Possible role of xanthobaccins produced by Stenotrophomonas sp. strain SB-K88 in suppression of sugar beet damping-off disease.

Three antifungal compounds, designated xanthobaccins A, B, and C, were isolated from the culture fluid of Stenotrophomonas sp. strain SB-K88, a rhizobacterium of sugar beet that suppresses damping-off disease. Production of xanthobaccin A in culture media was compared with the disease suppression activities of strain SB-K88 and less suppressive strains that were obtained by subculturing. Strain SB-K88 was applied to sugar beet seeds, and production of xanthobaccin A in the rhizosphere of seedlings was confirmed by using a test tube culture system under hydroponic culture conditions; 3 microg of xanthobaccin A was detected in the rhizosphere on a per-plant basis. Direct application of purified xanthobaccin A to seeds suppressed damping-off disease in soil naturally infested by Pythium spp. We suggest that xanthobaccin A produced by strain SB-K88 plays a key role in suppression of sugar beet damping-off disease.     

Suppression of Damping-Off Disease in Host Plants by the Rhizoplane Bacterium Lysobacter sp. Strain SB-K88 Is Linked to Plant Colonization and Antibiosis against Soilborne Peronosporomycetes

We previously demonstrated that xanthobaccin A from the rhizoplane bacterium Lysobacter sp. strain SB-K88 suppresses damping-off disease caused by Pythium sp. in sugar beet. In this study we focused on modes of Lysobacter sp. strain SB-K88 root colonization and antibiosis of the bacterium against Aphanomyces cochlioides, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of Lysobacter colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This Lysobacter strain also efficiently colonized the roots of several plants, including spinach, tomato, Arabidopsis thaliana, and Amaranthus gangeticus. Plants grown from both sugar beet and spinach seeds that were previously treated with Lysobacter sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by A. cochlioides. Interestingly, zoospores of A. cochlioides became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 μg/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that Lysobacter sp. strain SB-K88 has a direct inhibitory effect on A. cochlioides, suppressing damping-off disease. Furthermore, this inhibitory effect of Lysobacter sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.     

Suppression of damping-off disease in host plants by the rhizoplane bacterium Lysobacter sp. strain SB-K88 is linked to plant colonization and antibiosis against soilborne Peronosporomycetes

We previously demonstrated that xanthobaccin A from the rhizoplane bacterium Lysobacter sp. strain SB-K88 suppresses damping-off disease caused by Pythium sp. in sugar beet. In this study we focused on modes of Lysobacter sp. strain SB-K88 root colonization and antibiosis of the bacterium against Aphanomyces cochlioides, a pathogen of damping-off disease. Scanning electron microscopic analysis of 2-week-old sugar beet seedlings from seeds previously inoculated with SB-K88 revealed dense colonization on the root surfaces and a characteristic perpendicular pattern of Lysobacter colonization possibly generated via development of polar, brush-like fimbriae. In colonized regions a semitransparent film apparently enveloping the root and microcolonies were observed on the root surface. This Lysobacter strain also efficiently colonized the roots of several plants, including spinach, tomato, Arabidopsis thaliana, and Amaranthus gangeticus. Plants grown from both sugar beet and spinach seeds that were previously treated with Lysobacter sp. strain SB-K88 displayed significant resistance to the damping-off disease triggered by A. cochlioides. Interestingly, zoospores of A. cochlioides became immotile within 1 min after exposure to a SB-K88 cell suspension, a cell-free supernatant of SB-K88, or pure xanthobaccin A (MIC, 0.01 microg/ml). In all cases, lysis followed within 30 min in the presence of the inhibiting factor(s). Our data indicate that Lysobacter sp. strain SB-K88 has a direct inhibitory effect on A. cochlioides, suppressing damping-off disease. Furthermore, this inhibitory effect of Lysobacter sp. strain SB-K88 is likely due to a combination of antibiosis and characteristic biofilm formation at the rhizoplane of the host plant.     

Mode of antagonism of a biocontrol bacterium Lysobacter sp. SB-K88 toward a damping-off pathogen Aphanomyces cochlioides

The biocontrol bacterium Lysobacter sp. SB-K88 suppresses damping-off disease in sugar beet and spinach caused by Aphanomyces cochlioides and Pythium sp. through characteristic plant colonization and antibiosis against the pathogens. This study aimed to unravel further details on mode of antagonism of SB-K88 against a damping-off pathogen A. cochlioides AC-5. The SB-K88 substantially inhibited growth and decomposed AC-5 mycelia and suppressed the release of zoospores from the hyphae. The excised root tips of sugar beet seedlings from seeds previously inoculated with SB-K88 were less attractive to AC-5 zoospores. Although aerial growth was not affected, however, root hairs of SB-K88 inoculated sugar beet seedlings were remarkably shorter and thicker than those of uninoculated control. When exposed to zoospores, the SB-K88 inhibited motility of zoospores and/or caused lysis, and then aggregated around the dead cystospores or lysed residues within 3–6 h likely to be micro-predatory behavior to a eukaryotic organism. Confocal laser scanning microscopic analysis revealed that number of lipid bodies and activities of mitochondria were markedly increased in the affected hyphae compared with control hyphae as visualized by established vital stains. Taken together, these results suggest that Lysobacter sp. SB-K88 suppresses damping-off diseases through exerting multifaceted antagonistic effects against the peronosporomycetes.     

Pseudomonas fluorescens DR54 Reduces Sclerotia Formation,Biomass Development,and Disease Incidence of Rhizoctonia solani Causing Damping-Off in Sugar Beet

Effects of the biocontrol strain, Pseudomonas fluorescens DR54, on growth and disease development by Rhizoctonia solani causing damping-off in sugar beet were studied in soil microcosms and in pot experiments with natural, clay-type soil. In pot experiments with P. fluorescens DR54-treated seeds, significantly fewer Rhizoctonia-challenged seedlings showed damping-off symptoms than when not inoculated with the biocontrol agent. In the rhizosphere of P. fluorescens DR54 inoculated seeds, the bacterial inoculant was present in high numbers as shown by dilution plating and immunoblotting. By the ELISA antibody technique and direct microscopy of the fungal pathogen grown in soil microcosms, it was shown that the presence of P. fluorescens DR54 on the inoculated seeds had a strong inhibitory effect on development of both mycelium biomass and sclerotia formation by R. solani. In the field experiment, plant emergence was increased by treatment with P. fluorescens DR54 and the inoculant was found to be the dominating rhizosphere colonizing pseudomonad immediately after seedling emergence.     

Effect of the polysaccharide ofAgrobacterium radiobacter on the growth of plants and occurrence of damping-off in sugar beet

Agrobacterium radiobacter, strain B6 (a strain isolated in this laboratory, which limited the occurrence of damping-off of sugar beet and influenced growth of plants in hot-house and field experiments) was found to produce an acidic exopolysaccharide in a mineral medium with various carbon sources. Hydrolyzates of the polysaccharide contained glucose, galactose, glycerol, succinic acid and pyruvic acid, whose quantitative content varied according to the carbon source used. The polysaccharide isolated from the medium containing glucose exhibited the highest physiological activity. Seeds germinated best and sugar beet roots were found to grow most rapidly in a medium containing 0.2 % (W/W) of the polysaccharide. The roots exposed for 3 d in this medium grew 2.7-fold as compared with non-treated plants. Higher sumbers of microorganisms were detected on the surface of roots treated with the polysaccharide. Growth of roots was also stimulated when immersing the seeds (30 min) in a 0.2 –0.4 % solution of this polysaccharide. After a two-fold treatment the roots were less damaged by the fungusPythium ultimum. Plants from seeds treated with the polysaccharide grew in the field soil more rapidly than the non-treated plants but worse than after bacterization of the seeds byA. radiobacter B6 and were only partially protected against the damping-off of sugar beet.     

Control of Pythium damping-off of sugar beet by seed treatment with crop straw powders and a biocontrol agent

Seed treatment with non-sterilized powdered straws from 39 crops was tested for the control of Pythium damping-off of sugar beet. Four straws, including flax, coriander, pea, and lentil were effective in controlling the disease in soil artificially infested with Pythium sp. “group G.” Sterilizing flax and pea straws eliminated the efficacy of these straws. Wheat straw powder coated on sugar beet seeds increased the incidence of Pythium damping-off but this effect was reversed by the co-inoculation of wheat straws with the biocontrol agent Pseudomonas fluorescens 708. Coating sugar beet seeds with P. fluorescens 708 and flax or pea straws also increased the efficiency of the bacterial strain for the control of Pythium damping-off. Pea straws and to a lesser extent lentil straws produced volatile substances that affected mycelial growth of Pythium sp. “group G” on potato dextrose agar in Petri plates when the straws were mixed with water and left to ferment for two days. Fermentation of pea straws led to the accumulation of volatile ammonia, which was produced by the reduction of the large amount of nitrate stored in the straw. Reduction of nitrate and therefore the release of volatile ammonia did not occur in sterilized pea straws. However, fermenting sterile pea straws with bacteria from different genera restored nitrate reduction and the release of volatile ammonia, suggesting that microorganisms associated with pea straws are responsible for the conversion of nitrate into volatile ammonia which in turn control Pythium damping-off disease in sugar beet.     

Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.)

Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain.

Tn5 mutagenesis and complementation analysis were used to clone a 6-kb genomic fragment required for biosynthesis of 2,4-diacetylphloroglucinol (Phl) from fluorescent Pseudomonas sp. strain F113. A recombinant plasmid, pCU203, containing this region partially complemented a Phl production-negative mutant (F113G22) derived from strain F113. When sugar beet seeds were sown into an unsterilized soil, in which sugar beet was subject to damping-off by Pythium ultimum, the emergence of sugar beet seeds inoculated with strain F113 was significantly greater than that of seeds inoculated with F113G22. Transfer of pCU203 into eight other Pseudomonas strains conferred the ability to synthesize Phl in only one of these strains, Pseudomonas sp. strain M114. Strain M114(pCU203) showed enhanced antagonism towards P. ultimum in vitro and significantly increased the emergence of sugar beet seeds in the same soil compared with emergence induced by the parent strain M114.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g⁻¹) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ~2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.     

Mutation of rpiA in Enterobacter cloacae decreases seed and root colonization and biocontrol of damping-off caused by Pythium ultimum on cucumber

Strains of Enterobacter cloacae show promise as biocontrol agents for Pythium ultimum-induced damping-off on cucumber and other crops. E. cloacae A145 is a mini-Tn5 Km transposon mutant of strain 501R3 that was significantly reduced in suppression of damping-off on cucumber caused by P. ultimum. Strain A145 was deficient in colonization of cucumber, sunflower, and wheat seeds and significantly reduced in colonization of corn and cowpea seeds relative to strain 501R3. Populations of strain A145 were also significantly lower than those of strain 501R3 at all sampling times in cucumber, wheat, and sunflower rhizosphere. Populations of strain A145 were not detectable in any rhizosphere after 42 days, while populations of strain 501R3 remained at substantial levels throughout all experiments. Molecular characterization of strain A145 indicated mini-Tn5 Km was inserted in a region of the E. cloacae genome with a high degree of DNA and amino acid sequence similarity to rpiA, which encodes ribose-5-phosphate isomerase. In Escherichia coli, RpiA catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate and is a key enzyme in the pentose phosphate pathway. Ribose-5-phosphate isomerase activity in cell lysates from strain A145 was approximately 3.5% of that from strain 501R3. In addition, strain A145 was a ribose auxotroph, as expected for an rpiA mutant. Introduction of a 1.0-kb DNA fragment containing only the rpiA homologue into strain A145 restored ribose phosphate isomerase activity, prototrophy, seedling colonization, and disease suppression to levels similar to those associated with strain 501R3. Experiments reported here indicate a key role for rpiA and possibly the pentose phosphate pathway in suppression of damping-off and colonization of subterranean portions of plants by E. cloacae.     

Ecological basis for biocontrol of damping-off disease by Pseudomonas fluorescens 54/96

Pseudomonas fluorescens 54/96, originally isolated from the rhizosphere of sugar beet, has been shown to be commercially effective in field trials for the suppression of a number of fungal diseases of seedlings. In vitro and microcosm-based assays revealed that both the timing and method of application of bacteria were important for effective control of Pythium ultimum , the causative agent of damping-off disease. Following transposon mutagenesis (Tn 5lac ), mutants deficient for the suppression of Pythium ultimum infections of peas were isolated. Three major classes of insertional mutants of Ps. fluorescens 54/96 were identified which either inhibited sporulation, reduced mycelial growth or affected the regulation of bacterial metabolic activity. Evaluation of the metabolic capability of pathogen and antagonist revealed evidence for direct competition, as both the fungus and bacterium had similar sole carbon source nutrient utilization profiles. Further comparisons of the activity of the transposon mutants indicated that although the mechanisms of disease control were multifactorial, the most significant factor was the prevention of rapid spore germination in the presence of pea seeds.     

Enhanced activity of introduced biocontrol agents in solarized soils and its implications on the integrated control of tomato damping-off caused by Pythium spp

Soil solarization in combination with introduction of biocontrol agents (BCA) was evaluated as a potential disease management strategy for tomato damping-off caused by Pythium spp. A rifampicin resistant Pseudomonas fluorescens strain (PfT-8) and a carbendazim resistant Trichoderma harzianum strain (ThM-1) were introduced into soil following solarization. Tomato seeds were planted into treated field plots. The influence of soil solarization and application of biocontrol agents on damping-off incidence, plant biomass, rhizosphere population of introduced antagonists, and native Pythium spp. was assessed by two consecutive field trials. Damping-off incidence was significantly reduced in solarized plots compared to control. Soil inoculation of biocontrol agents into solarized plots resulted in the highest suppression of damping-off incidence (PfT-8 up to 92%; ThM-1 up to 83%), and increase in plant biomass (PfT-8 up to 66%; ThM-1 up to 48%) when compared to un-solarized control plots. Rhizosphere population of introduced biocontrol agents gradually increased (PfT-8 up to 102% and ThM-1 up to 84%) in solarized soils when compared to unsolarized control. The population of Pythium spp in rhizosphere soil was reduced up to 55% in solarized plots; whereas, application of BCA to solarized soils reduced the rhizosphere population of Pythium spp. by 86 and 82% in P. fluorescens and T. harzianum applied plots respectively.     

Einfluß der Behandlung von Rübensaatgut mit Rhizosphärebakterien auf den Befall durch Pilze der Gattung Pythium I. Antagonistische Wirkung verschiedener Bakterienisolate gegenüber Pythium spp.

Abstract The influence of a seed-dressing with rhizosphere bacteria on the infection of sugarbeet by fungi of the genus Pythium I. Antagonistic effect of different bacterial isolates towards Pythium spp.
Seed treatment with selectively isolated rhizosphere bacteria from the fluorescent pseudomonad group can protect sugar beet seedlings from damping-off caused by species of Pythium. The antagonistic rhizobacteria were equally effective in different soil substrates, both unsterilized and steam-sterilized. Antagonistic activity of an isolate was similar within seeds of a sugarbeet cultivar but different when different cultivars were compared. The number of bacteria adhering to the seed of eachcultivar which influenced the level of antagonism to Pythium infection, varied with seed morphology. A mixture of the three different isolates did not increase antagonistic activity when compared to the activity of the isolates individually.     

Lipopeptide Production in Pseudomonas sp. Strain DSS73 Is Regulated by Components of Sugar Beet Seed Exudate via the Gac Two-Component Regulatory System

Pseudomonas sp. strain DSS73 isolated from the sugar beet rhizosphere produces the cyclic lipopeptide amphisin, which inhibits the growth of plant-pathogenic fungi. By Tn5::luxAB mutagenesis, we obtained two nonproducing mutant strains, DSS73-15C2 and DSS73-12H8. The gene interrupted by the transposon in strain DSS73-15C2 (amsY) encoded a protein with homology to peptide synthetases that was designated amphisin synthetase. DSS73-12H8 carried the transposon in a regulatory gene encoding a protein with homology to the sensor kinase GacS. Growth of strain DSS73-15C2 (amsY) was impaired during the transition to stationary phase in a minimal medium amended with an exudate of sugar beet seeds. This growth phenotype could be complemented by purified amphisin. Seed exudate further induced expression of bioluminescence from the amsY::luxAB reporter during the transition to stationary phase. This agreed with an increase in amphisin production by the DSS73 wild-type strain during early stationary phase. Amphisin synthesis in DSS73 was strictly dependent on GacS, and even induction by seed exudate depended on a functional gacS locus. Hence, a signal triggering the GacS/GacA two-component system appeared to be present in the seed exudate.     

Timelapse scanning reveals spatial variation in tomato (Solanum lycopersicum L.) root elongation rates during partial waterlogging

Aims

Effects of soil amendments with crop residues on suppression of damping-off of sugar beet were examined by growing the seedlings in pasteurized, Rhizoctonia solani (AG2-2 IIIB)-infested soil at different temperatures. Dried residues of five dasheen or taro (Colocasia esculenta (L.) Schott) cultivars were compared with those of peanut (Arachys hypogaea L.) and Brassica rapa Olsson for their effects on disease suppression.

Methods and Results

When the seedlings were grown at 17/12 °C (day/night), all residues equally suppressed the disease when amended into the soil just before sowing. At 22/17 or 32/27 °C, damping-off developed in non-treated soil within 10 days, and differential suppressive effects by the residues became apparent by 21 days. When non-pasteurized, non-treated soil was infested with the pathogen, seedling survival was markedly better than in the same but pasteurized, infested soil. Yet, the effect was not different within the entire temperature ranges. Growth of both R. solani and the seedlings peaked near 25 °C and leveled off at higher temperatures.

Conclusions

These results suggest that damping-off was suppressed by antagonistic soil microorganisms, and individual residues elevated their effects differently. Under cool conditions, the antagonists dominated the pathogen to suppress the disease. Under warmer conditions, pathogenesis overcame antagonism depending on the residue, resulting in differential effects of disease suppression.     

Genes Involved in Cyclic Lipopeptide Production Are Important for Seed and Straw Colonization by Pseudomonas sp. Strain DSS73

Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.     

Secondary Metabolite- and Endochitinase-Dependent Antagonism toward Plant-Pathogenic Microfungi of Pseudomonas fluorescens Isolates from Sugar Beet Rhizosphere

Forty-seven isolates representing all biovars of Pseudomonas fluorescens (biovars I to VI) were collected from the rhizosphere of field-grown sugar beet plants to select candidate strains for biological control of preemergence damping-off disease. The isolates were tested for in vitro antagonism toward the plant-pathogenic microfungi Pythium ultimum and Rhizoctonia solani in three different plate test media. Mechanisms of fungal inhibition were elucidated by tracing secondary-metabolite production and cell wall-degrading enzyme activity in the same media. Most biovars expressed a specific mechanism of antagonism, as represented by a unique antibiotic or enzyme production in the media. A lipopeptide antibiotic, viscosinamide, was produced independently of medium composition by P. fluorescens bv. I, whereas the antibiotic 2,4-diacetylphloroglucinol was observed only in glucose-rich medium and only in P. fluorescens bv. II/IV. Both pathogens were inhibited by the two antibiotics. Finally, in low-glucose medium, a cell wall-degrading endochitinase activity in P. fluorescens bv. I, III, and VI was the apparent mechanism of antagonism toward R. solani. The viscosinamide-producing DR54 isolate (bv. I) was shown to be an effective candidate for biological control, as tested in a pot experiment with sugar beet seedlings infested with Pythium ultimum. The assignment of different patterns of fungal antagonism to the biovars of P. fluorescens is discussed in relation to an improved selection protocol for candidate strains to be used in biological control.