Alterations in Epiphytic Bacterial Communities during the Occurrence of Green Rot Disease in <i>Saccharina japonica</i> Seedlings

Bacteria are one of the causes of green rot disease (GRD) in Saccharina japonica mariculture, which may lead to complete failure of seedling production. However, the association between bacterial community and host disease severity remains largely unknown. Therefore, in this study, the bacterial communities associated with GRD-infected seedlings with naturally varying disease severity from two seedling hatcheries in Northern China were analyzed to investigate the interactions between bacterial communities and GRD. The results indicated incorrect nutrient supply in both sites. Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes were prevalent in all samples. Significant structural alterations were detected for epibacterial communities, which were further evidenced by differently abundant bacterial taxa associated with seedlings with varying disease severity. The predicted pathways of bacterial adhesion and antimicrobial compounds biosynthesis were significantly enriched in less severely diseased seedlings, whereas glutathione metabolism and lipopolysaccharide biosynthesis were significantly increased in more severely diseased seedlings. The predicted categories of a two-component system, flagellar assembly, bacterial chemotaxis, and biofilm formation were significantly enriched in the bacterioplankton in more severely infected seawater. The differential bacterial community compositions and predicted functions provide new clues to elucidate the mechanism underlying the interaction between GRD occurrence and bacterial communities

Alterations in Epiphytic Bacterial Communities during the Occurrence of Green Rot Disease in Saccharina japonica Seedlings

Bacteria are one of the causes of green rot disease (GRD) in Saccharina japonica mariculture, which may lead to complete failure of seedling production. However, the association between bacterial community and host disease severity remains largely unknown. Therefore, in this study, the bacterial communities associated with GRD-infected seedlings with naturally varying disease severity from two seedling hatcheries in Northern China were analyzed to investigate the interactions between bacterial communities and GRD. The results indicated incorrect nutrient supply in both sites. Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes were prevalent in all samples. Significant structural alterations were detected for epibacterial communities, which were further evidenced by differently abundant bacterial taxa associated with seedlings with varying disease severity. The predicted pathways of bacterial adhesion and antimicrobial compounds biosynthesis were significantly enriched in less severely diseased seedlings, whereas glutathione metabolism and lipopolysaccharide biosynthesis were significantly increased in more severely diseased seedlings. The predicted categories of a two-component system, flagellar assembly, bacterial chemotaxis, and biofilm formation were significantly enriched in the bacterioplankton in more severely infected seawater. The differential bacterial community compositions and predicted functions provide new clues to elucidate the mechanism underlying the interaction between GRD occurrence and bacterial communities

Correction: De Novo Assembly of the Japanese Flounder (Paralichthys olivaceus) Spleen Transcriptome to Identify Putative Genes Involved in Immunity.

[This corrects the article DOI: 10.1371/journal.pone.0117642.]

Dynamics of Planktonic Microbial Community Associated with <i>Saccharina japonica</i> Seedling

Macroalgae interact with planktonic microbes in seawater. It remains unclear how planktonic microbes interact with the environment and each other during the cultivation processes of commercially important algal species. Such an interaction is important for developing environment-friendly mariculture methods. In this study, the dynamics of the planktonic microbial community associated with Saccharina japonica were profiled during the seedling production stage, with its environmental correlation and co-occurrence pattern determined simultaneously. Microbial richness increased and positively correlated with light intensity and contents of NO3− and PO43−. A clear temporal succession of the community was observed, which coincided with changes in light intensity, dissolved oxygen, pH, and NO3− content. α-Proteobacteria, Bacteroidetes, γ-Proteobacteria, and the genera prevalent in these taxa dominated the planktonic microbial community, and their relative abundance temporally changed. A profile of keystone taxa that is different from prevalent genera was identified based on betweenness centrality scores. A modularized co-occurrence pattern was determined, in addition to intensified species-to-species interactions at the core of the co-occurrence network. These findings expanded our cognization of the planktonic microbial community in response to S. japonica cultivation

Screening and Identification of Antagonistic Bacteria Against Pythium Causing Red Rot Disease in Neopyropia

China is the largest producer of Neopyropia yezoensis, ranking first in the world for cultivation area and yield production. In N. yezoensis production, diseases occur frequently every year due to increased farming density, environmental deterioration, and germplasm degeneration, resulting in serious economic losses to farmers. Red rot disease is caused by Pythium sp. and is one of the most common diseases during N. yezoensis farming, leading to empty nets and harvest loss. Air-dry, cold storage, and acid wash are common methods to counteract red rot disease in N. yezoensis farming. These physical or chemical disinfection methods, however, are not completely effective, and some have serious consequences. For example, refrigeration equipment and space will greatly increase costs, and acid wash treatments can cause environmental pollution. Although research has attempted to select or cultivate disease-resistant strains of laver, there remains no laver strain completely immune to red rot disease. Biocontrol is an effective method that is widely used in disease control of land crops. Biocontrol is potentially an environment-friendly and effective control method for macroalgal diseases. However, limited information exists on biocontrol in macroalgal diseases. During the growth and development of macroalgae, a variety of metabolites are produced on their surfaces, which provide suitable substrates for microbial colonization. The microbial community attached to the surface of algae is highly diverse and can produce many kinds of biologically active compounds. These compounds not only play a major role in normal morphology, growth, and development of algae, but also have antibacterial, antiviral, antiparasitic, and other activities to protect the host from harmful organisms. Therefore, the epiphytic microorganisms of algae provide good sources of microorganisms for biological screening. This study aimed to screen and identify bacteria with antagonistic ability towards Pythium sp.. A total of 385 bacterial strains, isolated from farming algae and their culturing environments, were screened. In the first round of screening, the plate confrontation method was used and repeated twice and confirmed that nine strains had antagonistic effects on the growth of Pythium sp.. The diameter of the bacteriostatic zone was approximately 1.65–16.54 mm. In the second round of screening, three strains (assigned as P3, P6, and P19) were further investigated using the toxic medium method for inhibitory activities in their extracellular products. Repeated experiments showed that the bacteriostatic rate was approximately 20.04%–30.09%. The antibacterial spectrum was determined by the plate confrontation method. Strains P3, P6, and P19 all had antagonistic effects on the eight tested strains of Pythium preserved in our laboratory. The inhibition rates reached 52.09%–97.95% for P3, 26.81%–78.04% for P6, 10.47%–41.91% for P19, respectively. The Pythium hyphae on the confrontation edge were further investigated by lactic acid phenol cotton blue staining. When compared with Pythium hyphae in a control group, the density and color of Pythium hyphae against strains P3 and P19 became sparse and lighter. There were no significant changes in Pythium hyphae against strain P6. Strains P3 and P6 were identified as Pseudoalteromonas piscicida, and P19 as P. peptidolytica, based on 16S rRNA gene identification and multilocus sequences analysis of 16S rRNA-dnaA-dnaN-recA. The bacterial strains of P3, P6, and P19 had significant antagonistic capabilities against the pathogenic Pythium strains. This indicates they are potential biocontrol probiotics for the control of red rot disease in N. yezoensis. The present study provides the foundation for research on the evaluation and application of antagonistic bacterial strains in the biocontrol of red rot disease of N. yezoensis

Isolation and Identification of Aeromonas salmonicida from Thamnaconus septentrionalis and Sebastes schlegeli

The greenfin horse-faced filefish (Thamnaconus septentrionalis) and rockfish (Sebastes schlegeli) occupy important positions in the offshore net fishery in Shandong Province. Interest in their mariculture has been developing rapidly in recent years as candidates for submerged cage open-sea aquaculture. With the development of breeding techniques and the expansion of large- scale farming, fish disease may become a serious constraint that limits sustainable aquaculture and leads to great economic losses. Epidemiological investigation is the basis of disease control and should be carried out throughout the culture process. In this study, we describe the diseases of T. septentrionalis and S. schlegeli caused by Aeromonas salmonicida subsp. masoucida. In November 2018, an outbreak of T. septentrionalis disease was observed in a farm located in Penglai, Shandong Province, and an outbreak of S. schlegeli disease occurred in the same farm in April 2019, with daily mortalities of 0.4%~1% and about 1%, respectively. The main symptoms in the diseased fish were ulcers, redness, swelling, and bleeding in the mouth. Most diseased fish in the ponds showed "red mouth". No parasites were observed by the naked eye or light microscope. From the liver, spleen, and kidney of all the diseased fish, many homogeneous colonies were observed after three days incubation on TSA and 2216E agar plates. All strains had the same shape, color, and size, and the 16S rRNA genes of all strains were the same, with high identity with A. salmonicida. The virulence of the isolates was tested experimentally via injection with T. septentrionalis (infected by 2018TS-1) and S. schlegeli (infected by 2019SS-1) in the laboratory to calculate the median lethal dose (LD50). The results showed that the LD50 of 2018TS-1 to T. septentrionalis was 1.78×105 CFU/fish, and that of 2019SS-1 to S. schlegeli was 0.89×105 CFU/fish. The dead fish of the experimentally infected group showed ulcers and red mouth, the same symptoms as in naturally infected fish. Dominant colonies isolated from experimentally infected fish were all identified as A. salmonicida by 16S rRNA gene sequencing, which indicated that 2018TS-1 and 2019SS-1 were the pathogens of T. septentrionalis and S. schlegeli, respectively. Bacterial identification was carried out by 16S rRNA gene analysis and Biolog Gen Ⅲ characterization. The 16S rRNA gene sequences of 2018TS-1 and 2019SS-1 (Gene Bank: OK258319 and OK258320) isolated from T. septentrionalis and S. schlegeli were analyzed using MEGA5, and the phylogenetic tree derived from 16S rRNA gene sequences clustered the isolates with A. salmonicida. Among the Biolog Gen Ⅲ tests, 31 produced positive reactions or weak positive reactions for both strains (Dextrin, D-Maltose, D-Trehalose, D-Cellobiose, Sucrose, β-Methyl-D-Glucoside, D-Salicin, α-D-Glucose, D-Mannose, D-Fructose, D-Mannitol, Glycerol, Gelatin, Glycyl-L-Proline, L-Arginine, D-Gluconic Acid, Methyl Pyruvate, L-Malic Acid, Bromo-Succinic Acid, Tween 40, α-Keto-Butyric Acid, Acetoacetic Acid, Propionic Acid, Acetic Acid, pH 6, 1% NaCl, 1% Sodium Lactate, L-Aspartic Acid, L-Glutamic Acid, L-Histidine, and L-Serine), and two weak positive reactions for 2019SS-1, while the others were negative. According to the Biolog database, both strains were identified as A. salmonicida. Based on the molecular analysis of 16S rRNA genes and Biolog Gen Ⅲ phenotype results, the isolates were identified as A. salmonicida. The vapA gene, which encodes the outer membrane protein (A-layer protein) and causes the auto-aggregation of bacteria, is a conserved gene with some variation region in A. salmonicida. vapA gene typing is an effective and important method for classifying the molecular types and subspecies of this fish. vapA gene typing was also used in this study to identify subspecies of strains isolated from T. septentrionalis and S. schlegeli. The vapA gene sequences of 2018TS-1 and 2019SS-1 (Gene Bank: OK300094 and OK300095) were analyzed using MEGA5 with type strains obtained from Gene Bank. The phylogenetic tree derived from the vapA gene sequences clustered 2018TS-1 and 2019SS-1 with type strain ATCC 27013, indicating that the strains isolated from T. septentrionalis and S. schlegeli belonged to A. salmonicida subsp. masoucida, similar to the A-layer type Ⅶ strains, which are all from the northeast Asian and Canadian coasts in the Pacific Ocean. Based on the experimental infection, 16S rRNA sequence analysis, Biolog Gen Ⅲ characterization, and vapA gene typing, we confirmed that A. salmonicida subsp. masoucida is the pathogen of T. septentrionalis and S. schlegeli, and the cause of these two diseases on the farm. This is the first report of T. septentrionalis and S. schlegeli infected by A. salmonicida in industrial aquaculture, as well as the first report of a disease of T. septentrionalis in culture. It has been reported that A. salmonicida subsp. masoucida can infect Atlantic salmon (Salmo salar), turbot (Scophthalmus maximus), sablefish (Anoplopoma fimbria), and tongue sole (Cynoglossus semilaevis) cultured in Shandong Province. In this study, we expanded the host list of A. salmonicida subsp. masoucida to include two new species in aquaculture, T. septentrionalis and S. schlegeli, on the same farm, indicating that A. salmonicida subsp. masoucida may translate and adapt to a new host in a short period. Considering the increasing host and economic losses caused by A. salmonicida in fish culture, the prevention of A. salmonicida subsp. masoucida should be an important objective for mariculture in the future

Venn diagram showing the comparison among <i>P. olivaceus</i> transcriptomic sequences with the known sequences from <i>D. rerio</i> and <i>P. olivaceus</i> EST deposited in the NCBI database.

<p>Venn diagram showing the comparison among <i>P. olivaceus</i> transcriptomic sequences with the known sequences from <i>D. rerio</i> and <i>P. olivaceus</i> EST deposited in the NCBI database.</p

Cluster of Orthologous Groups (COG) annotations of annotated unigenes.

<p>19,547 unigenes were classified into 26 COG categories.</p

Gene ontology (GO) annotations of the annotated unigenes.

<p>12,503 unigenes were assigned to three GO categories containing 52 functional subcategories.</p