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Viscosinamide-producing Pseudomonas fluorescens DR54 exerts a biocontrol effect on Pythium ultimum in sugar beet rhizosphere
FEMS Microbiology Ecology 33 (2000) 139^146
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Viscosinamide-producing Pseudomonas £uorescens DR54 exerts a biocontrol e¡ect on Pythium ultimum in sugar beet rhizosphere Charlotte Thrane *, Tommy Harder Nielsen, Mette Neiendam Nielsen, Jan SÖrensen, Stefan Olsson Section of Genetics and Microbiology, Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark Received 7 January 2000; received in revised form 15 May 2000; accepted 18 May 2000
Abstract Growth inhibition of the root pathogen Pythium ultimum by the biocontrol agent Pseudomonas fluorescens DR54 inoculated on sugar beet seeds was studied in a soil microcosm. Plant emergence was followed, together with bacterial rhizosphere colonization, antibiotic production and effects on fungal growth. P. fluorescens DR54 inoculation of the P. ultimum-challenged seeds improved plant emergence after 7 days compared to a control without the biocontrol strain. At this time, P. fluorescens DR54 was the dominating colony-forming pseudomonad in rhizosphere soil samples from inoculated seedlings as shown by immuno-staining with a strain specific antibody. Viscosinamide, a cyclic lipopeptide, which has previously been identified as a major antagonistic determinant produced by P. fluorescens DR54 and shown to induce physiological changes in P. ultimum in vitro, could be detected in the rhizosphere samples. The impact of P. fluorescens DR54 on the growth and activity of P. ultimum was studied by direct microscopy after staining with the vital fluorescent dyes Calcofluor white and fluorescein diacetate. P. fluorescens DR54 caused reduction in P. ultimum mycelial density, oospore formation and intracellular activity. Further, Pythium oospore formation was absent in the presence of P. fluorescens DR54. A striking effect on zoosporeforming indigenous fungi was also observed in microcosms with P. fluorescens DR54 and, thus, where viscosinamide could be detected; a large number of encysted zoospores were seen in such microcosms both with and without P. ultimum infections. In vitro studies confirmed that purified viscosinamide induced encystment of Pythium zoospores. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Biological control ; Pseudomonas £uorescens ; Pythium ultimum; Soil microcosm; Viscosinamide ; Zoospore encystment
1. Introduction In biological control of plant pathogenic microorganisms, evaluation of the e¤cacy is often conducted by monitoring plant health. Thus, there is a general lack of direct observations demonstrating inhibitory e¡ects on the disease-causing target organisms in soil. Detection of antagonistic compounds from biocontrol agents in soil, such as phenazine produced by Pseudomonas £uorescens 2-79 controlling Gaumannomyces graminis var. tritici, has been re-
* Corresponding author. Tel.: +45 (35) 282649; Fax: +45 (35) 282606; E-mail : [email protected]
ported [1]. However, a direct in situ monitoring of the biocontrol agent, the antimicrobial metabolites and the e¡ects on target organisms in the rhizosphere has, to our knowledge, not been reported. P. £uorescens DR54 was isolated from sugar beet rhizosphere and has shown biocontrol of Pythium in planta [2]. This bacterial isolate produces the antifungal cell-associated lipopolypeptide, viscosinamide [3], which induces physiological changes in Pythium ultimum and Rhizoctonia solani in vitro and in soil as studied by £uorescence microscopy [4,5]. The purpose of this work was to study e¡ects of seed-inoculated P. £uorescens DR54 on the growth pattern of P. ultimum in soil by direct microscopy. For these experiments, a soil microcosm was developed to study the fate and activity of interacting microorganisms in a biocontrol experiment using P. £uorescens DR54 as biocontrol agent focusing on the early development (1 week) of pre-emergence damping-o¡ disease in sugar
0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 0 5 4 - 4
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beet caused by P. ultimum. The microcosm allowed us to study: (1) plant health as monitored by seedling emergence; (2) rhizosphere colonization and production of antifungal metabolites by seed-inoculated P. £uorescens DR54 and (3) response by the plant pathogenic P. ultimum when a¡ected by the bacterial antagonist. 2. Materials and methods 2.1. Plant and microorganisms Sugar beet (cv. `Madison', Danisco Seed A/S, Holeby, Denmark) was used as test plant. The biocontrol agent was P. £uorescens DR54 isolated from sugar beet [2]. The bacterium was stored at 380³C in glycerol (1:1 w/v) and for short periods in the laboratory on LB agar (Luria^ Bertani agar; 1% Bacto Tryptone (Difco Laboratories, Detroit, MI, USA), 0.5% yeast extract (Difco), 1% NaCl, 0.01% glucose, 2% agar (Difco) ; pH 7.2). For production of bacterial inoculum, the strain was cultivated in LB broth for 16^24 h. Inoculum was added to the seeds in a 10-Wl volume (5U107 colony-forming units (cfu) was added per seed). The pathogen P. ultimum Trow isolate HB2 (var. ultimum) was originally isolated from the HÖjbakkegaard ¢eld station in Denmark (Plant Pathology Section, Royal Veterinary and Agricultural University, Copenhagen ; Danisco Seed strain 92001). For maintenance and production of inoculum, P. ultimum was grown on potato dextrose agar (PDA) (Difco). For speci¢c studies on fungal zoospore production a well-characterized zoospore-producing Pythium isolate P11 was used. Pythium isolate P11 (Pythium `Group F' according to van der Plaats-Niterink (1981)) was isolated from diseased pepper plants grown in a hydroponic system by C. Rosendahl, University of Copenhagen, Denmark. To isolate indigenous Pythium spp., a soil extract was made by sonicating 100 g test soil in 75 ml 0.9% NaCl for 3 min in a water bath. The suspension was vortexed and the soil was allowed to settle for 4 min before 5 ml of the liquid phase was sampled. Two hundred Wl of this sample was spread on each of 20 plates with Pythium selective P10 -AR agar [6] made up as follows : 17 g corn meal (Difco), 40 g agar (Difco), 10 mg l31 pimaricin (Sigma P-0440), 250 mg l31 ampicillin (Sigma A-9518), 10 mg l31 rifampicin (Sigma R-3501) and 1000 ml H2 O. The antibiotics were added after autoclaving. The plates were incubated at 20³C for 4 days. At this time colonies of Pythium spp. had grown into the agar medium whereas colonies of other fungi grew only on the agar surface and could easily be washed o¡ with tap water. Twenty of the indigenous Pythium spp. isolates were subcultured twice for 2 days on selective medium (P10 -AR), before they were transferred to PDA medium. Zoospores from the Pythium isolate P11 were obtained according to Thrane et al. [7] except that the solution for induction of zoospore
formation (`Petri' solution) was made up containing : 0.4 g Ca(NO3 )2 W4H2 O, 0.15 g MgSO4 W7H2 O, 0.15 g KH2 PO4 and 0.06 g KCl. This solution was kept at 4³C and ¢lter sterilized (0.2 Wm) before use. 2.2. Soil microcosm The soil microcosm developed is shown in Fig. 1. A sandy loam from a fallow ¢eld (pH 6.4) at the HÖjbakkegaard ¢eld station was used as test soil. After sieving (4 mm mesh) the water content was adjusted to 13% using a perfusing spray technique and the soil was kept at 4³C for 24 h before use. This water content was 85% of the ¢eld capacity of the soil. This speci¢c water content was chosen because a suitable disease pressure by the pathogenic fungus could be obtained under these conditions. An inoculum of P. ultimum was applied as a 1U1 cm agar plug from a 3^4-day old colony grown on PDA medium. Glass ¢ber ¢lters (42.5 mm; GF/C, Whatman International) were prewetted in tap water and placed on top of the fungal inoculum. Each Petri dish representing one replicate contained three such GF/C ¢lters covering agar plugs with fungal inoculum. The sugar beet seed was placed in the center of each ¢lter. Bacterial inoculum (10 Wl containing 5U109 cells ml31 ) was then applied to the seed surface. The Petri dishes were ¢lled with soil and incubated for 0, 5 or 7 days in the dark at 15³C in plastic bags. Incubation times of 5 and 7 days were chosen as appropriate because the seeds germinated with visible root development after approximately 4 days. The incubation temperature of 15³C was close to that of the natural soil during seedling emergence in spring. Each experiment had 3^6 replicates of each treatment. Separate microcosms were used for di¡erent analyses as both microscopy and viscosinamide extraction were destructive methods. 2.3. Harvest and study of soil microcosms At the end of the experiment (days 5 or 7), soil microcosms were harvested to study the following parameters: plant emergence, numbers of pseudomonads (cfu) colonizing the rhizosphere using selective medium combined with P. £uorescens DR54 strain-speci¢c antibody staining, production of viscosinamide using high-performance liquid chromatography (HPLC) analysis, and changes in growth pattern, spore formation and activity of P. ultimum using vital £uorescent stains and direct microscopy. Plant health was estimated by quantifying root length and the number of seedlings emerging from the soil. Total populations of Pseudomonas spp. were enumerated using Gould's S1 selective medium [8]. Speci¢c determination of P. £uorescens DR54 was carried out using immuno-staining of colonies developed on Gould's S1. Enumerations were carried out as follows. Plants were harvested and soil adhering to a root after gentle shaking by hand was de¢ned as rhizosphere soil. All roots of the
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Fig. 1. Design of the microcosm. (A) The Petri dish seen from a side view. Only one agar plug with fungal inoculum is shown but three agar plugs with fungal inoculum were used in each Petri dish. Prewetted glass ¢ber ¢lters were placed on top of the agar plugs. Seeds were placed in the center on top of the ¢lters. Bacteria were added to the seeds in a 10-Wl volume (5U107 cells seed31 ). (B) Microcosm seen from the bottom of the Petri dish where fungal structures could be studied by an inverted microscope. Macroscopically, it was possible to observe the seed through the glass ¢ber ¢lter. (C) Detailed view of one glass ¢ber ¢lter observed from the bottom of the Petri dish. In a circular area (15 mm diameter) around the seed fungal mycelium was examined to study the antagonistic e¡ect of P. £uorescens DR54. The introduced fungus could be distinguished from indigenous fungi by following the outgrowth of the fungus from the point of inoculation (agar plug).
three seedlings from each Petri dish were pooled and bacteria associated with the rhizosphere soil were extracted in 2 ml 0.9% (w/v) NaCl by vortexing (30 s) and sonication in a water bath (30 s). After vortexing (30 s), the extract was left 3 min for sedimentation of particles and 1.5 ml of the liquid phase was removed for dilutions. In one set of experiments, drop-plating according to Hoben and Somasegaran [9] was carried out on whole roots. In another set of experiments the roots were divided into three pieces of approximately 1 cm length representing base, middle and tip of the root. In this case, 100 Wl of each dilution was plated on each Petri dish. In both sets of experiments, cfu counts were determined on Gould's S1 medium after the plates had been incubated for approximately 22 h at 28³C. Data are shown as means with standard error and t-tests were performed to compare treatments statistically. The blotting of colonies from Gould's S1 plates followed by immuno-staining with speci¢c antibodies targeting P. £uorescens DR54 was carried out according to Kan-
del et al. [10]. A polyclonal antibody speci¢c against P. £uorescens DR54 was made by immunizing rabbits (strain Ssc:CPH) with lipopolysaccharides prepared from Proteinase K digestion as described by Chart and Rowe [11]. Immunization, determination of speci¢city and removal of weak cross reactions were performed as described by Hansen et al. [12]. Viscosinamide produced by P. £uorescens DR54 was extracted from plants grown in the microcosms at each sampling day (days 0, 5 and 7). Thirty plants from 10 microcosms were pooled in one replicate and three such replicates were used for viscosinamide extraction. Viscosinamide was extracted from ¢lters or from rhizosphere soil acetonitrile (HPLC grade) mixed with 3.8 mM tri-£uoroacetic acid (4:1, v:v) as suggested by Asaka and Shoda [13]. Extractions included 1 h vigorous shaking of the organic phase on a rotary shaker before particles were allowed to settle for 15 min. The organic phase was subsequently collected in Te£on tubes before evaporation in a
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vacuum centrifuge (Maxy dry Iyo, Holm and Halby, Copenhagen, Denmark). After evaporation the extracts were dissolved in methanol and frozen for later analysis. HPLC analysis was carried out according to Nielsen et al. [3]. For determination of background signal, extracts were made from microcosms where seeds had been treated with 10 Wl of bacteria-free spent growth medium. At day 5, seed coats were still attached to the seedlings but viscosinamide was quanti¢ed separately for both seed coat and rhizosphere extract samples. At day 7, the seed coat had detached from the seedling and both plant parts were analyzed separately as on day 5. In another experiment, viscosinamide was quanti¢ed in the glass ¢ber ¢lters. The central part (15 mm diameter (Fig. 1)) and the outer rim of the ¢lters were analyzed separately. The fungal mycelium grew well within the glass ¢ber ¢lters and subsequent visualization by microscopy was possible after application of vital £uorescent stains directly to the ¢lter. For the microscopic studies, six separate replicates with three ¢lters each were used for each experiment. The stains di¡used easily within the ¢lters resulting in nearly homogeneous staining. Because the fungi were growing within the ¢lter matrix, spatial disturbance of fungal hyphae by stain application was minimal. Fluorescence microscopy of P. ultimum could be performed successfully in the soil microcosm using Calco£uor white (Sigma Fluorescent Brightner, Sigma F-3397) and £uorescein diacetate (FDA, Sigma F-7378). Calco£uor white may be used as a wall stain in fungi [14] and FDA may be used to study general fungal activity as represented by intracellular esterase activity [15]. Stock solutions of the stains were 1 mg ml31 in water (Calco£uor white) or acetone (FDA). The stains were applied in a water solution at a ¢nal concentration of 3 Wl ml31 (Calco£uor white) or 4 Wl ml31 (FDA) [4]. Three aliquots of 100 Wl stain solution were applied to each ¢lter in the Petri dishes using a pipette tip to penetrate the soil compartment. For £uorescence microscopy, a Nikon Eclipse TE 300 inverted microscope mounted with a high pressure Hg 100 W lamp was used. UV (330^380 nm excitation ¢lter and 420 nm barrier ¢lter) and blue (450^490 nm excitation ¢lter and 520 nm barrier ¢lter) ¢lter sets were used. Photographs were taken with 200 or 400 ISO Fujicolor ¢lms. Detailed examination by microscopy was carried out in the area close to the seed (Fig. 1). Hyphal length was recorded according to Hansen et al. [16] On each ¢lter an average of the recorded length was determined by counting the intersections in three areas of 6.25 mm2 . The number of oospores (approximately 15^20 Wm diameter) was determined as the total number counted in ten random 1-mm2 areas on each ¢lter within a 15-mm wide total area around the seed. Encysted zoospores (approximately 5 Wm diameter) were determined by counting the numbers of cysts in three random 0.25-mm2 areas on each ¢lter within the 15-mm wide total area around the seed. The experiment was repeated with the same result.
2.4. E¡ects of viscosinamide on zoospore production and encystment of Pythium The e¡ect of viscosinamide on zoospore production was studied using the zoospore-producing isolate Pythium P11. Ten Wl DMSO containing HPLC-puri¢ed viscosinamide was added to Petri dishes containing 10 ml `Petri solution' to give either 5 or 10 Wg ml31 ¢nal concentrations of the antibiotic. Each Petri dish was inoculated with 10 agar plugs (4 mm diameter) of mycelial inoculum. Ten Wl DMSO was added to the control dishes. Zoospore production was quanti¢ed by enumeration in a counting chamber after 18 h of incubation at 20³C. Three replicates were used per treatment. Finally, the e¡ect of viscosinamide on zoospore encystment was studied. Three small ¢lters (GF/C; 5 mm diameter) each containing 1 Wg viscosinamide [4] were placed in a Petri dish. Three-ml suspensions of freshly prepared zoospores from Pythium P11 were added to the Petri dishes (zoospore concentrations were 5U104 ^1U105 ml31 ). Zoospore encystment was quanti¢ed by counting the number of encysted, non-germinated zoospores after 24 h of incubation at 20³C. Three replicates were used per treatment. Data are shown as means with standard error and t-tests were performed to compare treatments statistically. The experiments were repeated with the same result. 3. Results 3.1. Plant emergence and biological control Fig. 2A shows that all plants emerged in the control experiment with neither P. ultimum nor P. £uorescens DR54 inoculation. By comparison, an average of 81% plants had emerged after 7 days in the presence of DR54. This indicated that the P. £uorescens DR54 biocontrol strain delayed seed germination or inhibited plant growth slightly by a phytotoxic e¡ect. The plant pathogenic e¡ect of P. ultimum was apparent from further reduction of seedling numbers to a low 55% emergence. However, when both P. ultimum and strain DR54 were present, a biocontrol e¡ect could be observed from a high (86%) emergence which was similar to the level when only P. £uorescens DR54 was present. Fig. 2B shows that root lengths after 7 days were approximately 3.5 cm on seedlings without both P. ultimum and P. £uorescens DR54 and on the seedlings with only DR54. The putative phytotoxic e¡ect of P. £uorescens DR54 of seedling emergence (Fig. 2A) was therefore not re£ected in root development of the emerging seedlings. However, the presence of Pythium reduced the average root lengths to approximately 3 cm on the emerging seedlings. Finally, despite the e¤cacy of P. £uorescens DR54 as a biocontrol strain improving seedling emergence, its presence together with Pythium further reduced the aver-
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age root lengths to approximately 2 cm on the emerging seedlings. 3.2. Rhizosphere colonization by P. £uorescens DR54 Fig. 3A shows P. £uorescens DR54 colonization of the sugar beet rhizosphere during the 7 days. Immediately after the seeds were placed in the soil microcosms (day 0), only a small fraction (2%) of the initial population of approximately 5U107 cells inoculated could be recovered as cfu from the seeds. It was clear that a very large proportion of the initial P. £uorescens DR54 population either was rapidly displaced to the soil surrounding the seeds or decreased in culturability. It was likely that at least a part of the bacteria inoculated on the seeds was a major source of P. £uorescens DR54 cells for rhizosphere colonization occurring from day 4 when roots began to develop from the seed. However, cell death, decreased culturability or decreased cell extraction due to adherence to the seed surface are likely explanations to the fact that only a small cell fraction could be recovered from the inoculated seeds. At day 5, the seedlings were approximately 1 cm long and the total Pseudomonas population was approximately 5U105 cells on the whole root as judged from colony formation on Gould's S1 medium; 93^100% of the Fig. 3. Microcosm experiment to study rhizosphere colonization and viscosinamide production by P. £uorescens DR54 in the absence of P. ultimum. (A) Cfu formed on Pseudomonas selective medium (Gould's S1) in treatments with seeds inoculated with P. £uorescens DR54. Cfu counts were determined at three sampling days; 0, 5 and 7. At day 0, only a small fraction (2%) of the added inoculum of 5U107 adhered to the seeds. At days 5 and 7, bacteria were extracted from the rhizosphere of the whole roots with lengths of approximately 1 and 3.5 cm, respectively. (B) Viscosinamide was extracted from the seed at day 0. At days 5 and 7 the two bars show the viscosinamide detected on the seed and in the rhizosphere of the approximately 1 and 3.5 cm long roots, respectively. The data shown are from one representative experiment. Bars show standard error (n = 3).
Fig. 2. Plant data from soil microcosm experiments after 7 days of incubation at 15³C. Sugar beet seeds were challenged with P. ultimum with or without protection with P. £uorescens DR54. (A) The percentage of plants emerged in four di¡erent treatments. (B) Plant root length in the same four treatments. Data are shown as the average from three microcosm experiments in which all plants in the control treatment emerged. Bars show standard error (n = 9).
colonies were identi¢ed as P. £uorescens DR54 by the immuno-assay. At day 7, when the seedling roots were approximately 3.5 cm long, the total Pseudomonas population was approximately 2U105 cells on the whole root ; again, 93^100% of the colonies were identi¢ed as P. £uorescens DR54. Finally, the cell distribution along the roots demonstrated that the rhizosphere of the region of root closest to the seed contained a large majority of the P. £uorescens DR54 cells (99%) at this time, while much smaller fractions were recovered from the intermediate (0.6%) and tip (0.01%) segments of the roots. On day 7, rhizosphere colonization of P. £uorescens DR54 was also studied in Pythium-infected microcosms. In these treatments (n = 9), the total Pseudomonas population was similar (P s 0.05) to that in rhizosphere developed from P. £uorescens DR54-treated roots (1.16U105 þ 0.24U105 cfu plant31 ). As in microcosms without Pythium infection, almost all (91^100%) the colonies
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Table 1 E¡ect of P. £uorescens DR54 inoculation on sugar beet seeds Seed inoculation
Viscosinamide (ng mm32 )
Total hyphal length (mm mm32 )
Oospores (numbers mm32 ) Encysted zoospores (numbers mm32 )
3DR54 +DR54
nda;b 0.22b
20.3 þ 2.0c 9.7 þ 0.2
12.8 þ 2.3c nda
36.7 þ 12.0c 3250.0 þ 150.0
Data are from 7-day-old seedlings and show viscosinamide concentration, total hyphal length and numbers of oospores (P. ultimum) and numbers of encysted zoospores (indigenous fungi). The parameters were quanti¢ed in a 15-mm-wide area surrounding the seed. Data shown are means þ S.E.M. a nd = not detected. b The value shown is the average viscosinamide content from 10 microcosms with three plants each (n = 1). After 5 days the viscosinamide content was 0.17 þ 0.0035 ng mm32 (n = 3). c n = 6.
were identi¢ed as P. £uorescens DR54 by the immunoassay. Hence, the presence of P. ultimum had no apparent signi¢cant e¡ect (P s 0.05) on the total population of DR54 cells colonizing the rhizosphere roots (0.90U105 þ 0.19U105 cfu plant31 ). In the absence of P. £uorescens DR54 treatment of the seeds, higher numbers (P 6 0.05) of indigenous pseudomonads colonized the rhizosphere of Pythium-challenged plants (5.44U 104 þ 2.11U104 cfu plant31 ) than control plants without Pythium (2.1U103 þ 0.78U103 cfu plant31 ). 3.3. Occurrence of viscosinamide on sugar beet roots The viscosinamide produced by P. £uorescens DR54 was found to be strongly cell-bound in batch cultures of P. £uorescens DR54 [3]. The P. £uorescens DR54 inoculum added to each seed (5U107 cfu per seed) contained approximately 400 ng viscosinamide. Fig. 3B shows that the viscosinamide detected on the seeds at day 0 was approximately 31 ng per seed which is approximately 8% of the added viscosinamide. However, this relates well to the low recovery of cells (2% of the total P. £uorescens DR54 inoculum) on the seeds at day 0 (see above). Thus, neither viscosinamide nor bacterial cells could be recovered in large amounts after the seed inoculation. It is possible that the soil surrounding the seeds thus contained both the majority of P. £uorescens DR54 cells and viscosinamide present at day 0. However, in the case of viscosinamide, adherence to particles on the seeds (or soil) is another possible explanation to the low recovery from seeds. At day 5, approximately 16 ng viscosinamide was detected in the rhizosphere (roots were approximately 1 cm long) and approximately 157 ng on the seed coats. At day 7, approximately 61 ng viscosinamide was detected in the rhizo-
sphere soil (roots were approximately 3.5 cm long) and approximately 270 ng on the seed coats. It was thus apparent that the total amount of viscosinamide associated with both the rhizosphere soil and the seed coats increased during the 7 days of incubation. 3.4. E¡ect of P. £uorescens DR54 and viscosinamide on P. ultimum Fungal growth and viscosinamide pools were determined in the central area of the ¢lter (Fig. 1 and below), where the seeds had been inoculated with P. £uorescens DR54. The amount of viscosinamide was approximately seven times larger in the 15-mm-wide center (0.22 ng mm2 ) than in the periphery of the ¢lter (Table 1). The introduced P. ultimum isolate has characteristic hyphae without septation and radial growth could easily be determined as the mycelium grew out from the point of inoculation (agar plug). Within the incubation time of 7 days, microscopy of the Pythium inoculum was made easier because mycelia of indigenous fungi were only sparsely developed. Table 1 shows that the total hyphal length per unit area was reduced to approximately 9.7 mm mm32 compared to approximately 20.3 mm mm32 in the controls without P. £uorescens DR54. Oospore formation in the Pythium mycelium was common, approximately 12.8 oospores mm32 immediately around the seed without P. £uorescens DR54 inoculum while oospores were completely absent from P. £uorescens DR54 inoculated seeds. In these hyphae close to the uninoculated seed, FDA staining was strong (data not shown) indicating that the fungus was alive and ¢lled with cytoplasm. Thus, the presence of P. £uorescens DR54 on the seeds partially inhibited fungal growth and inhibited oospore formation close
Table 2 In vitro zoospore encystment and zoospore production after treatment of Pythium P11 with viscosinamide Treatment
Zoospores produceda (numbers mm32 )
Encysted zoosporesb (numbers mm32 )
Control Viscosinamide-treated
212 þ 84 120 þ 28
312 þ 84 1876 þ 420
a The e¡ect of viscosinamide on zoospore production was studied after viscosinamide was added to the zoospore-inducing medium (5.0 Wg viscosinamide ml31 medium). b Zoospore encystment was studied by counting encysted zoospores on small glass ¢ber ¢lters placed in zoospore suspension (1 Wg of viscosinamide was initially adsorbed to each ¢lter). Data shown are mean þ S.E.M. (n = 3).
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to the seed dramatically as no oospores could be detected in the P. £uorescens DR54-inoculated microcosms. Interestingly, a large number of encysted zoospores were observed around seeds inoculated with P. £uorescens DR54. The encysted zoospores were thought to originate from indigenous zoospore-producing fungi since the introduced P. ultimum isolate did not produce zoospores. Furthermore, the encysted zoospores were also formed in microcosms without P. ultimum inoculum. The numbers of cysts mm32 was approximately 10 times higher when P. £uorescens DR54 was present (Table 1). The zoospore encystment was primarily detected in the center of the ¢lter (15 mm in diameter) indicating that the high viscosinamide content here was responsible for the pronounced zoospore encystment. Zoospore production under in vitro conditions was slightly inhibited by viscosinamide (P s 0.05), while encystment of zoospores was strongly stimulated by viscosinamide (P 6 0.005) (Table 2). 4. Discussion The biocontrol strain P. £uorescens DR54 increased plant emergence of sugar beet from P. ultimum-challenged seeds as was also shown by Nielsen et al. [2]. Such a result is in general interpreted to result from direct antagonistic interaction between the biocontrol agent and the pathogen, e.g. by antibiotic production, competition or release of cell wall degrading enzymes. In this work we developed a method for direct studies in soil microcosms of the growth and activity of the biocontrol strain and the pathogenic fungus and of the mechanism of interaction. An important prerequisite for the biocontrol e¡ect of P. £uorescens DR54 was the ability of P. £uorescens DR54 to establish in the rhizosphere. Population sizes of P. £uorescens DR54 in the rhizosphere were thus 10^100 times higher than for other, naturally-occurring Pseudomonas spp. in the rhizosphere of control plants during the 7 days of seedling emergence. P. £uorescens DR54 was even detected in the rhizosphere of the distal part of the roots indicating that the bacterium was able to actively colonize the plant rhizosphere by root attachment and growth. Lu«beck et al. [17] studied P. £uorescens DR54 colonization on the sugar beet rhizoplane by confocal laser scanning microscopy and found that P. £uorescens DR54 was the dominating organism a few days after the inoculation. During their 20-day study, active microcolonies of P. £uorescens DR54 could still be identi¢ed on all parts of the roots. The mechanism of Pythium biocontrol in soil microcosms was likely to be the viscosinamide antibiotic produced by P. £uorescens DR54. Viscosinamide was detected in increasing amounts on both seed coats and in rhizosphere soil surrounding the roots during the 7 days of incubation. The results indicated that viscosinamide was
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replenished from P. £uorescens DR54 colonizing the roots at an early stage of emergence. In this study a possible phytotoxic e¡ect of P. £uorescens DR54 in combination with P. ultimum was recorded by root length measurements. However, the measured root lengths of plants from microcosms with inoculation of P. £uorescens DR54 without co-inoculation with P. ultimum, indicated that the bacterium did not by itself cause this e¡ect. In pot experiments with a similar soil no signi¢cant reduction of root lengths of plants that have been co-inoculated with both P. £uorescens DR54 and P. ultimum was seen (Ba«renholdt-Scho«ler, unpublished). The presence of P. £uorescens DR54 seemed to have both short- and long-term e¡ects on the target fungus, P. ultimum. As studied by microscopy in the soil microcosms, mycelial density was signi¢cantly reduced and oospore formation was not detected in the presence of P. £uorescens DR54. One explanation could be that oospore formation occurred only during dense and rapid mycelium development in the control microcosms without P. £uorescens DR54. The absence of oospores could be a result of the sparse fungal biomass development due to competition in the presence of P. £uorescens DR54 [18]. However, the complete absence of oospores in the P. £uorescens DR54-inoculated microcosms indicated a speci¢c inhibition of oospore formation by the biocontrol strain. In general, formation of survival structures such as spores is thought to be one of several responses to physiological stress. Inhibition of oospore formation in turn suggested that this process was most sensitive to P. £uorescens DR54. A most intriguing observation was also that P. £uorescens DR54 strongly stimulated the encystment of zoospores formed by Pythium and indigenous fungi in the soil microcosms. Viscosinamide produced by P. £uorescens DR54 was likely to be directly responsible for the observed induction of zoospore encystment in the microcosms as in vitro experiments supported this observation. Supplementary studies supported the hypothesis that the structures observed could indeed be encysted zoospores. (1) Indigenous oomycetes could be isolated from the test soil. (2) Nile red, that is useful for staining of membranes and lipids in fungi [15], demonstrated the presence of both swimming zoospores and encysted zoospores. (3) Calco£uor white, which stains cell wall polysaccharides of fungal structures [14], also con¢rmed the presence of encysted zoospores. (4) Finally, the encysted zoospores were easily distinguished from other fungal spores in the microcosms because fungal sporangia could be seen and because the spores were of di¡erent size and shape. In a search for the mechanistic action of viscosinamide in fungal inhibition, Thrane et al. [4] suggested that the compound inhibits growth by formation of ion-channels in the fungal membrane. This has subsequently been examined by challenging an Aspergillus awamori transformant expressing the Ca2 -sensitive protein aequorin with visco-
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sinamide. The fungus responded to the viscosinamide by a large and immediate increase in cytoplasmic Ca2 -level (Kozlova et al., unpublished result). Warburton and Deacon [19] have shown that zoospores of Phytophthora parasitica take up Ca2 just before encystment. Increased permeability of the zoospore to Ca2 resulting in higher intracellular Ca2 levels could thus explain why viscosinamide triggered instant encystment of the fungal zoospores on non-plant surfaces in this study. Compounds with surfactant properties have been successfully applied in hydroponic systems to control zoospore-producing fungal pathogens [20]. The results of our study indicate that this may take place by induction of zoospore encystment in the absence of a plant and that P. £uorescens DR54 and comparable strains may have a large potential as biocontrol agents speci¢cally against zoospore-forming fungi. Encystment of zoospores in the rhizosphere soil prevents their motility towards the root surface. Colonization and proliferation of Pythium by zoospore infection on the root surface should thus be prevented. Thus, in the presence of viscosinamide, Pythium infection is antagonized by inhibition of both hyphal growth and zoospore motility in the rhizosphere. Acknowledgements The Danish Ministry of Agriculture (contract No. 93Sî 95-00788) supported this study in cooperation with 2466-A Danisco Seeds and Novo Nordisk a/s. Further the work was supported by grant No. 9313839 from the Danish Agricultural and Veterinary Research Council. We thank Ole Nybroe for providing the protocols for immuno-detection of the bacterial inoculant. The technical assistance by Dorte Rasmussen and Ulla Rasmussen is highly appreciated. Dan Funck Jensen kindly provided Pythium isolate P11. References [1] Thomashow, L.S., Weller, D.M., Bonsall, R.F. and Pierson, L.S. (1990) Production of the antibiotic phenazine-1-carboxylic acid by £uorescent Pseudomonas species in the rhizosphere of wheat. Appl. Environ. Microbiol. 56, 908^912. [2] Nielsen, M.N., SÖrensen, J., Fels, J. and Pedersen, H.C. (1998) Secondary metabolite- and endochitinase-dependent antagonism toward plant-pathogenic microfungi of Pseudomonas £uorescens isolates from sugar beet rhizosphere. Appl. Environ. Microbiol. 64, 3563^3569. [3] Nielsen, T.H., Christophersen, C., Anthoni, U. and SÖrensen, J. (1999) Structure and production of a new bacterial cyclic depsipeptide; viscosinamide antagonistic against Pythium ultimum and Rhizoctonia solani. J. Appl. Microbiol. 87, 80^90.
[4] Thrane, C., Olsson, S., Nielsen, T.H. and SÖrensen, J. (1999) Vital £uorescent stains for detection of stress in Pythium ultimum and Rhizoctonia solani challenged with viscosinamide from Pseudomonas £uorescens DR54. FEMS Microbiol. Ecol. 30, 11^23. [5] Hansen, M., Thrane, C., SÖrensen, J. and Olsson, S. (2000) Confocal imaging of living fungal hyphae challenged with the antifungal antagonist. Mycologia 92, 216^221. [6] Tsao, P.H. and Ocana, G. (1969) Selective isolation of species of Phytophtora from natural soils on improved antibiotic medium. Nature 223, 636^638. [7] Thrane, C., Tronsmo, A. and Jensen, D.F. (1997) Endo-1,3-L-glucanase and cellulase from Trichoderma harzianum: puri¢cation and partial characterization, induction of and biological activity against Pythium spp.. Eur. J. Plant. Pathol. 103, 331^344. [8] Gould, W.D., Hegedorn, C., Bardinelli, T.R. and Zablotowics, R.M. (1985) New selective media for enumeration and recovery of £uorescent pseudomonads from various habitats. Appl. Environ. Microbiol. 49, 28^32. [9] Hoben, H.J. and Somasegaran, P. (1982) Comparison of the pour, spread, and drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized peat. Appl. Environ. Microbiol. 44, 1246^1247. [10] Kandel, A., Nybroe, O. and Rasmussen, O.F. (1992) Survival of 2,4dichlorophenoxyacetic acid degrading Alcaligenes eutrophus AEO106 (pR0101) in lake water microcosms. Microb. Ecol. 24, 291^303. [11] Chart, H. and Rowe, B. (1992) A simple procedure for the preparation of lipopolysaccharides for the production of antisera. Lett. Appl. Microbiol. 14, 263^265. [12] Hansen, M., Kragelund, L., Nybroe, O. and SÖrensen, J. (1997) Early colonization of barley roots by Pseudomonas £uorescens studied by immuno£uorescence technique and confocal laser scanning microscopy. FEMS Microbiol. Ecol. 23, 353^360. [13] Asaka, O. and Shoda, M. (1996) Biocontrol of Rhizoctonia solani damping-o¡ of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol. 62, 4081^4085. [14] Bartnicki-Garcia, S., Persson, J. and Chanzy, H. (1994) An electron microscope and electron di¡raction study of the e¡ect of calco£uor and congo red on the biosynthesis of chitin in vitro. Arch. Biochem. Biophys. 310, 6^15. [15] Butt, T.M., Hoch, H.C., Staples, R.C. and St. Leger, R.J. (1989) Use of £uorochromes in the study of fungal cytology and di¡erentiation. Exp. Mycol. 13, 303^320. [16] Hansen, J.F., Thingstad, T.F. and Gokso«yr, J. (1974) Evaluation of hyphal lengths and fungal biomass by a membrane ¢lter technique. Oikos 25, 102^107. [17] Lu«beck, P.S., Hansen, M. and SÖrensen, J. (2000) Simultaneous detection of the establishment of seed-inoculated Pseudomonas £uorescens strain DR54 and native soil bacteria on sugar beet root surfaces using £uorescent antibody and in situ hybridization technique. FEMS Microbiol. Ecol. 33, 11^19. [18] Deacon, J.W. and Berry, L.A. (1992) Modes of action of mycoparasites in relation to biocontrol of soil-borne plant pathogens. In: Biological Control of Plant Diseases (Tjamos, E.S., Papavizas, G.C. and Cook, R.J., Eds.), pp. 157^165. Plenum Press, New York. [19] Warburton, A.J. and Deacon, J.W. (1998) Transmembrane Ca2 £uxes associated with zoospore encystment and cyst germination by the phytopathogen Phytophthora parasitica. Fungal Genet. Biol. 25, 54^62. [20] Stanghellini, M.E. and Miller, R.M. (1997) Biosurfactants : Their identity and potential e¤cacy in the biological control of zoosporic plant pathogens. Plant Disease 81, 4^12.
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Viscosinamide-producing Pseudomonas £uorescens DR54 exerts a biocontrol e¡ect on Pythium ultimum in sugar beet rhizosphere Charlotte Thrane *, Tommy Harder Nielsen, Mette Neiendam Nielsen, Jan SÖrensen, Stefan Olsson Section of Genetics and Microbiology, Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsenvej 40, DK-1871 Frederiksberg C, Denmark Received 7 January 2000; received in revised form 15 May 2000; accepted 18 May 2000
Abstract Growth inhibition of the root pathogen Pythium ultimum by the biocontrol agent Pseudomonas fluorescens DR54 inoculated on sugar beet seeds was studied in a soil microcosm. Plant emergence was followed, together with bacterial rhizosphere colonization, antibiotic production and effects on fungal growth. P. fluorescens DR54 inoculation of the P. ultimum-challenged seeds improved plant emergence after 7 days compared to a control without the biocontrol strain. At this time, P. fluorescens DR54 was the dominating colony-forming pseudomonad in rhizosphere soil samples from inoculated seedlings as shown by immuno-staining with a strain specific antibody. Viscosinamide, a cyclic lipopeptide, which has previously been identified as a major antagonistic determinant produced by P. fluorescens DR54 and shown to induce physiological changes in P. ultimum in vitro, could be detected in the rhizosphere samples. The impact of P. fluorescens DR54 on the growth and activity of P. ultimum was studied by direct microscopy after staining with the vital fluorescent dyes Calcofluor white and fluorescein diacetate. P. fluorescens DR54 caused reduction in P. ultimum mycelial density, oospore formation and intracellular activity. Further, Pythium oospore formation was absent in the presence of P. fluorescens DR54. A striking effect on zoosporeforming indigenous fungi was also observed in microcosms with P. fluorescens DR54 and, thus, where viscosinamide could be detected; a large number of encysted zoospores were seen in such microcosms both with and without P. ultimum infections. In vitro studies confirmed that purified viscosinamide induced encystment of Pythium zoospores. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Biological control ; Pseudomonas £uorescens ; Pythium ultimum; Soil microcosm; Viscosinamide ; Zoospore encystment
1. Introduction In biological control of plant pathogenic microorganisms, evaluation of the e¤cacy is often conducted by monitoring plant health. Thus, there is a general lack of direct observations demonstrating inhibitory e¡ects on the disease-causing target organisms in soil. Detection of antagonistic compounds from biocontrol agents in soil, such as phenazine produced by Pseudomonas £uorescens 2-79 controlling Gaumannomyces graminis var. tritici, has been re-
* Corresponding author. Tel.: +45 (35) 282649; Fax: +45 (35) 282606; E-mail : [email protected]
ported [1]. However, a direct in situ monitoring of the biocontrol agent, the antimicrobial metabolites and the e¡ects on target organisms in the rhizosphere has, to our knowledge, not been reported. P. £uorescens DR54 was isolated from sugar beet rhizosphere and has shown biocontrol of Pythium in planta [2]. This bacterial isolate produces the antifungal cell-associated lipopolypeptide, viscosinamide [3], which induces physiological changes in Pythium ultimum and Rhizoctonia solani in vitro and in soil as studied by £uorescence microscopy [4,5]. The purpose of this work was to study e¡ects of seed-inoculated P. £uorescens DR54 on the growth pattern of P. ultimum in soil by direct microscopy. For these experiments, a soil microcosm was developed to study the fate and activity of interacting microorganisms in a biocontrol experiment using P. £uorescens DR54 as biocontrol agent focusing on the early development (1 week) of pre-emergence damping-o¡ disease in sugar
0168-6496 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 0 ) 0 0 0 5 4 - 4
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beet caused by P. ultimum. The microcosm allowed us to study: (1) plant health as monitored by seedling emergence; (2) rhizosphere colonization and production of antifungal metabolites by seed-inoculated P. £uorescens DR54 and (3) response by the plant pathogenic P. ultimum when a¡ected by the bacterial antagonist. 2. Materials and methods 2.1. Plant and microorganisms Sugar beet (cv. `Madison', Danisco Seed A/S, Holeby, Denmark) was used as test plant. The biocontrol agent was P. £uorescens DR54 isolated from sugar beet [2]. The bacterium was stored at 380³C in glycerol (1:1 w/v) and for short periods in the laboratory on LB agar (Luria^ Bertani agar; 1% Bacto Tryptone (Difco Laboratories, Detroit, MI, USA), 0.5% yeast extract (Difco), 1% NaCl, 0.01% glucose, 2% agar (Difco) ; pH 7.2). For production of bacterial inoculum, the strain was cultivated in LB broth for 16^24 h. Inoculum was added to the seeds in a 10-Wl volume (5U107 colony-forming units (cfu) was added per seed). The pathogen P. ultimum Trow isolate HB2 (var. ultimum) was originally isolated from the HÖjbakkegaard ¢eld station in Denmark (Plant Pathology Section, Royal Veterinary and Agricultural University, Copenhagen ; Danisco Seed strain 92001). For maintenance and production of inoculum, P. ultimum was grown on potato dextrose agar (PDA) (Difco). For speci¢c studies on fungal zoospore production a well-characterized zoospore-producing Pythium isolate P11 was used. Pythium isolate P11 (Pythium `Group F' according to van der Plaats-Niterink (1981)) was isolated from diseased pepper plants grown in a hydroponic system by C. Rosendahl, University of Copenhagen, Denmark. To isolate indigenous Pythium spp., a soil extract was made by sonicating 100 g test soil in 75 ml 0.9% NaCl for 3 min in a water bath. The suspension was vortexed and the soil was allowed to settle for 4 min before 5 ml of the liquid phase was sampled. Two hundred Wl of this sample was spread on each of 20 plates with Pythium selective P10 -AR agar [6] made up as follows : 17 g corn meal (Difco), 40 g agar (Difco), 10 mg l31 pimaricin (Sigma P-0440), 250 mg l31 ampicillin (Sigma A-9518), 10 mg l31 rifampicin (Sigma R-3501) and 1000 ml H2 O. The antibiotics were added after autoclaving. The plates were incubated at 20³C for 4 days. At this time colonies of Pythium spp. had grown into the agar medium whereas colonies of other fungi grew only on the agar surface and could easily be washed o¡ with tap water. Twenty of the indigenous Pythium spp. isolates were subcultured twice for 2 days on selective medium (P10 -AR), before they were transferred to PDA medium. Zoospores from the Pythium isolate P11 were obtained according to Thrane et al. [7] except that the solution for induction of zoospore
formation (`Petri' solution) was made up containing : 0.4 g Ca(NO3 )2 W4H2 O, 0.15 g MgSO4 W7H2 O, 0.15 g KH2 PO4 and 0.06 g KCl. This solution was kept at 4³C and ¢lter sterilized (0.2 Wm) before use. 2.2. Soil microcosm The soil microcosm developed is shown in Fig. 1. A sandy loam from a fallow ¢eld (pH 6.4) at the HÖjbakkegaard ¢eld station was used as test soil. After sieving (4 mm mesh) the water content was adjusted to 13% using a perfusing spray technique and the soil was kept at 4³C for 24 h before use. This water content was 85% of the ¢eld capacity of the soil. This speci¢c water content was chosen because a suitable disease pressure by the pathogenic fungus could be obtained under these conditions. An inoculum of P. ultimum was applied as a 1U1 cm agar plug from a 3^4-day old colony grown on PDA medium. Glass ¢ber ¢lters (42.5 mm; GF/C, Whatman International) were prewetted in tap water and placed on top of the fungal inoculum. Each Petri dish representing one replicate contained three such GF/C ¢lters covering agar plugs with fungal inoculum. The sugar beet seed was placed in the center of each ¢lter. Bacterial inoculum (10 Wl containing 5U109 cells ml31 ) was then applied to the seed surface. The Petri dishes were ¢lled with soil and incubated for 0, 5 or 7 days in the dark at 15³C in plastic bags. Incubation times of 5 and 7 days were chosen as appropriate because the seeds germinated with visible root development after approximately 4 days. The incubation temperature of 15³C was close to that of the natural soil during seedling emergence in spring. Each experiment had 3^6 replicates of each treatment. Separate microcosms were used for di¡erent analyses as both microscopy and viscosinamide extraction were destructive methods. 2.3. Harvest and study of soil microcosms At the end of the experiment (days 5 or 7), soil microcosms were harvested to study the following parameters: plant emergence, numbers of pseudomonads (cfu) colonizing the rhizosphere using selective medium combined with P. £uorescens DR54 strain-speci¢c antibody staining, production of viscosinamide using high-performance liquid chromatography (HPLC) analysis, and changes in growth pattern, spore formation and activity of P. ultimum using vital £uorescent stains and direct microscopy. Plant health was estimated by quantifying root length and the number of seedlings emerging from the soil. Total populations of Pseudomonas spp. were enumerated using Gould's S1 selective medium [8]. Speci¢c determination of P. £uorescens DR54 was carried out using immuno-staining of colonies developed on Gould's S1. Enumerations were carried out as follows. Plants were harvested and soil adhering to a root after gentle shaking by hand was de¢ned as rhizosphere soil. All roots of the
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Fig. 1. Design of the microcosm. (A) The Petri dish seen from a side view. Only one agar plug with fungal inoculum is shown but three agar plugs with fungal inoculum were used in each Petri dish. Prewetted glass ¢ber ¢lters were placed on top of the agar plugs. Seeds were placed in the center on top of the ¢lters. Bacteria were added to the seeds in a 10-Wl volume (5U107 cells seed31 ). (B) Microcosm seen from the bottom of the Petri dish where fungal structures could be studied by an inverted microscope. Macroscopically, it was possible to observe the seed through the glass ¢ber ¢lter. (C) Detailed view of one glass ¢ber ¢lter observed from the bottom of the Petri dish. In a circular area (15 mm diameter) around the seed fungal mycelium was examined to study the antagonistic e¡ect of P. £uorescens DR54. The introduced fungus could be distinguished from indigenous fungi by following the outgrowth of the fungus from the point of inoculation (agar plug).
three seedlings from each Petri dish were pooled and bacteria associated with the rhizosphere soil were extracted in 2 ml 0.9% (w/v) NaCl by vortexing (30 s) and sonication in a water bath (30 s). After vortexing (30 s), the extract was left 3 min for sedimentation of particles and 1.5 ml of the liquid phase was removed for dilutions. In one set of experiments, drop-plating according to Hoben and Somasegaran [9] was carried out on whole roots. In another set of experiments the roots were divided into three pieces of approximately 1 cm length representing base, middle and tip of the root. In this case, 100 Wl of each dilution was plated on each Petri dish. In both sets of experiments, cfu counts were determined on Gould's S1 medium after the plates had been incubated for approximately 22 h at 28³C. Data are shown as means with standard error and t-tests were performed to compare treatments statistically. The blotting of colonies from Gould's S1 plates followed by immuno-staining with speci¢c antibodies targeting P. £uorescens DR54 was carried out according to Kan-
del et al. [10]. A polyclonal antibody speci¢c against P. £uorescens DR54 was made by immunizing rabbits (strain Ssc:CPH) with lipopolysaccharides prepared from Proteinase K digestion as described by Chart and Rowe [11]. Immunization, determination of speci¢city and removal of weak cross reactions were performed as described by Hansen et al. [12]. Viscosinamide produced by P. £uorescens DR54 was extracted from plants grown in the microcosms at each sampling day (days 0, 5 and 7). Thirty plants from 10 microcosms were pooled in one replicate and three such replicates were used for viscosinamide extraction. Viscosinamide was extracted from ¢lters or from rhizosphere soil acetonitrile (HPLC grade) mixed with 3.8 mM tri-£uoroacetic acid (4:1, v:v) as suggested by Asaka and Shoda [13]. Extractions included 1 h vigorous shaking of the organic phase on a rotary shaker before particles were allowed to settle for 15 min. The organic phase was subsequently collected in Te£on tubes before evaporation in a
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vacuum centrifuge (Maxy dry Iyo, Holm and Halby, Copenhagen, Denmark). After evaporation the extracts were dissolved in methanol and frozen for later analysis. HPLC analysis was carried out according to Nielsen et al. [3]. For determination of background signal, extracts were made from microcosms where seeds had been treated with 10 Wl of bacteria-free spent growth medium. At day 5, seed coats were still attached to the seedlings but viscosinamide was quanti¢ed separately for both seed coat and rhizosphere extract samples. At day 7, the seed coat had detached from the seedling and both plant parts were analyzed separately as on day 5. In another experiment, viscosinamide was quanti¢ed in the glass ¢ber ¢lters. The central part (15 mm diameter (Fig. 1)) and the outer rim of the ¢lters were analyzed separately. The fungal mycelium grew well within the glass ¢ber ¢lters and subsequent visualization by microscopy was possible after application of vital £uorescent stains directly to the ¢lter. For the microscopic studies, six separate replicates with three ¢lters each were used for each experiment. The stains di¡used easily within the ¢lters resulting in nearly homogeneous staining. Because the fungi were growing within the ¢lter matrix, spatial disturbance of fungal hyphae by stain application was minimal. Fluorescence microscopy of P. ultimum could be performed successfully in the soil microcosm using Calco£uor white (Sigma Fluorescent Brightner, Sigma F-3397) and £uorescein diacetate (FDA, Sigma F-7378). Calco£uor white may be used as a wall stain in fungi [14] and FDA may be used to study general fungal activity as represented by intracellular esterase activity [15]. Stock solutions of the stains were 1 mg ml31 in water (Calco£uor white) or acetone (FDA). The stains were applied in a water solution at a ¢nal concentration of 3 Wl ml31 (Calco£uor white) or 4 Wl ml31 (FDA) [4]. Three aliquots of 100 Wl stain solution were applied to each ¢lter in the Petri dishes using a pipette tip to penetrate the soil compartment. For £uorescence microscopy, a Nikon Eclipse TE 300 inverted microscope mounted with a high pressure Hg 100 W lamp was used. UV (330^380 nm excitation ¢lter and 420 nm barrier ¢lter) and blue (450^490 nm excitation ¢lter and 520 nm barrier ¢lter) ¢lter sets were used. Photographs were taken with 200 or 400 ISO Fujicolor ¢lms. Detailed examination by microscopy was carried out in the area close to the seed (Fig. 1). Hyphal length was recorded according to Hansen et al. [16] On each ¢lter an average of the recorded length was determined by counting the intersections in three areas of 6.25 mm2 . The number of oospores (approximately 15^20 Wm diameter) was determined as the total number counted in ten random 1-mm2 areas on each ¢lter within a 15-mm wide total area around the seed. Encysted zoospores (approximately 5 Wm diameter) were determined by counting the numbers of cysts in three random 0.25-mm2 areas on each ¢lter within the 15-mm wide total area around the seed. The experiment was repeated with the same result.
2.4. E¡ects of viscosinamide on zoospore production and encystment of Pythium The e¡ect of viscosinamide on zoospore production was studied using the zoospore-producing isolate Pythium P11. Ten Wl DMSO containing HPLC-puri¢ed viscosinamide was added to Petri dishes containing 10 ml `Petri solution' to give either 5 or 10 Wg ml31 ¢nal concentrations of the antibiotic. Each Petri dish was inoculated with 10 agar plugs (4 mm diameter) of mycelial inoculum. Ten Wl DMSO was added to the control dishes. Zoospore production was quanti¢ed by enumeration in a counting chamber after 18 h of incubation at 20³C. Three replicates were used per treatment. Finally, the e¡ect of viscosinamide on zoospore encystment was studied. Three small ¢lters (GF/C; 5 mm diameter) each containing 1 Wg viscosinamide [4] were placed in a Petri dish. Three-ml suspensions of freshly prepared zoospores from Pythium P11 were added to the Petri dishes (zoospore concentrations were 5U104 ^1U105 ml31 ). Zoospore encystment was quanti¢ed by counting the number of encysted, non-germinated zoospores after 24 h of incubation at 20³C. Three replicates were used per treatment. Data are shown as means with standard error and t-tests were performed to compare treatments statistically. The experiments were repeated with the same result. 3. Results 3.1. Plant emergence and biological control Fig. 2A shows that all plants emerged in the control experiment with neither P. ultimum nor P. £uorescens DR54 inoculation. By comparison, an average of 81% plants had emerged after 7 days in the presence of DR54. This indicated that the P. £uorescens DR54 biocontrol strain delayed seed germination or inhibited plant growth slightly by a phytotoxic e¡ect. The plant pathogenic e¡ect of P. ultimum was apparent from further reduction of seedling numbers to a low 55% emergence. However, when both P. ultimum and strain DR54 were present, a biocontrol e¡ect could be observed from a high (86%) emergence which was similar to the level when only P. £uorescens DR54 was present. Fig. 2B shows that root lengths after 7 days were approximately 3.5 cm on seedlings without both P. ultimum and P. £uorescens DR54 and on the seedlings with only DR54. The putative phytotoxic e¡ect of P. £uorescens DR54 of seedling emergence (Fig. 2A) was therefore not re£ected in root development of the emerging seedlings. However, the presence of Pythium reduced the average root lengths to approximately 3 cm on the emerging seedlings. Finally, despite the e¤cacy of P. £uorescens DR54 as a biocontrol strain improving seedling emergence, its presence together with Pythium further reduced the aver-
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age root lengths to approximately 2 cm on the emerging seedlings. 3.2. Rhizosphere colonization by P. £uorescens DR54 Fig. 3A shows P. £uorescens DR54 colonization of the sugar beet rhizosphere during the 7 days. Immediately after the seeds were placed in the soil microcosms (day 0), only a small fraction (2%) of the initial population of approximately 5U107 cells inoculated could be recovered as cfu from the seeds. It was clear that a very large proportion of the initial P. £uorescens DR54 population either was rapidly displaced to the soil surrounding the seeds or decreased in culturability. It was likely that at least a part of the bacteria inoculated on the seeds was a major source of P. £uorescens DR54 cells for rhizosphere colonization occurring from day 4 when roots began to develop from the seed. However, cell death, decreased culturability or decreased cell extraction due to adherence to the seed surface are likely explanations to the fact that only a small cell fraction could be recovered from the inoculated seeds. At day 5, the seedlings were approximately 1 cm long and the total Pseudomonas population was approximately 5U105 cells on the whole root as judged from colony formation on Gould's S1 medium; 93^100% of the Fig. 3. Microcosm experiment to study rhizosphere colonization and viscosinamide production by P. £uorescens DR54 in the absence of P. ultimum. (A) Cfu formed on Pseudomonas selective medium (Gould's S1) in treatments with seeds inoculated with P. £uorescens DR54. Cfu counts were determined at three sampling days; 0, 5 and 7. At day 0, only a small fraction (2%) of the added inoculum of 5U107 adhered to the seeds. At days 5 and 7, bacteria were extracted from the rhizosphere of the whole roots with lengths of approximately 1 and 3.5 cm, respectively. (B) Viscosinamide was extracted from the seed at day 0. At days 5 and 7 the two bars show the viscosinamide detected on the seed and in the rhizosphere of the approximately 1 and 3.5 cm long roots, respectively. The data shown are from one representative experiment. Bars show standard error (n = 3).
Fig. 2. Plant data from soil microcosm experiments after 7 days of incubation at 15³C. Sugar beet seeds were challenged with P. ultimum with or without protection with P. £uorescens DR54. (A) The percentage of plants emerged in four di¡erent treatments. (B) Plant root length in the same four treatments. Data are shown as the average from three microcosm experiments in which all plants in the control treatment emerged. Bars show standard error (n = 9).
colonies were identi¢ed as P. £uorescens DR54 by the immuno-assay. At day 7, when the seedling roots were approximately 3.5 cm long, the total Pseudomonas population was approximately 2U105 cells on the whole root ; again, 93^100% of the colonies were identi¢ed as P. £uorescens DR54. Finally, the cell distribution along the roots demonstrated that the rhizosphere of the region of root closest to the seed contained a large majority of the P. £uorescens DR54 cells (99%) at this time, while much smaller fractions were recovered from the intermediate (0.6%) and tip (0.01%) segments of the roots. On day 7, rhizosphere colonization of P. £uorescens DR54 was also studied in Pythium-infected microcosms. In these treatments (n = 9), the total Pseudomonas population was similar (P s 0.05) to that in rhizosphere developed from P. £uorescens DR54-treated roots (1.16U105 þ 0.24U105 cfu plant31 ). As in microcosms without Pythium infection, almost all (91^100%) the colonies
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Table 1 E¡ect of P. £uorescens DR54 inoculation on sugar beet seeds Seed inoculation
Viscosinamide (ng mm32 )
Total hyphal length (mm mm32 )
Oospores (numbers mm32 ) Encysted zoospores (numbers mm32 )
3DR54 +DR54
nda;b 0.22b
20.3 þ 2.0c 9.7 þ 0.2
12.8 þ 2.3c nda
36.7 þ 12.0c 3250.0 þ 150.0
Data are from 7-day-old seedlings and show viscosinamide concentration, total hyphal length and numbers of oospores (P. ultimum) and numbers of encysted zoospores (indigenous fungi). The parameters were quanti¢ed in a 15-mm-wide area surrounding the seed. Data shown are means þ S.E.M. a nd = not detected. b The value shown is the average viscosinamide content from 10 microcosms with three plants each (n = 1). After 5 days the viscosinamide content was 0.17 þ 0.0035 ng mm32 (n = 3). c n = 6.
were identi¢ed as P. £uorescens DR54 by the immunoassay. Hence, the presence of P. ultimum had no apparent signi¢cant e¡ect (P s 0.05) on the total population of DR54 cells colonizing the rhizosphere roots (0.90U105 þ 0.19U105 cfu plant31 ). In the absence of P. £uorescens DR54 treatment of the seeds, higher numbers (P 6 0.05) of indigenous pseudomonads colonized the rhizosphere of Pythium-challenged plants (5.44U 104 þ 2.11U104 cfu plant31 ) than control plants without Pythium (2.1U103 þ 0.78U103 cfu plant31 ). 3.3. Occurrence of viscosinamide on sugar beet roots The viscosinamide produced by P. £uorescens DR54 was found to be strongly cell-bound in batch cultures of P. £uorescens DR54 [3]. The P. £uorescens DR54 inoculum added to each seed (5U107 cfu per seed) contained approximately 400 ng viscosinamide. Fig. 3B shows that the viscosinamide detected on the seeds at day 0 was approximately 31 ng per seed which is approximately 8% of the added viscosinamide. However, this relates well to the low recovery of cells (2% of the total P. £uorescens DR54 inoculum) on the seeds at day 0 (see above). Thus, neither viscosinamide nor bacterial cells could be recovered in large amounts after the seed inoculation. It is possible that the soil surrounding the seeds thus contained both the majority of P. £uorescens DR54 cells and viscosinamide present at day 0. However, in the case of viscosinamide, adherence to particles on the seeds (or soil) is another possible explanation to the low recovery from seeds. At day 5, approximately 16 ng viscosinamide was detected in the rhizosphere (roots were approximately 1 cm long) and approximately 157 ng on the seed coats. At day 7, approximately 61 ng viscosinamide was detected in the rhizo-
sphere soil (roots were approximately 3.5 cm long) and approximately 270 ng on the seed coats. It was thus apparent that the total amount of viscosinamide associated with both the rhizosphere soil and the seed coats increased during the 7 days of incubation. 3.4. E¡ect of P. £uorescens DR54 and viscosinamide on P. ultimum Fungal growth and viscosinamide pools were determined in the central area of the ¢lter (Fig. 1 and below), where the seeds had been inoculated with P. £uorescens DR54. The amount of viscosinamide was approximately seven times larger in the 15-mm-wide center (0.22 ng mm2 ) than in the periphery of the ¢lter (Table 1). The introduced P. ultimum isolate has characteristic hyphae without septation and radial growth could easily be determined as the mycelium grew out from the point of inoculation (agar plug). Within the incubation time of 7 days, microscopy of the Pythium inoculum was made easier because mycelia of indigenous fungi were only sparsely developed. Table 1 shows that the total hyphal length per unit area was reduced to approximately 9.7 mm mm32 compared to approximately 20.3 mm mm32 in the controls without P. £uorescens DR54. Oospore formation in the Pythium mycelium was common, approximately 12.8 oospores mm32 immediately around the seed without P. £uorescens DR54 inoculum while oospores were completely absent from P. £uorescens DR54 inoculated seeds. In these hyphae close to the uninoculated seed, FDA staining was strong (data not shown) indicating that the fungus was alive and ¢lled with cytoplasm. Thus, the presence of P. £uorescens DR54 on the seeds partially inhibited fungal growth and inhibited oospore formation close
Table 2 In vitro zoospore encystment and zoospore production after treatment of Pythium P11 with viscosinamide Treatment
Zoospores produceda (numbers mm32 )
Encysted zoosporesb (numbers mm32 )
Control Viscosinamide-treated
212 þ 84 120 þ 28
312 þ 84 1876 þ 420
a The e¡ect of viscosinamide on zoospore production was studied after viscosinamide was added to the zoospore-inducing medium (5.0 Wg viscosinamide ml31 medium). b Zoospore encystment was studied by counting encysted zoospores on small glass ¢ber ¢lters placed in zoospore suspension (1 Wg of viscosinamide was initially adsorbed to each ¢lter). Data shown are mean þ S.E.M. (n = 3).
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to the seed dramatically as no oospores could be detected in the P. £uorescens DR54-inoculated microcosms. Interestingly, a large number of encysted zoospores were observed around seeds inoculated with P. £uorescens DR54. The encysted zoospores were thought to originate from indigenous zoospore-producing fungi since the introduced P. ultimum isolate did not produce zoospores. Furthermore, the encysted zoospores were also formed in microcosms without P. ultimum inoculum. The numbers of cysts mm32 was approximately 10 times higher when P. £uorescens DR54 was present (Table 1). The zoospore encystment was primarily detected in the center of the ¢lter (15 mm in diameter) indicating that the high viscosinamide content here was responsible for the pronounced zoospore encystment. Zoospore production under in vitro conditions was slightly inhibited by viscosinamide (P s 0.05), while encystment of zoospores was strongly stimulated by viscosinamide (P 6 0.005) (Table 2). 4. Discussion The biocontrol strain P. £uorescens DR54 increased plant emergence of sugar beet from P. ultimum-challenged seeds as was also shown by Nielsen et al. [2]. Such a result is in general interpreted to result from direct antagonistic interaction between the biocontrol agent and the pathogen, e.g. by antibiotic production, competition or release of cell wall degrading enzymes. In this work we developed a method for direct studies in soil microcosms of the growth and activity of the biocontrol strain and the pathogenic fungus and of the mechanism of interaction. An important prerequisite for the biocontrol e¡ect of P. £uorescens DR54 was the ability of P. £uorescens DR54 to establish in the rhizosphere. Population sizes of P. £uorescens DR54 in the rhizosphere were thus 10^100 times higher than for other, naturally-occurring Pseudomonas spp. in the rhizosphere of control plants during the 7 days of seedling emergence. P. £uorescens DR54 was even detected in the rhizosphere of the distal part of the roots indicating that the bacterium was able to actively colonize the plant rhizosphere by root attachment and growth. Lu«beck et al. [17] studied P. £uorescens DR54 colonization on the sugar beet rhizoplane by confocal laser scanning microscopy and found that P. £uorescens DR54 was the dominating organism a few days after the inoculation. During their 20-day study, active microcolonies of P. £uorescens DR54 could still be identi¢ed on all parts of the roots. The mechanism of Pythium biocontrol in soil microcosms was likely to be the viscosinamide antibiotic produced by P. £uorescens DR54. Viscosinamide was detected in increasing amounts on both seed coats and in rhizosphere soil surrounding the roots during the 7 days of incubation. The results indicated that viscosinamide was
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replenished from P. £uorescens DR54 colonizing the roots at an early stage of emergence. In this study a possible phytotoxic e¡ect of P. £uorescens DR54 in combination with P. ultimum was recorded by root length measurements. However, the measured root lengths of plants from microcosms with inoculation of P. £uorescens DR54 without co-inoculation with P. ultimum, indicated that the bacterium did not by itself cause this e¡ect. In pot experiments with a similar soil no signi¢cant reduction of root lengths of plants that have been co-inoculated with both P. £uorescens DR54 and P. ultimum was seen (Ba«renholdt-Scho«ler, unpublished). The presence of P. £uorescens DR54 seemed to have both short- and long-term e¡ects on the target fungus, P. ultimum. As studied by microscopy in the soil microcosms, mycelial density was signi¢cantly reduced and oospore formation was not detected in the presence of P. £uorescens DR54. One explanation could be that oospore formation occurred only during dense and rapid mycelium development in the control microcosms without P. £uorescens DR54. The absence of oospores could be a result of the sparse fungal biomass development due to competition in the presence of P. £uorescens DR54 [18]. However, the complete absence of oospores in the P. £uorescens DR54-inoculated microcosms indicated a speci¢c inhibition of oospore formation by the biocontrol strain. In general, formation of survival structures such as spores is thought to be one of several responses to physiological stress. Inhibition of oospore formation in turn suggested that this process was most sensitive to P. £uorescens DR54. A most intriguing observation was also that P. £uorescens DR54 strongly stimulated the encystment of zoospores formed by Pythium and indigenous fungi in the soil microcosms. Viscosinamide produced by P. £uorescens DR54 was likely to be directly responsible for the observed induction of zoospore encystment in the microcosms as in vitro experiments supported this observation. Supplementary studies supported the hypothesis that the structures observed could indeed be encysted zoospores. (1) Indigenous oomycetes could be isolated from the test soil. (2) Nile red, that is useful for staining of membranes and lipids in fungi [15], demonstrated the presence of both swimming zoospores and encysted zoospores. (3) Calco£uor white, which stains cell wall polysaccharides of fungal structures [14], also con¢rmed the presence of encysted zoospores. (4) Finally, the encysted zoospores were easily distinguished from other fungal spores in the microcosms because fungal sporangia could be seen and because the spores were of di¡erent size and shape. In a search for the mechanistic action of viscosinamide in fungal inhibition, Thrane et al. [4] suggested that the compound inhibits growth by formation of ion-channels in the fungal membrane. This has subsequently been examined by challenging an Aspergillus awamori transformant expressing the Ca2 -sensitive protein aequorin with visco-
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sinamide. The fungus responded to the viscosinamide by a large and immediate increase in cytoplasmic Ca2 -level (Kozlova et al., unpublished result). Warburton and Deacon [19] have shown that zoospores of Phytophthora parasitica take up Ca2 just before encystment. Increased permeability of the zoospore to Ca2 resulting in higher intracellular Ca2 levels could thus explain why viscosinamide triggered instant encystment of the fungal zoospores on non-plant surfaces in this study. Compounds with surfactant properties have been successfully applied in hydroponic systems to control zoospore-producing fungal pathogens [20]. The results of our study indicate that this may take place by induction of zoospore encystment in the absence of a plant and that P. £uorescens DR54 and comparable strains may have a large potential as biocontrol agents speci¢cally against zoospore-forming fungi. Encystment of zoospores in the rhizosphere soil prevents their motility towards the root surface. Colonization and proliferation of Pythium by zoospore infection on the root surface should thus be prevented. Thus, in the presence of viscosinamide, Pythium infection is antagonized by inhibition of both hyphal growth and zoospore motility in the rhizosphere. Acknowledgements The Danish Ministry of Agriculture (contract No. 93Sî 95-00788) supported this study in cooperation with 2466-A Danisco Seeds and Novo Nordisk a/s. Further the work was supported by grant No. 9313839 from the Danish Agricultural and Veterinary Research Council. We thank Ole Nybroe for providing the protocols for immuno-detection of the bacterial inoculant. The technical assistance by Dorte Rasmussen and Ulla Rasmussen is highly appreciated. Dan Funck Jensen kindly provided Pythium isolate P11. References [1] Thomashow, L.S., Weller, D.M., Bonsall, R.F. and Pierson, L.S. (1990) Production of the antibiotic phenazine-1-carboxylic acid by £uorescent Pseudomonas species in the rhizosphere of wheat. Appl. Environ. Microbiol. 56, 908^912. [2] Nielsen, M.N., SÖrensen, J., Fels, J. and Pedersen, H.C. (1998) Secondary metabolite- and endochitinase-dependent antagonism toward plant-pathogenic microfungi of Pseudomonas £uorescens isolates from sugar beet rhizosphere. Appl. Environ. Microbiol. 64, 3563^3569. [3] Nielsen, T.H., Christophersen, C., Anthoni, U. and SÖrensen, J. (1999) Structure and production of a new bacterial cyclic depsipeptide; viscosinamide antagonistic against Pythium ultimum and Rhizoctonia solani. J. Appl. Microbiol. 87, 80^90.
[4] Thrane, C., Olsson, S., Nielsen, T.H. and SÖrensen, J. (1999) Vital £uorescent stains for detection of stress in Pythium ultimum and Rhizoctonia solani challenged with viscosinamide from Pseudomonas £uorescens DR54. FEMS Microbiol. Ecol. 30, 11^23. [5] Hansen, M., Thrane, C., SÖrensen, J. and Olsson, S. (2000) Confocal imaging of living fungal hyphae challenged with the antifungal antagonist. Mycologia 92, 216^221. [6] Tsao, P.H. and Ocana, G. (1969) Selective isolation of species of Phytophtora from natural soils on improved antibiotic medium. Nature 223, 636^638. [7] Thrane, C., Tronsmo, A. and Jensen, D.F. (1997) Endo-1,3-L-glucanase and cellulase from Trichoderma harzianum: puri¢cation and partial characterization, induction of and biological activity against Pythium spp.. Eur. J. Plant. Pathol. 103, 331^344. [8] Gould, W.D., Hegedorn, C., Bardinelli, T.R. and Zablotowics, R.M. (1985) New selective media for enumeration and recovery of £uorescent pseudomonads from various habitats. Appl. Environ. Microbiol. 49, 28^32. [9] Hoben, H.J. and Somasegaran, P. (1982) Comparison of the pour, spread, and drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized peat. Appl. Environ. Microbiol. 44, 1246^1247. [10] Kandel, A., Nybroe, O. and Rasmussen, O.F. (1992) Survival of 2,4dichlorophenoxyacetic acid degrading Alcaligenes eutrophus AEO106 (pR0101) in lake water microcosms. Microb. Ecol. 24, 291^303. [11] Chart, H. and Rowe, B. (1992) A simple procedure for the preparation of lipopolysaccharides for the production of antisera. Lett. Appl. Microbiol. 14, 263^265. [12] Hansen, M., Kragelund, L., Nybroe, O. and SÖrensen, J. (1997) Early colonization of barley roots by Pseudomonas £uorescens studied by immuno£uorescence technique and confocal laser scanning microscopy. FEMS Microbiol. Ecol. 23, 353^360. [13] Asaka, O. and Shoda, M. (1996) Biocontrol of Rhizoctonia solani damping-o¡ of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol. 62, 4081^4085. [14] Bartnicki-Garcia, S., Persson, J. and Chanzy, H. (1994) An electron microscope and electron di¡raction study of the e¡ect of calco£uor and congo red on the biosynthesis of chitin in vitro. Arch. Biochem. Biophys. 310, 6^15. [15] Butt, T.M., Hoch, H.C., Staples, R.C. and St. Leger, R.J. (1989) Use of £uorochromes in the study of fungal cytology and di¡erentiation. Exp. Mycol. 13, 303^320. [16] Hansen, J.F., Thingstad, T.F. and Gokso«yr, J. (1974) Evaluation of hyphal lengths and fungal biomass by a membrane ¢lter technique. Oikos 25, 102^107. [17] Lu«beck, P.S., Hansen, M. and SÖrensen, J. (2000) Simultaneous detection of the establishment of seed-inoculated Pseudomonas £uorescens strain DR54 and native soil bacteria on sugar beet root surfaces using £uorescent antibody and in situ hybridization technique. FEMS Microbiol. Ecol. 33, 11^19. [18] Deacon, J.W. and Berry, L.A. (1992) Modes of action of mycoparasites in relation to biocontrol of soil-borne plant pathogens. In: Biological Control of Plant Diseases (Tjamos, E.S., Papavizas, G.C. and Cook, R.J., Eds.), pp. 157^165. Plenum Press, New York. [19] Warburton, A.J. and Deacon, J.W. (1998) Transmembrane Ca2 £uxes associated with zoospore encystment and cyst germination by the phytopathogen Phytophthora parasitica. Fungal Genet. Biol. 25, 54^62. [20] Stanghellini, M.E. and Miller, R.M. (1997) Biosurfactants : Their identity and potential e¤cacy in the biological control of zoosporic plant pathogens. Plant Disease 81, 4^12.
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