Community Structure of Bacteria Associated With Drifting Sargassum horneri, the Causative Species of Golden Tide in the Yellow Sea

Abstract

Golden tides dominated by Sargassum spp. are occurring at an accelerated rate worldwide. In China, Sargassum has started to bloom in the Yellow Sea and led to tremendous economic losses, but the underlying biological causes and mechanisms are still unclear. Although algae-associated bacteria were suggested to play crucial roles in algal blooms, the profiles of bacteria associated with drifting Sargassum remain unexplored. In this study, the community structures and functions of Sargassum-associated bacteria were analyzed using the high-throughput sequencing data of the V5-V7 hypervariable region of the 16S rRNA gene. Molecular identification revealed that the golden tide analyzed in the Yellow Sea was dominated by a single species, Sargassum horneri. They were a healthy brown color nearshore but were yellow offshore with significantly decreased chlorophyll contents (P < 0.01), which indicates that yellow S. horneri was under physiological stress. The structural and functional analyses of bacterial communities indicated that the drifting S. horneri had an obvious selectivity on their associated bacteria against surrounding seawater. Although the bacterial communities phylogenetically differed between brown and yellow S. horneri (P < 0.01), their dominant functions were all nitrogen and iron transporters, which strongly indicates microbial contribution to blooming of the algal host. For the first time, potential epiphytic and endophytic bacteria associated with Sargassum were independently analyzed by a modified co-vortex method with silica sand. We showed that the composition of dominant endophytes, mainly Bacillus and Propionibacterium, was relatively consistent regardless of host status, whereas the epiphytic operational taxonomic units (OTUs) greatly varied in response to weakness of host status; however, dominant functions were consistent at elevated intensities, which might protect the host from stress related to nitrogen or iron deficiency. Thus, we propose that host physiological status at different intensities of functional demands, which were related to variable environmental conditions, may be a critical factor that influences the assembly of epiphytic bacterial communities. This study provided new insight into the structure and potential functions of associated bacteria with golden tide blooms.

Keywords: 16S rRNA gene; Sargassum horneri; bacterial community; golden tide; high-throughput sequencing; the Yellow Sea.

FIGURE 1

Sites for sampling in this study during Ke Xue III cruise. A and C represented nearshore sites with lower transparency where floating seaweeds were in brown color; B and D represented offshore sites with higher transparency where floating seaweeds were in yellow color.

FIGURE 2

The status in field, morphology and tissues of drifting Sargassum. (A) In light yellow from offshore site. (B) In dark brown from nearshore site. (C) Thallus. (D) Blades. (E) Stalks. (F) Vesicles. Scale bars = 1 cm.

FIGURE 3

Phylogenetic NJ tree based on ITS2 alignment. Numbers at internal nodes indicate support values obtained using the bootstrap method with NJ. Scale indicates substitutions per site. G1 and G2 represented two genotypes of ITS2 sequences in S. horneri, representing intra-individual polymorphism in both yellow and brown samples.

FIGURE 4

Comparison of chlorophyll contents between brown and yellow S. horneri thalli. The error bar represented the standard deviation, the asterisk above represented the significant differences.

FIGURE 5

Culture method detection for removal effects of epiphytic bacteria from three kinds of S. horneri tissues.

FIGURE 6

SEM detection for removal effects of epiphytic bacteria from three kinds of S. horneri tissues. (A) Blade blank control. (B) Blade vortexed for 5 times. (C) Blade vortexed for 10 times. (D) Stalk blank control. (E) Stalk vortexed for 5 times. (F) Stalk vortexed for 10 times. (G) Vesicle blank control. (H) Vesicle vortexed for 5 times. (I) Vesicle vortexed for 10 times. Scale bars = 5 μm.

FIGURE 7

Dendogram of similarity of all samples clustered by the UPGMA method. (A) Unweighted. (B) Weighted. The scale bar shows the distance between clusters in UniFrac units.

FIGURE 8

Bar-plot of bacterial composition of S. horneri and surrounding seawater at genus levels. (A) Nearshore. (B) Offshore. 1A: total bacteria isolated from brown S. horneri, 1W: total bacteria isolated from nearshore seawater, 2A: total bacteria isolated from yellow S. horneri, 2W: total bacteria isolated from offshore seawater.

FIGURE 9

Principal Component Analysis (PCA) of the bacterial communities from all samples based on Euclidean distance at genus level.

FIGURE 10

Principal Component Analysis (PCA) of the bacterial communities from blade, stalk, and vesicle of S. horneri based on Euclidean distance at genus level. (A) Brown S. horneri. (B) Yellow S. horneri.

FIGURE 11

Potential dominant epiphytic and endophytic bacterial genera in brown and yellow S. horneri. (A) Epiphytic bacterial genera. (B) Endophytic bacterial genera. The number above column indicated the rank in abundance. 1ep: all epiphytic bacteria associated with brown thallus, 1en: all endophytic bacteria associated with brown thallus, 2ep: all epiphytic bacteria associated with yellow thallus, 2en: all endophytic bacteria associated with yellow thallus.

FIGURE 12

Heat-map of top 50 abundant functional proteins combined with the phylogenetic distribution of samples clustered by Hierarchical cluster. The color key scale represented the abundance of corresponding proteins. Each row represented the abundance distribution of right KEGG orthologous category (KO) among samples, and each column represented the abundance distribution of various KO in each sample whose name was shown at bottom. Lateral topology was contributed by the similarity of KO abundance distribution among different samples, and longitudinal topology was contributed by similarity of different KO species distribution in each sample.