Impact of siderophore production by Pseudomonas syringae pv. syringae 22d/93 on epiphytic fitness and biocontrol activity against Pseudomonas syringae pv. glycinea 1a/96

Abstract

The use of naturally occurring microbial antagonists to suppress plant diseases offers a favorable alternative to classical methods of plant protection. The soybean epiphyte Pseudomonas syringae pv. syringae strain 22d/93 shows great potential for controlling P. syringae pv. glycinea, the causal agent of bacterial blight of soybean. Its activity against P. syringae pv. glycinea is highly reproducible even in field trials, and the suppression mechanisms involved are of special interest. In this work we demonstrated that P. syringae pv. syringae 22d/93 produced a significantly larger amount of siderophores than the pathogen P. syringae pv. glycinea produced. While P. syringae pv. syringae 22d/93 and P. syringae pv. glycinea produce the same siderophores, achromobactin and pyoverdin, the regulation of siderophore biosynthesis in the former organism is very different from that in the latter organism. The epiphytic fitness of P. syringae pv. syringae 22d/93 mutants defective in siderophore biosynthesis was determined following spray inoculation of soybean leaves. The population size of the siderophore-negative mutant P. syringae pv. syringae strain 22d/93DeltaSid was 2 orders of magnitude lower than that of the wild type 10 days after inoculation. The growth deficiency was compensated for when wound inoculation was used, indicating the availability of iron in the presence of small lesions on the leaves. Our results suggest that siderophore production has an indirect effect on the biocontrol activity of P. syringae pv. syringae 22d/93. Although siderophore-defective mutants of P. syringae pv. syringae 22d/93 still suppressed development of bacterial blight caused by P. syringae pv. glycinea, siderophore production enhanced the epiphytic fitness and thus the competitiveness of the antagonist.

FIG. 1.

Influence of culture media on siderophore production. P. syringae pv. syringae 22d/93 (filled bars) and P. syringae pv. glycinea 1a/96 (open bars) were cultivated in various low-iron media at 28°C for 48 h. The siderophore activity of supernatants was determined by the CAS assay and normalized to an OD₆₀₀ of 1.0. The data are the means and standard deviations of three independent experiments.

FIG. 2.

Isoelectric focusing and CAS overlay for detection of siderophores in P. syringae. Siderophores were extracted from concentrated culture supernatants using XAD-4 resin at pH 3.0. After separation by isoelectric focusing, the polyacrylamide gel was overlaid with a thin layer of CAS agar to visualize siderophore activity. Lane 1, P. syringae pv. syringae 22d/93ΔSid; lane 2, purified pyoverdin of P. syringae; lane 3, P. syringae pv. syringae 22d/93; lane 4, P. syringae pv. glycinea 1a/96; lane 5, P. syringae pv. syringae 22d/93ΔPvd.

FIG. 3.

(A) Siderophore production by P. syringae pv. syringae 22d/93 and siderophore mutants of this strain on CAS agar. CAS agar plates were inoculated with 5-μl portions of suspensions (>10⁶ CFU/ml) of the different strains and incubated at 28°C for 48 h. Siderophore production is indicated by the formation of haloes. 1, P. syringae pv. syringae 22d/93; 2, P. syringae pv. syringae 22d/93ΔAch; 3, P. syringae pv. syringae 22d/93ΔSid; 4, P. syringae pv. syringae 22d/93ΔPvd. (B) Growth of P. syringae pv. syringae 22d/93 and siderophore mutants of this strain in PIPES medium at 28°C, as determined by measurement of the OD₆₀₀. The data are the means and standard deviations of three independent cultures. □, P. syringae pv. syringae 22d/93; ▿, P. syringae pv. syringae 22d/93ΔAch; ▵, P. syringae pv. syringae 22d/93ΔPvd; ⧫, P. syringae pv. syringae 22d/93ΔSid.

FIG. 4.

Population dynamics of P. syringae pv. glycinea 1a/96, P. syringae pv. syringae 22d/93, and P. syringae pv. syringae 22d/93 siderophore mutants after single inoculation (solid lines) and coinoculation (dashed lines) into soybean leaves using wound inoculation. Single inoculations were used to evaluate the epiphytic fitness of the strains and as controls for comparison with the coinoculation experiment. (A) Coinoculation of P. syringae pv. glycinea 1a/96 (□) with P. syringae pv. syringae 22d/93 (▪) and controls; (B) coinoculation of P. syringae pv. glycinea 1a/96 (□) with P. syringae pv. syringae 22d/93ΔPvd (▪) and controls; (C) coinoculation of P. syringae pv. glycinea 1a/96 (□) with P. syringae pv. syringae 22d/93ΔAch (▪) and controls; (D) coinoculation of P. syringae pv. glycinea 1a/96 (□) with P. syringae pv. syringae 22d/93ΔSid (▪) and controls. The data are the means and standard deviations of four independent experiments.