Fig 1.
Proteobacteria (A) and Gammaproteobacteria (B) community analyses of diseased and healthy salmon eggs by PhyloChip-based analyses.
Salmon eggs were sampled from a commercial hatchery [22]. Indicated is the average number of OTUs at different taxonomic levels: the phylum Proteobacteria (A) and the class Gammaproteobacteria (B). Error bars represent S.E.M. (N = 6).
Fig 2.
Phenotypic and genotypic analysis of Pseudomonas isolates.
The isolates were obtained from healthy salmon egg samples and salmon eggs infected with Saprolegnia. (A) Mean percentage of the number of bacterial isolates that were inhibitory to hyphal growth of Saprolegnia diclina VS20 and Saprolegnia parasitica CBS 223.65, tested by observation of an inhibition halo around the bacterial colony (right panel insert). (B) Mean percentage of the number of bacterial isolates that produce biosurfactants based on the drop collapse assay (right panel insert). Error bars represent the S.E.M. (N = 6). The asterisk indicates a statistically significant difference (P<0.05, Student’s t-test). (C) Genotypic BOX-PCR grouping of bacterial isolates that inhibiting Saprolegnia hyphal growth and/or producing biosurfactants. The Venn diagram (left panel insert) shows the total number of BOX groups obtained for isolates from either diseased, healthy or both samples. An example of the DNA profiles obtained by BOX-PCR is shown in agarose gel (right panel insert).
Fig 3.
Phylogenetic tree of 16S rRNA sequences of Pseudomonas strains representative of 18 BOX-PCR groups.
The 16S rRNA sequences were approximately 960 bp. The BOX-PCR groups were identified among the Pseudomonas isolates from healthy and diseased salmon eggs (S1 Table). A total of 29 reference Pseudomonas species/strains were included to delineate the 27 aquatic Pseudomonas strains obtained in this study. Bootstrap values at the nodes are based on 1000 replications. Only those branch values higher than 80% are shown. Asterisks indicate the isolates selected for salmon egg bioassays.
Fig 4.
In vivo activity of 11 Pseudomonas strains against S. diclina 1152F4.
In bioassay 2, the mean percentage of eggs to which of S. diclina 1152F4 hyphae attached was determined at 20 days post inoculation (dpi) [22]. Bacterial strains were introduced at an initial cell density of 107 CFU ml-1. The incubation temperature was 5–7°C. Error bars represent S.E.M. (N = 6). Asterisks indicate statistically significant differences with the control, S. diclina only, based on a one-way analysis of variance and post hoc LSD analysis (P<0.05).
Fig 5.
Live colony mass spectrometry and MS/MS networking.
(A) Molecular network of Pseudomonas H6, P. fluorescens SS101, P. fluorescens SBW25 and 18 other Pseudomonads and 42 Bacilli [30]. The viscosin-like cluster was boxed and enlarged. (B) Selection of nodes of Pseudomonas H6, P. fluorescens SS101 and SBW25 for MS/MS raw spectra analyses and generation of amino acid sequence tag. The sequence tag is created by analysing the mass shifts between adjacent ions in the MS/MS spectra, which are corresponding to the mass of an amino acid. The value of the parent mass ions for Pseudomonas H6 and P. fluorescens SBW25 in Fig 5B is not completely matching to the parent mass ion as indicated in Fig 5A, because during network analyses a mass tolerance setting of 0.3 Da was used. (C) Reversed-phase HPLC chromatograms of cell-free culture extracts of lipopeptide surfactants from Pseudomonas H6, P. fluorescens SS101 and SBW25.
Fig 6.
Effect of biosurfactants from Pseudomonas H6 and P. fluorescens SS101 on S. diclina 1152F4.
(A) S. diclina 1152F4 hyphal plugs were incubated for 3 days in 1/5PDB supplemented with biosurfactants from Pseudomonas H6 or P. fluorescens SS101 (massetolide A) at concentrations ranging from 15 to 200 μg ml-1. (B) The activity of the biosurfactants against S. diclina under in vivo condition was assessed by determining the mean percentage of salmon eggs to which S. diclina 1152F4 hyphae were attached at 18 dpi. Salmon eggs were treated with biosurfactants from Pseudomonas H6 and P. fluorescens SS101 at 15±3 μg ml-1 and 40±8 μg ml-1. Eggs exposed to S. diclina only were used as negative control. Eggs treated with 2.5±0.5 μg ml-1 (ppm) malachite green and S. diclina were used as the chemical reference. Error bars represent S.E.M. (N = 9). The asterisk indicates a statistically significant difference from the negative control (S. diclina only) based on one-way analysis of variance and post hoc LSD analysis (P<0.05).
Fig 7.
Effect of biosurfactants from Pseudomonas H6 and P. fluorescens SS101 on S. parasitica CBS 223.65.
The effect was according to hyphal growth (A) and hyphal morphology (B). S. parasitica hyphal plugs were grown for 48 hours in 1/5PDB supplemented with biosurfactants from Pseudomonas H6 or P. fluorescens SS101 (massetolide A) at concentrations ranging from 15 to 200 μg ml-1. The bottom row shows the enlargement of the area indicated in the top row. White arrows indicate branching hyphae. The scale bars in the pictures of panel B represent 100 μm.