultimum, such as oomycin A, pyoluteorin, and phl (Howell and Stipanovic, 1980; Howie and Suslow, 1991; Keel et al., 1992). An increase in fungal cellular GSH content was noticed when the fungus was exposed to phl (10 ␮g/ml) (Table 3). Furthermore, a decrease in the cellular concentrations of cysteine, a GSH precursor (Meister, 1988), was observed (Table 3). This suggests an increased production of GSH as a response to the exposure. When the fungus was exposed to the higher concentration of phl (50 ␮g/ml), the content of GSH was lowered and finally growth was inhibited. Maurhofer et al. (1992) observed initiation of growth inhibition of P. ultimum at 160 ␮M (34 ␮g/ml) of phl, a result that is in agreement with the findings of the present study. Toxicity, as measured by influence on the cellular GSH content, is observed at lower concentrations (10 ␮g/ml). Comparing GSH concentrations in the broth from the different treatments suggests that P. ultimum is exporting GSH to the growth medium and the export is significantly decreased when phl is added to the broth. Little is known about the occurrence and metabolism of GSH among different fungal species, and this topic should be investigated further as outlined by Sherrill

and Fahey (1998) to obtain information about transport mechanisms of GSH. However, the current study clearly demonstrates that the oomycete P. ultimum contains GSH. To our knowledge, this is the first report on GSH content in Oomycetes. The results obtained in the present study indicate that GSH is involved in cellular protection. The suppressive effect of siderophores on Pythium spp. in vitro, which has been well documented (Paulitz and Loper, 1991), is further supported by this work (Table 2). However, the importance of siderophores for disease reduction in vivo has been questioned (Paulitz and Loper, 1991). Notably, the bioassay used in the present study was sensitive enough to show the differences in the production of siderophores by the strains, in contrast to the visual determination of emittance of fluorescent light. Notably, Sexton et al. (1996) reported that in P. fluorescens M114 the production of a pseudobactin, its receptor, and a casein protease was coordinately regulated in response to the intracellular iron concentration. A possible interpretation of the finding of a lower production of siderophores and protease in strain 5-2/4 could therefore be an adverse effect on this regulation system due to the transformation. The HPLC analysis showed the presence of two, possibly three, pseudobactin/pyoverdine-type siderophores in the bacterial growth medium. Dowling et al. (1996) stated that an individual Pseudomonas strain usually produces only one type of pseudobactin. However, Nowak-Thompson and Gould (1994) found that several pseudobactin-related compounds were produced by a single strain. They suggested that this is due to structural differences in the peptide chain of the siderophore. It is, however, not possible to exclude the presence of degradation products of the initially produced siderophore in the present study. The incomplete reversal of growth inhibition after addition of Fe 3⫹ for the mutant may have been caused by phl production in KB broth or, as discussed above, is due to an affected response to the iron concentration. No differences were detected between the bacterized and the nonbacterized seedlings when the production of siderophores in the rhizosphere was estimated. This is probably related to the iron acquisition of the plant. The method used, CAS assay, has been developed for measurement of microbial siderophores and has not been evaluated in a system including plants. It is possible that the method does not work optimally under these conditions. Extracellular protease production by rhizosphere bacteria has previously been shown to be of less importance for survival in the rhizosphere (O’Sullivan et al., 1991). However, for suppression of Pythium spp., inoculation with lytic bacteria that produce glucanase and protease was shown to significantly reduce dampingoff (Mitchell and Hurwitz, 1964). Also, Dunne et al. (1997) found that protection provided by a Stenotroph-

SUPPRESSION OF Pythium BY P. fluorescens

omonas strain against P. ultimum was related to production of extracellular protease, results which recently have been further confirmed (Dunne et al., 1998). In the present study, strain 5-2/4, with a lower protease expression, protected the seedlings to the same extent as 5.014, indicating that extracellular protease was not a major metabolite responsible for disease reduction in this study. This is further supported by dual inoculation of the bacteria and fungi on skimmilk plates, which did not result in any obvious alterations in fungal growth, when the colony reached the halos. This result is in agreement with that reported by Elad and Chet (1987), who demonstrated that no lytic enzymes were involved in the in vitro interactions between rhizosphere bacteria and Pythium aphanidermatum. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Council of Forestry and Agricultural Research, which are gratefully acknowledged. We thank Karl-Erik Gustavsson, Håkan Asp, Jan-Erik Englund, and Annelie Ahlman for assistance with HPLC analysis, statistics, and laboratory work. Paul Je`nsen is thanked for his valuable criticism of earlier versions of the manuscript. The determination of glutathione and cell protein in P. ultimum was performed at the Department of Clinical Chemistry, University Hospital Lund. Antibiotic standard was kindly provided by Dr. L. S. Thomashow and Dr. A. Natsch.

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