Phenotypic and Genomic Characterization of Bacterial Strain TAM1, a Potential Biocontrol Agent Against Tetranychus urticae

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This paper presents the isolation and characterization of the bacterial strain Kosakonia sacchari TAM1 with acaricidal potential against Tetranychus urticae. The topic is timely and important in the context of the search for biological methods for mite control, but the manuscript contains a number of significant methodological and interpretative shortcomings.

The most serious problem is the lack of a clear link between the observed mite mortality and a specific mechanism of bacterial action. The authors suggest the role of enzymes (chitinases and collagenases), but their involvement in the infection process has not been directly demonstrated. Furthermore, enzymatic activity was detected primarily in cell lysate, not in the supernatant, which undermines the hypothesis of action via enzyme secretion and direct contact with the host cuticle. Functional experiments (e.g., enzyme inhibition, protein fraction assays) that would confirm the mechanism are lacking.

The bioassay methodology also raises concerns. The number of individuals (20 per replicate) is relatively low, and the lack of information on randomization and control for biological variability limits the reliability of the results. The potential influence of the culture medium (NB vs. TSB) as an independent factor was also not considered – differences in mortality may be due to medium metabolites rather than bacterial activity. Furthermore, the lack of LC₅₀/LD₅₀ analysis significantly limits the application value of the results.

The microscopic section is technically well-developed (SEM, cryo-SEM), but its interpretation is excessively speculative. Observed changes in cuticle structure are not quantitatively analyzed or correlated with biological data. Controls to exclude sample preparation artifacts are also lacking, although the authors partially acknowledge this.

In terms of strain identification, the correct approach (16S rRNA + WGS + ANI) was used, but the interpretation of the results is inconsistent. The authors themselves point out taxonomic ambiguity (close similarity to Enterobacter), yet they assign the strain to K. sacchari without sufficient justification. This requires more careful formulation of conclusions.

The genomic analysis is a strength of the work, but here too there is a significant inconsistency: enzymatic activity (chitinase) was demonstrated, but the corresponding genes in the genome were not identified. The authors suggest an imperfection in the annotation, which is possible but requires deeper analysis (e.g., manual annotation, analysis of protein domains).

 

Furthermore, the work fails to address key application aspects:

-lack of assessment of specificity against other organisms (environmental safety),

-lack of testing in semi-field or field conditions,

-lack of preparation stability and bacterial survival in environmental conditions.

In summary, the work has potential, but requires significant additions. The most important of these are:

(1) experimental confirmation of the mechanism of action,

(2) expansion of toxicity and dose-response analysis,

(3) clarification of taxonomic identification,

(4) reduction of speculation in the interpretation of microscopic and genomic data.

In their current form, the conclusions are partially overinterpreted in relation to the data presented.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript titled – Phenotypic and Genomic Characterization of Bacterial Strain TAM1, a Potential Biocontrol Agent Against Tetranychus urticae by Shu-Chen Chang et al, describes isolation, pathogenicity of bacterial strain TAM1 against T. urticae. microbiological characterization, whole genome sequencing and analysis of TAM.

Overall, the manuscript is relevant and interesting in terms of research topic and content.

Comments

Page 2                Line 61 – 62

A bacterial strain was isolated from naturally deceased T. urticae collected in Taichung, Taiwan …

Did any of the naturally deceased T. urticae mites exhibit any distinct symptoms?

Suggestions for improvement:

Scientific names must be italicized.

Strengthening the literature review/citations in the introduction (Acinetobacter sp. on T. urticae) and in the discussion (Chitinase genes among members of the Kosakonia genus and Enterobater sp.).

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents a compelling and well-structured study on the isolation and characterization of Kosakonia sacchari strain TAM1 as a novel biocontrol agent against Tetranychus urticae. The integration of phenotypic bioassays, detailed microscopy, enzymatic activity assays, and whole-genome sequencing provides a robust, multi-faceted line of evidence supporting the strain's acaricidal potential. The clear demonstration of over 90% mortality within 48 hours, coupled with the observed cuticular degradation and the identification of chitinolytic and collagenolytic activities, strongly suggests a promising multi-target mode of action.

However, several points require clarification or revision to strengthen the manuscript prior to publication. First, there are significant inconsistencies between the enzyme activity results and the genomic analysis. The authors report high chitinase and collagenase activities but state that no corresponding genes were found in the current genome annotation. While the text suggests these genes may be identified with future annotations, this is a major discrepancy that needs to be addressed now. The authors should thoroughly re-examine their genome annotation using specialized tools like dbCAN or a focused manual BLAST search against characterized chitinase families (GH18, GH19) and collagenase families (M9, etc.) before concluding the genes are absent. It is more plausible that they are present but were not identified by the initial automated pipeline. If they remain absent, this paradox must be discussed more critically as a central finding of the study.

Second, the taxonomic assignment of strain TAM1 is presented with unnecessary ambiguity. The ANI value of 98.67% to K. sacchari DSM107661 is well above the established 95-96% species boundary, firmly placing it within this species. The mention of a 98.60% similarity to an uncharacterized Enterobacter sp. R4-368 does not create genuine taxonomic uncertainty, as many strains have close relatives. The statement that the assignment is a "matter of practical convenience" undermines the validity of the authors' own robust ANI analysis. We recommend revising this to definitively assign the strain as Kosakonia sacchari and, if the authors wish to highlight the high similarity to R4-368, it can be noted as a close relative without questioning the primary classification.

Furthermore, the manuscript contains several minor errors and formatting issues. The Materials and Methods section has incorrectly duplicated numbering (two sections labeled "2.5"), which must be corrected. There are also typographical errors (e.g., "ian extension" on page 5, "antidietran" in reference 25) and inconsistent formatting of in-text citations. The legends for Figures 4 and 5 are partially placed on separate pages from the figures themselves, which should be adjusted in the final layout. Addressing these technical details will improve the overall professionalism of the manuscript. Finally, the discussion would benefit from a more balanced perspective; while the multi-target mechanism is a key strength, the authors should acknowledge the limitations, such as the lack of field data, the uncertain role of the identified toxin-antitoxin systems, and the unresolved discrepancy between the enzymatic and genomic data.

Comments on the Quality of English Language

good

Author Response

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Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant topic and presents a bacterial strain with potential as a biocontrol agent against Tetranychus urticae. However, the manuscript still requires significant revisions. Specific comments are noted directly in the document.

Comments for author File: Comments.pdf

Author Response

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Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Taxonomy of strain TAM1 requires resolution (Section 3.9)
The ANI analysis shows TAM1 shares 98.67% identity with K. sacchari DSM107661 and 98.60% with Enterobacter sp. R4-368. Both values fall within the accepted species boundary (>96%), yet the authors assign TAM1 to K. sacchari without explaining why the Enterobacter alternative is rejected. Given that R4-368 may represent a misclassified Kosakonia (a known issue in Enterobacteriaceae), the authors should:

• Perform ANI between DSM107661 and R4-368 to clarify whether R4-368 is itself a K. sacchari strain.

• Provide a core-genome or concatenated marker gene phylogeny to resolve the taxonomic position.

• Discuss the boundary issue explicitly rather than making a tentative assignment without justification.

The enzyme localization data contradict the proposed infection mechanism (Sections 3.6 and 4)
Chitinase and gelatinase activities were detected predominantly in the cell lysate, not in the culture supernatant. Despite this, the Discussion repeatedly attributes cuticular damage to extracellular enzymatic degradation. The authors must:

• Explicitly acknowledge that the current data do not demonstrate extracellular secretion under the tested conditions.

• Discuss why intracellular enzyme localization is inconsistent with a model of external cuticle digestion.

• Consider alternative mechanisms (contact-dependent delivery, cell lysis on the cuticle surface, intracellular pathogenesis, or host-induced secretion) and present external enzymatic degradation as a hypothesis requiring experimental validation.

• Validate secretome predictions experimentally (e.g., proteomics of supernatant under inducing conditions) or clearly label all secretion-related claims as predicted, not demonstrated.

Microscopy interpretation should be qualified (Section 3.2)
SEM images show crest-line disruption in TAM1-treated mites. However, the temporal data show TAM1 cells present on the cuticle at 4 h but "remarkably scarce" by 24 h, while damage is observed at 24 h. This temporal disconnect (bacteria disappear, damage appears later) is not adequately explained. Alternative explanations—host autolytic processes triggered by infection, dehydration artifacts accentuated by pre-existing cuticular compromise—should be discussed. The interpretation of "bacterial-mediated structural damage" should be appropriately tempered.

Probit analysis shows poor model fit (Section 3.1.2)
The chi-square test was significant (P=0.01475), necessitating a heterogeneity factor. The non-monotonic deviation at 10⁴ CFU mL⁻¹ should be discussed as possibly reflecting biological complexity (e.g., quorum-sensing effects, bacterial aggregation) rather than simply noise.

Paragraph Formation and Textual Issues

Merged draft versions causing incoherent text
Several locations contain residual text from unmerged drafts, making sentences unintelligible:

• Abstract (Page 1): "The strong- Refined functional profiling..." contains the remnant "strong-".

• Introduction (Page 2): "Managing In Taiwan's subtropical climate, consistently high temperatures facilitate the year- round, rapid proliferation of T. urticae often requires, necessitating frequent applications of chemical acaricides due to the mite's short life cyclenterventions." This sentence is incomprehensible and appears to be two alternative phrasings merged together.

• Introduction (Page 2): "Biological control, specifically the use of microbial Microbial pesticides..." contains duplicated and inconsistently capitalized words.

• Section 3.6 (Page 13): "From this culture, the chitinase . . . and collagenasegelatinase activities..." contains a stray period and merged terminology.

The entire manuscript requires careful proofreading to remove all editing artifacts and produce a single clean draft.

Overly dense paragraphs lacking thematic unity
The second paragraph of the Introduction attempts to cover fungal biocontrol, emerging bacterial acaricides, specific strains, commercial products, the Taiwan context, and the genus Kosakonia in a single block. Similarly, the Discussion contains paragraphs that mix enzymatic results, genomic data, microscopy, and mechanistic speculation without logical breaks.

Recommendation: Restructure long paragraphs so each addresses one main idea with a clear topic sentence. For the Introduction second paragraph, suggested breaks are: (a) advantages of microbial pesticides and fungal biocontrol status, (b) emerging bacterial acaricides with specific examples, (c) the Taiwan context, limited options, and the Kosakonia knowledge gap.

Fragmented and duplicate headings in Methods
Section 2.3 contains both "2.3.1. Comparative Efficacy..." and an unnumbered "2.3. Evaluation of the Acaricidal Activity..." with overlapping content. These should be consolidated into a single coherent subsection hierarchy.

Missing introductory sentences before data
Some paragraphs jump directly into statistics or table references without orienting the reader. For example, Section 3.1.2 immediately discusses Abbott's correction without first summarizing the experimental design. Tables and figures should be preceded by a sentence alerting the reader to the key finding.

 Inconsistent terminology
The manuscript alternates between "gelatinase" and "collagenase" activity. One consistent term should be adopted and justified, or the distinction clearly explained.

Minor Comments

• Page 1, Keywords: "ecoli- genus- gelatinase" appears to be a placeholder error.

• Page 3 (Section 2.2): The sentence "Although 20 bacterial isolates were obtained, they exhibited nearly identical colony morphotypes" conflicts with the subsequent statement that the five most virulent were selected. The text should clarify whether all 20 were conspecific and why selection was necessary.

• Page 12 (Figure 4 legend): The doubling time is stated as "1.53±0.2 min" in the legend but estimated at ~70.2 min in the text. Correct this discrepancy. A doubling time of 1.53 minutes is biologically implausible.

• Data Availability: The plasmid accession is given as both CP102955 and CP120955. Verify which is correct.

Comments on the Quality of English Language

good

Author Response

Response to Reviewer III Comments
We would like to express our sincere gratitude to the reviewers for their meticulous evaluation and constructive feedback on our manuscript. We have carefully addressed all concerns, with particular emphasis on enhancing the logical rigor of our mechanistic discussions and improving the overall clarity of the text. The key revisions are summarized as follows:
1.    Taxonomic Clarification: We have expanded our discussion on the taxonomic status of strain TAM1. Based on comprehensive ANI analysis, we clarified the differentiation between Kosakonia sacchari groups and justified our tentative assignment of TAM1 to this species while explicitly acknowledging the current complexities within the Enterobacteriaceae phylogeny.
2.    Refinement of the Infection Mechanism: Addressing the concerns regarding enzyme localization, we have explicitly stated that chitinase and gelatinase activities were detected in the cell lysate rather than the supernatant under tested conditions. We have tempered our conclusions by relabeling all secretion-related claims as "predicted" or "hypothesized" and added a nuanced discussion on alternative delivery mechanisms, such as contact-dependent systems and host-induced secretion.
3.    Qualifying Microscopy Interpretations: To address the "temporal disconnect" observed in SEM analysis, we have revised the Discussion to present the 24-h cuticular damage as a cumulative pathological outcome. We now account for potential secondary processes, including host autolytic responses and dehydration artifacts, thereby avoiding over-inference of direct bacterial action.
4.    Statistical Rigor and Dose-Response Dynamics: To enhance the robustness of the Probit analysis, we redid the bioassays and increased the biological replicates from four to five (n = 100 per concentration). We have strictly applied a heterogeneity factor ( h= 9.55) to adjust the 95% confidence intervals in response to the significant chi-square result. Furthermore, we have added a conservative discussion regarding the biological complexity of the dose-response relationship, interpreting the mortality plateau at sub-lethal concentrations as a concentration-dependent establishment phase while avoiding over-inference of unverified mechanisms.
5.    Linguistic and Structural Optimization: The entire manuscript has undergone thorough professional proofreading. We have eliminated all editing artifacts, standardized inconsistent terminology (e.g., universal adoption of "gelatinase activity"), and restructured dense paragraphs to improve thematic unity and readability.
6.    Data Accuracy: All identified discrepancies in data—including genomic accession numbers (CP120955), bacterial doubling time, and keyword placeholders—have been corrected to ensure the highest standards of technical accuracy.
We believe these comprehensive revisions have significantly strengthened the scientific integrity and clarity of our manuscript.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

I appreciate the effort you put into reviewing the manuscript. I have carefully reviewed the new version and confirm that all of my comments and suggestions have been taken into account and addressed appropriately. I have no further comments.

Author Response

We would like to sincerely thank the reviewer for the final assessment and for the positive feedback. We are pleased to hear that the revisions have addressed all concerns to your satisfaction. We truly appreciate the time and effort you dedicated to reviewing our manuscript; your insightful suggestions have significantly enhanced the clarity and quality of this work.