Recent bacterial induced bleaching disease events of the commercially farmed brown seaweed Saccharina japonica has resulted in significant reduction in healthy sporeling supply. However, to date the host associated epimicrobial community shifts with the disease progression have not been characterized. We investigated the dynamic shifts in the composition of the epibacterial communities of S. japonica with disease progression using 16S rRNA gene amplicon sequencing. We found that the alpha diversity was significantly higher in the control group than in the infected group over disease progression (P < 0.01). There was a significant shift in the composition and predicted functions of the epibacterial communities in both control and infected groups. Indicator species, belonging to Stenotrophomonas, Pseudomonas and Sphingomona were significantly higher in the control group than infected group, suggesting that these taxa are associated with healthy S. japonica. In contrast, Vibrio, Pseudoalteromonas and Colwellia were keystone taxa in the infected group at 24 h, and thus maybe considered to be the secondary opportunistic pathogens. Our study describes the changes of epibacterial communities associated with the progression of bleaching disease in S. japonica. This new information not only extends our baseline knowledge of the S. japonica epimicrobiome, but also paves the way for developing measures to mitigate disease outbreaks for the sustainable aquaculture of S. japonica.

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Pathogenic bacteria can trigger active defense responses in higher plants, leading to hypersensitive programmed cell death (PCD) to against those bacteria. However, related research on seaweeds is very limited. Pseudoalteromonas piscicida X-8 (PpX-8) has been identified as the pathogen that causes bleaching disease in commercially farmed Saccharina japonica. In this study, using an inoculation assay and microscopic observations, we found that the proportion of bleaching tissue pieces inoculated with PpX-8 extracellular compounds was significantly higher (p < 0.05) than that inoculated with heated extracellular compounds, indicating that the virulence factors of PpX-8 exist in extracellular compounds and they are heat-sensitive. Using TEM, we observed typical morphological characteristics of PCD after inoculation with extracellular compounds, including chloroplast shrinkage, cytoplasmic vacuolation, and intact mitochondrial structures. Moreover, we detected biochemical characteristics of PCD, such as 3′-OH ends resulting from DNA cleavage and caspase-3-like enzymatic activity, using a TUNEL assay and fluorescence staining. Therefore, PpX-8 extracellular compounds can induce PCD, thus triggering active defense responses in S. japonica. These results indicate that seaweeds and higher plants are conservative in their active defense responses against pathogenic bacteria. The results of this study lay the foundation for further investigation of the virulence mechanisms of PpX-8.

Diseases of the commercially cultivated brown seaweed Saccharina japonica have been noticed since the 1960s in China. However, the usually opportunistic nature of the pathogenic bacteria of seaweeds is still a main obstacle for isolating and identifying pathogenic bacteria responsible for causing diseases of the cultivated S. japonica. In this study, a Gram-positive bacterium XP-2 was isolated from the abnormal diseased mature sporophytes of S. japonica. Pathogenicity test indicated that XP-2 could cause green-rotten disease in healthy juvenile sporophytes and significant algal ultrastructural changes were observed after S. japonica was infected by XP-2. The cells shrank, and the thylakoids of the chloroplasts became blurred after 12 h of re-infection. At 24 h slight deformation was observed in the structure of the mitochondria, chloroplasts, and nucleus. There also were many vacuoles in the infected cells. After 31 h of re-infection, mitochondrial structure and thylakoids became more indistinct. SEM and TEM observations indicated that XP-2 was rod-shaped with no flagella. Analysis of the full length 16S rRNA gene sequence and physiological and chemical characteristics indicated that XP-2 was closely related to Exiguobacterium oxidotolerans 12280 T . Based on these results, XP-2 was designated Exiguobacterium oxidotolerans XP-2. The pathogenicity of E. oxidotolerans XP-2 was identified by Koch's Postulate under laboratory conditions. These results enrich the growing list of pathogenic bacteria in commercially cultivated seaweeds and also provide a foundation for investigating the virulence mechanisms of pathogenic bacteria of commercially cultivated seaweeds in the near future.

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