Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The presented manuscript concerns the characterization of a novel endophytic bacterial species, Rhizobium moroccans sp. nov., isolated from the roots of Peganum harmala. The topic fits well within environmental and agricultural microbiology and is relevant to studies on plant–microorganism interactions. The manuscript has strong publication potential; however, in its current form it requires several important revisions. Below I provide my main substantive comments along with a few minor editorial remarks:

1. The authors demonstrated plant growth–promoting traits solely on the basis of qualitative assays, such as halo formation or changes in medium color. While such observations are useful for preliminary screening, they do not allow for a reliable assessment of the intensity or efficiency of the studied processes. Quantitative data are lacking, which would enable comparison of the strain’s activity with that of other microorganisms, for example measurements of solubilized phosphorus expressed in mass units or concentration in solution.
2. The authors infer the ability of the strain to fix nitrogen based on the presence of nifHDK genes in the genome. This approach is insufficient, as the mere presence of genes does not imply their expression or functional enzymatic activity. The study does not provide any direct evidence confirming nitrogenase activity, such as an acetylene reduction assay or measurements of atmospheric nitrogen assimilation. The absence of such data represents a significant methodological gap, particularly since nitrogen fixation is one of the key traits attributed to bacteria of the genus Rhizobium. In its current form, statements regarding nitrogen fixation remain hypothetical and should be treated as such.
3. In several parts of the discussion, the authors formulate conclusions suggesting a real impact of the studied strain on plant growth and its ecological significance. These statements are too categorical in relation to the presented data, as they are not supported by experiments conducted on plants or by analyses under conditions resembling natural environments. Interpretations based solely on genomic analyses and in vitro assays should not be presented as evidence of actual biological function. In its current form, there is a risk of overinterpretation and overestimation of the strain’s significance. It is recommended to moderate the language throughout the manuscript by using conditional expressions that clearly indicate the potential rather than confirmed nature of the observed traits.

 

Minor editorial comments:

Line 271 – “Growth occurred on potato dextrose agar (PDA, ×0.1)” – the notation “×0.1” is unclear.

Line 284 – “ANI/closest relative: 79.9% %.” – the duplicate “%” should be removed.

Line 288 – “The metabolic profile generated borderline positive reactions” – consider replacing with “yielded mostly weak or ambiguous reactions”.

Lines 344–345 – the sentence “The presence of a complete allantoin utilization operon further supports adaptation to nitrogen-limited root environments” is repeated and should be corrected.

Line 419 – “Data availability statement: Data Availability Statement:” – remove the duplication.

Author Response

Comment 1: The authors demonstrated plant growth–promoting traits solely on the basis of qualitative assays, such as halo formation or changes in medium color. While such observations are useful for preliminary screening, they do not allow for a reliable assessment of the intensity or efficiency of the studied processes. Quantitative data are lacking, which would enable comparison of the strain’s activity with that of other microorganisms, for example measurements of solubilized phosphorus expressed in mass units or concentration in solution.

Response 1: We thank the reviewer for this important observation. We fully agree that quantitative measurements are necessary to assess the efficiency of plant growth–promoting (PGP) traits. In the present study, the assays were conducted as part of an initial screening of a larger collection of bacterial isolates obtained from P. harmala, with the goal of identifying strains of potential interest. Strain AGC32 was subsequently selected for further taxonomic and genomic characterization based on its distinctive features. To clarify this point, we have revised the manuscript to explicitly state:
“The plant growth–promoting traits were evaluated using qualitative assays as part of an initial screening approach.”

Comment 2: The authors infer the ability of the strain to fix nitrogen based on the presence of nifHDK genes in the genome. This approach is insufficient, as the mere presence of genes does not imply their expression or functional enzymatic activity. The study does not provide any direct evidence confirming nitrogenase activity, such as an acetylene reduction assay or measurements of atmospheric nitrogen assimilation. The absence of such data represents a significant methodological gap, particularly since nitrogen fixation is one of the key traits attributed to bacteria of the genus Rhizobium. In its current form, statements regarding nitrogen fixation remain hypothetical and should be treated as such

Response 2: We thank the reviewer for raising this critical point. We agree that the presence of nifHDK genes alone does not confirm nitrogen fixation. In our study, the potential for nitrogen fixation is supported not only by genomic identification of nifHDK but also by physiological evidence: strain AGC32 was able to grow under nitrogen-limited conditions, indicating its capacity to utilize atmospheric nitrogen as a nitrogen source. Nevertheless, we acknowledge that direct confirmation of nitrogenase activity (e.g., via an acetylene reduction assay) is required. Accordingly, all statements regarding nitrogen fixation have been revised to reflect their hypothetical nature.

Comment 3: In several parts of the discussion, the authors formulate conclusions suggesting a real impact of the studied strain on plant growth and its ecological significance. These statements are too categorical in relation to the presented data, as they are not supported by experiments conducted on plants or by analyses under conditions resembling natural environments. Interpretations based solely on genomic analyses and in vitro assays should not be presented as evidence of actual biological function. In its current form, there is a risk of overinterpretation and overestimation of the strain’s significance. It is recommended to moderate the language throughout the manuscript by using conditional expressions that clearly indicate the potential rather than confirmed nature of the observed traits.

Response 3: We thank the reviewer for this important comment. We have carefully revised the Discussion and Conclusion to avoid overinterpretation of functional traits. Statements implying demonstrated effects on plant growth or ecological performance have been reformulated using conditional language (e.g., “may,” “could,” “suggests,” “is consistent with”). We now explicitly state that the functional inferences are based on genomic annotation and in vitro assays and do not constitute evidence of activity under natural or in planta conditions. In addition, we emphasize the need for experimental validation to confirm the ecological relevance and plant-associated functions of strain AGC32.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Authors concentrated on characterization of an endophytic bacterium obtained from surface-sterilized roots of the desert medicinal plant Peganum harmala.

Introduction subchapter gives the reader sufficient background to analyze Author’s obtained results.

Authors clearly describe the aim of the studies and tested hypothesis.

Materials and methods are described in detail and in a repetitive way.

Authors results demonstrated that phylogenomic analyses placed AGC32 within the genus Rhizobium, but clearly distinct from described species, supporting its designation as a novel species for which the name Rhizobium moroccans sp. nov. is proposed. Moreover, phenotypic assays demonstrating the ability to metabolize diverse organic acids and carbohydrates and to express multiple plant growth–promoting traits, including nitrogen fixation and the solubilization of phosphorus, potassium, and silicon.

In my opinion the reader should have a chance to show analysed plants as well as plant roots from which analyzed bacteria were isolated – it should be added; It is important part, especially ,when a lot of data are the predicted analyses using in silico methods. The simplest solution - Please, rethink adding Figure S1 and Figure S2 in appropriate place into the main text.

In my opinion very interesting part of data Authors received based on secondary metabolites gene clusters (subchapter);

I would like to ask Authors for deeply explanation of plant growth promoting obtained results taking into account presented figure 6.

Authors declare sequencing data effect- Therefore, I would like to ask about qPCR confirmation analyses of these results?

On the other side, in my opinion the discussion part is the weakest subpart of manuscript, which should be definitely strengthened. For example, the part describing statement that “AGC32 exhibits a functional genome architecture optimized for survival and plant interaction in arid and semi-arid environments” should be expanded for sure.

Minor aspect:

• Please, check all citation as well as some of them are marked by names and the other by numbers- please, unify the text.

Author Response

Comment 1: Please, rethink adding Figure S1 and Figure S2 in appropriate place into the main text.

Response 1: We thank the reviewer for this valuable suggestion. Figures S1 and S2 have now been integrated into the main manuscript, providing visual context for:

• The host plant (Peganum harmala).
• The root system from which strain AGC32 was isolated.

This integration improves the biological relevance and clarity of the study.

 

Comment 2: In my opinion very interesting part of data Authors received based on secondary metabolites gene clusters (subchapter);

Response 2: We thank the reviewer for highlighting this point. We have substantially expanded the description and interpretation of Figure 6. The revised text now reads:

“Plant growth–promoting traits of strain AGC32 were assessed using qualitative plate assays. Fresh bacterial colonies were streaked onto media designed to detect solubilization of phosphate, silicate, zinc, and potassium, as well as growth under nitrogen-limited conditions. Positive activity was indicated by visible halo formation around colonies and/or color changes in the medium, reflecting mobilization of insoluble mineral forms. Compared to the negative control, strain AGC32 exhibited clear solubilization zones and medium alterations, supporting its potential to mobilize essential nutrients.”

In addition, we now explicitly state:
“These assays provide qualitative evidence of functional potential and should be interpreted as preliminary indicators.”

 

Comment 3: Authors declare sequencing data effect- Therefore, I would like to ask about qPCR confirmation analyses of these results?

Response 3: We thank the reviewer for this suggestion. Whole-genome sequencing and annotation were performed using high-coverage sequencing and validated pipelines widely accepted for gene identification. As the primary objective of this study is taxonomic and genomic characterization, gene expression validation (e.g., qPCR) was beyond the scope of this work. However, we acknowledge its importance and have added the following statement:

“Future studies involving gene expression analyses (e.g., qPCR) will be conducted to confirm the functional activity of key genes identified in the genome.”

 

Comment 4: In my opinion the discussion part is the weakest subpart of manuscript, which should be definitely strengthened. For example, the part describing statement that “AGC32 exhibits a functional genome architecture optimized for survival and plant interaction in arid and semi-arid environments” should be expanded for sure

Response 4: We appreciate the reviewer’s suggestion to strengthen the Discussion. This section has been substantially revised and expanded to improve clarity and depth of interpretation. In particular, the paragraph describing the “functional genome architecture” has been reworked to provide a more balanced and detailed interpretation of genomic features, including stress response, transport systems, and metabolic traits, while clearly distinguishing between inferred potential and demonstrated function. We also improved the integration between genomic data, phenotypic assays, and ecological context, and added clearer links to existing literature.

Reviewer 3 Report

Comments and Suggestions for Authors

My comments are presented in the attached file.

Comments for author File: Comments.pdf

Author Response

Comment 1: How many plant root samples were used for bacterial isolation? How many strains were obtained, and how was AGC32 selected?

Response 1: We thank the reviewer for requesting clarification. A total of 50 root specimens of Peganum harmala were collected from the sampling site. Each root sample was processed independently, and bacterial isolation was performed in triplicate, following the protocol described in our previous work (https://doi.org/10.3390/microorganisms13122843). Multiple bacterial isolates were obtained; among these, only one isolate affiliated with the genus Rhizobium (strain AGC32) and one additional isolate identified as Sinorhizobium meliloti were recovered. Strain AGC32 was selected for further analysis due to its potential novelty within the genus, showing <98% sequence homology with known Rhizobium species.

Comment 2: For the Biolog GEN III MicroPlate system analysis, which culture medium was used?

Response 2: We thank the reviewer for this request. Bacterial cultures used for the Biolog GEN III MicroPlate analysis were grown on Tryptic Soy Agar (TSA) prior to inoculation, following standard Biolog procedures.

Comment 3: In the phylogenetic analyses, the GenBank accession numbers of the strains retrieved from NCBI should be provided.

Response 3: We thank the reviewer for pointing out this omission. The GenBank accession numbers of all reference strains used in the phylogenetic analyses have now been added to the revised figure 2 and are referenced in the main manuscript.

Comment 4: In the Plant-Growth-Promoting (PGP) characterization, the study is limited to in vitro experiments; therefore, the lack of in vivo validation may be considered an important limitation of the study.

Response 4: We thank the reviewer for this important observation. We acknowledge that the PGP traits described are based solely on in vitro assays and thus provide preliminary evidence of functional potential. The primary objective of this study was the taxonomic and genomic characterization of a novel Rhizobium species associated with a non-legume host, and to make this strain available to the scientific community. Further functional validation under in planta conditions is currently underway in a collaborative study, which will specifically investigate the ecological role and plant interaction mechanisms of strain AGC32.

Comment 5: Latin names are not written in italics in the reference list. In addition, there is a lack of consistency in the formatting of the references; in some entries, the first letters of words are capitalized, while in others they are not.

Response 5: We thank the reviewer for this observation. The reference list has been carefully revised to ensure that all Latin names are italicized and that formatting is consistent throughout, including capitalization, style, and punctuation. These corrections have been applied across the manuscript.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have reviewed the revised version of the paper. The authors have put significant effort into improving it, and in my opinion, in its current form the article is suitable for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors significanlt improved tha manuscript [especially the discussion part and some part in presenting results] and adressed almost all my sugestions.