Phaeobacter and Ruegeria species of the Roseobacter clade colonize separate niches in a Danish Turbot (Scophthalmus maximus)-rearing farm and antagonize Vibrio anguillarum under different growth conditions

Abstract

Members of the Roseobacter clade colonize a Spanish turbot larval unit, and one isolate (Phaeobacter strain 27-4) is capable of disease suppression in in vivo challenge trials. Here, we demonstrate that roseobacters with antagonistic activity against Vibrio anguillarum also colonize a Danish turbot larval farm that relies on a very different water source (the Danish fiord Limfjorden as opposed to the Galician Atlantic Ocean). Phylogenetic analyses based on 16S rRNA and gyrase B gene sequences revealed that different species colonized different niches in the larval unit. Phaeobacter inhibens- and Phaeobacter gallaeciensis-like strains were primarily found in the production sites, whereas strains identified as Ruegeria mobilis or Ruegeria pelagia were found only in the algal cultures. Phaeobacter spp. were more inhibitory against the general microbiota from the Danish turbot larval unit than were the Ruegeria spp. Phaeobacter spp. produced tropodithietic acid (TDA) and brown pigment and antagonized V. anguillarum when grown under shaking (200 rpm) and stagnant (0 rpm) conditions, whereas Ruegeria spp. behaved similarly to Phaeobacter strain 27-4 and expressed these three phenotypes only during stagnant growth. Both genera attached to an inert surface and grew in multicellular rosettes after stagnant growth, whereas shaking conditions led to single cells with low attachment capacity. Bacteria from the Roseobacter clade appear to be universal colonizers of marine larval rearing units, and since the Danish Phaeobacter spp. displayed antibacterial activity under a broader range of growth conditions than did Phaeobacter strain 27-4, these organisms may hold greater promise as fish probiotic organisms.

FIG. 1.

Phylogenetic trees constructed using the 16S rRNA gene (a) and gyrase B (gyrB) gene (b) sequences. Numbers at the nodes are bootstrap values from 100 replicates. Type strains Rhodobacter capsulatus ATCC 11166 and Rhodobacter sphaeroides ATCC 17023 served as outgroups in the 16S rRNA tree, and Roseobacter denitrificans Och114 and Roseobacter litoralis Och149 served as outgroups in the gyrB gene trees (not shown). and ▪, Danish and Spanish turbot-rearing farm strains, respectively. ^T, type strains.

FIG. 2.

Attachment (a), production of pigment (b), production of TDA (c), and ability to inhibit Vibrio anguillarum 90-11-287 in well diffusion assay (d) of Roseobacter clade strains and Vibrio anguillarum 90-11-287 grown under shaking (200 rpm) (black bars) or stagnant (0 rpm) (gray bars) conditions. The attachment experiment was conducted in duplicate, and error bars represent 1 standard deviation. When measuring TDA, the HPLC-tandem mass spectrometry peak area from the m/z 213 to 151 transition was used. In the well diffusion assay, the diameter of the well itself has been subtracted from the diameter of the inhibition zone.

FIG. 3.

Cell morphology of and pigment formation by Roseobacter clade strains grown in MB under static (0 rpm) (a, c, and e) or shaking (200 rpm) (b, d, and f) conditions. The Danish Phaeobacter sp. strain M23-3.1 (a and b), the Spanish Phaeobacter sp. strain 27-4 (c and d), and the Danish Ruegeria sp. strain M43-2.3 (e and f) are shown. Microscopy pictures are from phase-contrast microscopy at ×1,000 magnification. Bars, 10 μm.