Isolation and Identification of Pseudoalteromonas agarivorans LJ53, a Pathogenic Bacterium Causing Bleaching Disease in Saccharina japonica

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is well written and provides a thorough description of all aspects of the study. It is ready for publication. The study offers clear evidence linking Pseudoalteromonas agarivorans LJ53 to disease symptoms in S. japonica, strengthening its relevance for mariculture health management.The integration of metagenomic and ultrastructural analyses adds depth to the findings and enhances the manuscript’s scientific rigor.I do not have any further suggestions.

Comments on the Quality of English Language

should be improved

Author Response

We thank the reviewer for the valuable comments and considering our manuscript for revision. We provide a line-by-line response to the comments below (line numbers refer to the final manuscript file without tracked changes (filename: Manusript_1216_without tracked changes)).

• Comments 1: The manuscript is well written and provides a thorough description of all aspects of the study. It is ready for publication. The study offers clear evidence linking Pseudoalteromonas agarivorans LJ53 to disease symptoms in japonica, strengthening its relevance for mariculture health management. The integration of metagenomic and ultrastructural analyses adds depth to the findings and enhances the manuscript’s scientific rigor.I do not have any further suggestions.

Reply: We sincerely appreciate the reviewer’s comments and encouragements, we will strive to improve the quality of our manuscript in detail.

Reviewer 2 Report

Comments and Suggestions for Authors

This study isolated a dominant bacterial strain, Pseudoalteromonas agarivorans LJ53, from diseased farmed S. japonica. Based on microscopic observation and 16S rRNA gene amplicon analysis, the authors investigated potential disease mechanisms. The experimental work is generally sound; however, I have several concerns regarding the presentation and interpretation of the results, as well as the discussion.

 

First, genomic sequencing of this bacterium is recommended. Such data would refine the phylogenetic analysis and provide a basis for metabolic reconstruction. Furthermore, genomic analysis could help elucidate infection mechanisms by identifying potential virulence factors, such as type VI secretion system (T6SS) effectors.

 

Second, while the authors mention disease control in the discussion, the potential role of novel antimicrobial agents, such as antimicrobial peptides from marine microorganisms (e.g., those discussed in “Bioprospecting of culturable marine biofilm bacteria for novel antimicrobial peptides” and “Embracing the era of antimicrobial peptides with marine organisms”), could be addressed to strengthen this section.

 

Third, since Vibrio species were also identified through network analysis, their potential role in the disease should be discussed alongside Pseudoalteromonas. Do these genera share similar pathogenic mechanisms?

 

Minor comments:

 

Line 144: “Operational Taxonomic Unit” does not need to be capitalized.

 

Line 230: “Bacillus halodurans” should be italicized.

 

Line 232: The title of Fig. 5, “Ecological role of P. agarivorans in the S. japonica microbiome during disease,” appears overstated given that the figure mainly shows co-occurrence and relative abundance.

 

Line 273: “ROS” is used only once; therefore, the abbreviation is unnecessary.

 

Line 274: A reference citation is missing in the brackets.

 

Line 357: “Scientific Reports” should be italicized.

Author Response

We thank the reviewer for the valuable comments and considering our manuscript for revision. We provide a line-by-line response to the comments below (line numbers refer to the final manuscript file without tracked changes (filename: Manusript_1216_without tracked changes)).

• Comments 1: Genomic sequencing of this bacterium is recommended. Such data would refine the phylogenetic analysis and provide a basis for metabolic reconstruction. Furthermore, genomic analysis could help elucidate infection mechanisms by identifying potential virulence factors, such as type VI secretion system (T6SS) effectors.

Reply: Thank you for the reviewer’s valuable suggestions. We fully agree that the genomic sequencing of strain LJ53 would provide not only more accurate phylogenetic profile, but also further information about its potential virulence mechanisms. However, the genomic information in company with other experiments’ data was designed as one part of another manuscript, which focused on the study of pathogenic mechanism. Thank you again for your kindly reminding, and we will dig the genomic information for our further study.

• Comments 2: While the authors mention disease control in the discussion, the potential role of novel antimicrobial agents, such as antimicrobial peptides from marine microorganisms (e.g., those discussed in “Bioprospecting of culturable marine biofilm bacteria for novel antimicrobial peptides” and “Embracing the era of antimicrobial peptides with marine organisms”), could be addressed to strengthen this section.

Reply: Thanks for the reviewer’s suggestions. We have thoroughly reviewed the relevant literatures (Fan et al., 2024 and Chen et al., 2024). Accordingly, we have revised and expanded the discussions about the potential application of marine-derived AMPs as a sustainable and targeted biocontrol strategy against Pseudoalteromonas agarivorans LJ53 and similar pathogens in mariculture. And also cited these two recommended references to support our discussion.” (Line 443-446).

The following are the revisions: “Furthermore, although probiotic-based biocontrol strategy is mainly applicable to controllable stages such as seedling cultivation, its utility is significantly limited for mature S. japonica in open-sea farming systems that are subject to stochastic pathogen invasion. Thus, a highly promising alternative strategy involves the identification and characterization of novel antimicrobial agents, e.g., antimicrobial peptides (AMPs) [38, 39], secreted by these beneficial bacterial strains followed by introduction of the complete biosynthetic gene cluster encoding such AMPs into the host S. japonica to enable endogenous and continuous disease resistance. To date, AMP biosynthetic gene clusters derived from marine actinomycetes (e.g., the gene cluster responsible for mathermycin synthesis) have been successfully identified [40]. These gene clusters can be designed and synthesized before being transferred into the host to direct post-translational modifications (e.g., hydroxylation and cyclization) of precursor peptides within the plants to achieve in situ production of mature AMPs with high efficacy. This genetic engineering-based strategy for endogenous AMP production may represent a highly specific, environmentally friendly, and application-independent biocontrol approach that not only directly neutralizing pathogens but also durably enhances microbial ecological resilience of the host S. japonica.” (Line 314-330).

Fan, S.; Qin, P.; Lu, J.; Wang, S.; Zhang, J.; Wang, Y.; Cheng, A.; Cao, Y.; Ding, W.; Zhang, W. Bioprospecting of culturable marine biofilm bacteria for novel antimicrobial peptides. Imeta 2024, 3, e244.

Chen, P.; Ye, T.; Li, C.; Praveen, P.; Hu, Z.; Li, W.; Shang, C. Embracing the era of antimicrobial peptides with marine organisms. Nat. Prod. Rep. 2024, 41, 331-346.

• Comments 3: Since Vibrio species were also identified through network analysis, their potential role in the disease should be discussed alongside Pseudoalteromonas. Do these genera share similar pathogenic mechanisms.

Reply: We thank the reviewer for pointing this issue out. As known to us, Vibrio spp. are recognized as established pathogens in aquaculture, however, their ecological functions are highly environment-dependent. For instance, certain strains, such as Vibrio alginolyticus X-2, have been reported to act as beneficial bacteria that protect seaweed from bleaching disease (Ma et al.,2023). This illustrates the complex and host-specific nature of Vibrio-host interactions. Importantly, our microbial co-occurrence network analysis (Fig. 5) did not reveale a direct ecological linkage between the genus Vibrio and our focal pathogen Pseudoalteromonas agarivorans LJ53. This structural separation suggests that in the diseased microbiome investigated here, Vibrio populations functioned within an ecological niche distinct from that of LJ53. Therefore, given the current topology of the network with no direct connection, we focused our discussion the genus Pseudoalteromonas with which there is a direct experimentally validated link to the disease.

Ma, M.Y.; Zhuang, Y.R.; Chang, L.R.; Xiao, L.Y.; Lin, Q.; Qiu, Q.Y.; Chen, D.F.; Egan, S.; Wang, G.G. Naturally occurring beneficial bacteria Vibrio alginolyticus X-2 protects seaweed from bleaching disease. mBio 2023, 14, e00065–23.

• Comments 4: Line 144: “Operational Taxonomic Unit” does not need to be capitalized.

Reply: Thank you for your kindly reminding. “Operational Taxonomic Unit” has been corrected as “Operational taxonomic unit” (Line 157).

• Comments 5: Line 230: “Bacillus halodurans” should be italicized.

Reply: Thank you for your reminding. The species name “Bacillus halodurans” has been italicized (Line 227).

• Comments 6: Line 232: The title of Fig. 5, “Ecological role of agarivorans in the S. japonica microbiome during disease,” appears overstated given that the figure mainly shows co-occurrence and relative abundance.

Reply: Thanks for the reviewer’s kindly reminding. The title of Figure 5 has been revised as “Figure 5. Comparative microbial relative abundance and co-occurrence networks of Pseudoalteromonas in healthy and diseased S. japonica (Line 244-245).

• Comments 7: Line 273: “ROS” is used only once; therefore, the abbreviation is unnecessary.

Reply: Thanks to the reviewer’s reminding. The abbreviation “ROS” has been removed (Line 285).

• Comments 8: Line 274: A reference citation is missing in the brackets.

Reply: Sorry for our negligence. The appropriate reference citation ([34]) has now been added into the manuscript (Line 286).

Roussin-Léveillée, C.; St-Amand, M.; Desbiens-Fortin, P.; Perreault, R.; Pelletier, A.; Gauthier, S.; Gaudreault-Lafleur, F.; Laforest-Lapointe, I.; Moffett, P. Co-occurrence of chloroplastic ROS production and salicylic acid induction in plant immunity. New Phytol. 2025, 248, 1989–2004.

• Comments 9: Line 357: “Scientific Reports” should be italicized.

Reply: We thank the reviewer for the correction. The journal name “Scientific Reports” has been italicized as required (Line 390).

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Ouyang et al. reports the identification of a novel strain, Pseudoalteromonas agarivorans LJ53, causes the bleaching disease of Saccharina japonica. While the topic is relevant, the manuscript requires substantial revision.

Major comments:

1. Figures should be distributed throughout the text and placed close to their first citation, rather than all being grouped at the end of the Results section.
2. The authors did not perform a metagenomic analysis. The study is based on 16S rRNA gene amplicon sequencing or microbial community profiling. Metagenomic analysis implies random shotgun sequencing of genomic DNA without targeted primers, which is not the case here.
3. Line 82: in triplicate
4. Section 2.5: Please specify the similarity threshold used for OTU clustering, the similarity threshold applied for taxonomic assignment against the SILVA database, and the number of biological and technical replicates included in the analysis.
5. Section 3.4: This section should start with general sequencing statistics, including the total number of reads obtained, the number of reads retained after quality filtering, and the major bacterial phyla detected and their relative abundances.

Only after this overview should the authors focus on Pseudoalteromonas and P. agarivorans, indicating their relative contribution to the total microbial community and the number of OTUs affiliated with this genus/species. In addition, the results of the network analysis must be described explicitly in the text rather than only referenced via the figure.

1. In Figure 5d, the panel on the right is partially cut off and not fully visible.
2. In its current form, the manuscript does not provide sufficient data for publication in Water and should be strengthened by an additional experimental block. Possible options include whole-genome sequencing of strain LJ53, genome annotation, and calculation of ANI and GGDC values in comparison with P. agarivorans DSM 14585. This would also allow testing the hypothesis proposed by the authors that “LJ53 might possess conserved effector molecules—analogous to those in Pseudomonas syringae—that target the photosynthetic apparatus in chloroplasts, triggering a burst of reactive oxygen species”.

Or phenotypic characterization of the new strain using the Biolog Gen III MicroPlate, as performed by Zhang et al. (2022), which is already cited in the manuscript, or a comparable standardized test system for strain differentiation and characterization.

1. The next-generation sequencing data have not been deposited in any publicly available database, and no accession numbers are provided.

Author Response

We thank the reviewer for the valuable comments and considering our manuscript for revision. We provide a line-by-line response to the comments below (line numbers refer to the final manuscript file without tracked changes (filename: Manusript_1216_without tracked changes)).

• Comments 1: Figures should be distributed throughout the text and placed close to their first citation, rather than all being grouped at the end of the Results section.

Reply: Thank you for the reviewer’s suggestion. We have redistributed all the figures in the manuscript (Figures 1–5) according to the reviewer’s recommendation.

• Comments 2: The authors did not perform a metagenomic analysis. The study is based on 16S rRNA gene amplicon sequencing or microbial community profiling. Metagenomic analysis implies random shotgun sequencing of genomic DNA without targeted primers, which is not the case here.

Reply: We apologize for the inaccuracies in our initial description of the methodology. The title of Section 2.7 has been revised to “Microbial community profiling analysis,” and the structure of the microbial community was analyzed using metagenomic data. A detailed description of the method has also been added to this section in the revised manuscript (Line 139-160).

• Comments 3: Line 82: in triplicate.

Reply: Thank you for pointing this mistake out. We have corrected the word “treplicate” as “triplicate”(Line 82 in the revised manuscript).

• Comments 4: Section 2.5: Please specify the similarity threshold used for OTU clustering, the similarity threshold applied for taxonomic assignment against the SILVA database, and the number of biological and technical replicates included in the analysis.

Reply: We thank the reviewer for raising these essential methodological details. We acknowledge that the initial description in Section 2.5 was incorrect due to the conflation of 16S amplicon and metagenomic sequencing approaches, as addressed in our response to Comment 2. Regarding the method for taxonomic annotation, please see section 2.7. Furthermore, we have added the number of biological and technical replicates in the revised section 2.7.

• Comments 5: Section 3.4: This section should start with general sequencing statistics, including the total number of reads obtained, the number of reads retained after quality filtering, and the major bacterial phyla detected and their relative abundances. Only after this overview should the authors focus on Pseudoalteromonas and agarivorans, indicating their relative contribution to the total microbial community and the number of OTUs affiliated with this genus/species. In addition, the results of the network analysis must be described explicitly in the text rather than only referenced via the figure.

Reply: Thank you for the reviewer’s suggestions. We have added the related information of the sequencing statistics, including the total number of reads obtained per sample and the number of reads retained after quality filtering (Table S1 and Line 150-151), as well as the general community composition: “Analysis of microbial community composition revealed that the phyla Pseudomonadota and Bacteroidota constituted the most abundant taxonomic groups in both diseased and healthy S. japonica samples” (Figure S1 and Line 230-232).

• Comments 6: In Figure 5d, the panel on the right is partially cut off and not fully visible.

Reply: Thanks for the reviewer’s reminding. We have revised Figure 5 to ensure that all the panels are fully visible (Figure 5).

• Comments 7: In its current form, the manuscript does not provide sufficient data for publication in Water and should be strengthened by an additional experimental block. Possible options include whole-genome sequencing of strain LJ53, genome annotation, and calculation of ANI and GGDC values in comparison with agarivorans DSM 14585. This would also allow testing the hypothesis proposed by the authors that “LJ53 might possess conserved effector molecules—analogous to those in Pseudomonas syringae—that target the photosynthetic apparatus in chloroplasts, triggering a burst of reactive oxygen species”. Or phenotypic characterization of the new strain using the Biolog Gen III MicroPlate, as performed by Zhang et al. (2022), which is already cited in the manuscript, or a comparable standardized test system for strain differentiation and characterization.

Reply: We sincerely appreciate the reviewer’s constructive and insightful suggestions, and also fully recognize the importance of these proposed experiments for identifing a novel species. However, Our strain LJ53 showed the highest similarity (99.86%) to Pseudoalteromonas agarivorans DSM 14585^T, meaning that this isolate was not belonging to a novel species (far beyond the threshold of 97% that would correspond to the novel species borderline), but a new strain or subspecies. The main object of our study here was to illuminate its pathogenic characteristic, so we didn’t perform so much work on the identification of the strain LJ65.Thank you for your understanding.

• Comments 8: The next-generation sequencing data have not been deposited in any publicly available database, and no accession numbers are provided.

Reply: Thank you for the reviewer’s reminding. All raw sequence data generated in this study have been deposited in the NCBI’s Sequence Read Archive (SRA) database. The raw reads of kelp metagenomes and seawater metagenomes can be accessed under the BioProject PRJNA1390388.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed my concerns. 

Author Response

Comments:The authors have addressed my concerns.

Reply: We appreciate the reviewer's acknowledgment that our concerns have been addressed. Thank you for your valuable feedback.

Reviewer 3 Report

Comments and Suggestions for Authors

The comments have been taken into account. However, a few points remain:

1. If the data are metagenomic, the use of the term OTU is incorrect. Please use taxonomic features, taxonomic profiles or taxa instead.
2. Under BioProject accession PRJNA139038, there is currently no information available in the NCBI database, not even the basic project description that normally appears on the website prior to the release of the raw sequencing data.
3. Regarding the description of the taxonomic status and genome sequencing: many bacterial species show >97% similarity based on 16S rRNA gene sequences, yet comparative phenotypic and genomic analyses provide sufficient metrics to justify assignment to a new taxon. My suggestion was simply to provide more comprehensive data on this strain for the benefit of the scientific community. If this is difficult for you to implement at present, that is fine I do not insist.

Author Response

We thank the reviewer for the valuable comments and considering our manuscript for revision. We provide a line-by-line response to the comments below (line numbers refer to the final manuscript file without tracked changes (filename: Manusript_1222_without tracked changes)).

 

• Comments 1: If the data are metagenomic, the use of the term OTU is incorrect. Please use taxonomic features, taxonomic profiles or taxa instead.

Reply: We thank the reviewer for this reminder. As suggested, we have thoroughly checked our manuscript and replaced all incorrect descriptions related to “OTU” (Operational Taxonomic Unit) with “taxon” or “taxa” (Line 155-156, 236, 246).

 

• Comments 2: Under BioProject accession PRJNA139038, there is currently no information available in the NCBI database, not even the basic project description that normally appears on the website prior to the release of the raw sequencing data.

Reply: We thank the reviewer for pointing this out. The temporary inaccessibility of BioProject PRJNA139038 was likely due to a system delay during the NCBI processing and synchronization phase. All sequencing data associated with this project have now been fully released and are publicly accessible (Fig. 1).

Fig. 1 The public NCBI BioProject record (PRJNA139038)

 

• Comments 3: Regarding the description of the taxonomic status and genome sequencing: many bacterial species show >97% similarity based on 16S rRNA gene sequences, yet comparative phenotypic and genomic analyses provide sufficient metrics to justify assignment to a new taxon. My suggestion was simply to provide more comprehensive data on this strain for the benefit of the scientific community. If this is difficult for you to implement at present, that is fine I do not insist.

Reply: We sincerely appreciate the reviewer’s valuable suggestion, and fully agree that comprehensive genomic information and comparative analyses would provide more accurate evidence for a new taxon. As you rightly note, due to time constraints, we have had to leave this whole-genome sequencing work for future studies. We thank you again for your understanding.