Fig 1.
Categorization of the algal morphology of U. mutabilis.
(A) The tripartite community of U. mutabilis with the essential interactions investigated in this study using a standardized experimental set-up. One of the strains MS1, MS2 or MS3 along with MS6 recovers completely growth, development and morphogenesis of U. mutabilis: Strains MS1 (Halomonas sp.), MS2 (originally classified as Roseobacter sp.) and MS3 (Sulfitobacter sp.) induce cell division and growth, whereas MS6 (originally classified as Cytophaga sp.) promotes rhizoid and cell wall formation [11]. (B) “Ulva bioassay array” for morphogenesis assessment. The multiwell based survey of the morphogenetic activity using axenic gametes of U. mutabilis allows the fast determination of the various morphotypes categorized by a color code. Representative morphotypes I: callus like morphology of an axenic culture (yellow); II: cell divisions and growth of blade cells with malformed cell walls (black arrow) in the presence of only MS2 (pink); III: Rhizoid formation (white arrow) and normal cell wall formation in the presence of only MS6 (red) IV: Complete morphogenesis in the presence of both strains MS2 and MS6 (green). Images (I-IV) were taken from Wichard (2015) Front. Plant Sci. 6:86 (black bar = 100 μm).
Fig 2.
(A) Scheme and purpose of sampling. (B) The morphogenetic activity of sterile-filtered water collected from several sampling sites within the lagoon [(1–3: Ramalhete Marine Station, blue dot) in 2010, (1–11, red dots) in 2011 and (1–6, green dots) in 2012] and two control samples outside the lagoon was tested on axenic Ulva mutabilis gametes sl mt(+). The numbers of the sites represent the chronological order of the sampling.
Table 1.
Sampling sites and respective metadata.
Fig 3.
Bioassay of the morphogenetic activity with doubly sterile-filtered water from tidal pools and a main channel of the lagoon.
(A): Dilution series of the seawater samples with UCM were tested to estimate the potential morphogenetic activity on axenic Ulva gametes. Controls show the morphogenetic activity on gametes without bacteria, with the bacterial strain MS2, with the bacterial strain MS6 and the completely resembled morphology with both strains (MS2 and MS6). To estimate the MS2-like activity, the cell numbers of the germlings were counted 14 days after inoculation. (B) To estimate the MS6-like activity, the percentage of algae with a normal cell wall was evaluated 7 days after the first cell wall deformation was observed in axenic control cultures. Error bars represent (A) confidential intervals (P = 0.95; n = 60 individual algae) or (B) standard deviations (n = 60 individual algae). The dotted line indicates the maximum growth and development under axenic conditions.
Fig 4.
Screening of morphogenesis inducing bacteria in 96 microarray plates isolated from the surface of U. rigida.
Various morphotypes were identified: Known morphotypes I-IV (as also found in the standard assays with strains MS2 and MS6) and the novel morphotype V with enlarged cells or vacuoles.
Fig 5.
Phylogenetic tree of the isolated bacteria.
Maximum likelihood phylogram of isolated and cultivatable bacteria inferred from the 16S rRNA gene. Bacteria were collected through swapping from the surface of U. rigida collected in tidal pools in 2011. Halomonas sp. MS1, Roseobacter sp. MS2, Sulfitobacter sp. MS3 and Cytophaga sp. MS6 were used as reference strains isolated from U. mutabilis [11]. The algal morphotypes induced by the bacteria are annotated.
Fig 6.
Bioassay of the morphogenetic activity with doubly sterile-filtered water collected from various sampling sites across the lagoon.
(A) A dilution series of the seawater collected in 2011 with UCM was tested to estimate the potential morphogenetic activity on axenic Ulva gametes. The control of axenic growth is shown for comparison. To estimate the MS2-like activity, the cell numbers of the germlings were counted 14 days after inoculation. (B) To estimate the MS6-like activity, the percentage of algae with a normal cell wall was evaluated 7 days after the first cell wall deformation was observed in axenic control cultures. Error bars represent (A) confidential intervals (P = 0.95; n = 60 individual algae) or (B) standard deviations (n = 60 individual algae). The dotted line indicates the maximum growth and development under axenic conditions.