Comparative Analysis of Endophytic Curtobacterium Species Reveals Commonalities and Adaptations

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the writers' efforts in preparing this manuscript, which is well-structured and written in an easy-to-understand style. My thoughts, however, are as follows:

-This manuscript suggests the vertical transmission of endophytes from seeds and identifies common traits and adaptations among several Curtobacterium endophytes isolated from fruits, flower petal, and stem tissue of plants from various environments. Therefore, it would be highly beneficial to include an analysis of the environmental factors and conditions influencing the growth of these plants in the various habitats from which the samples are collected. Understanding their impact on the distribution and adaptive mechanisms of Curtobacterium species within the plant could provide valuable insights into plant-microbe interactions and their potential applications in agriculture.

-Please provide a reference for the method used for surface sterilization of plant parts, as it is crucial to ensure the effectiveness and reliability of the sterilization process.

-Please include a reference for Sections “2.5, 2.6, and 2.7”.

- In heat and cold tolerance tests, are the selected temperatures of 42°C, 25°C, and 6°C adequate to accurately assess the isolates' ability to withstand heat and cold stress?

-What was the rationale for selecting these six specific bacterial isolates from the overall collection of isolated bacteria? “As you reported in line 154”

-The laccase test is not included in Materials and Methods Section “2.6: Metabolic Tests of Bacterial Isolates”?

-In Figure 5B (Lower panel), the left chart is designated for 25°C, but the Y-axis title refers to 6/25°C. Please verify and correct if necessary.

Typo errors:

-Line 347: “[10],[42],[43], [44],[45],[46]”. Adjust font size

-Lines 351, 352: “polysaccharides or phosphorylated polysaccharides that may be phosphorylated”. It is repetitive phrasing. Check it

-Line 356: “(Kim et al., 2008, Saranya et al., 2013)”. The authors have chosen the numbering system for citations; therefore, reference names should be removed.

-Lines 364, 365: “ [14], 364 [38],[50],[51]”. Adjust font size

-Line 371: “°C[14, 38]”. Insert space

-Line 384: “C[10]”. Insert space

-In the reference list, all organism names should be written in italics.

- The attached file may be useful.

Comments for author File: Comments.pdf

Author Response

REVIEWER 1

I appreciate the writers' efforts in preparing this manuscript, which is well-structured and written in an easy-to-understand style. My thoughts, however, are as follows:

-This manuscript suggests the vertical transmission of endophytes from seeds and identifies common traits and adaptations among several Curtobacterium endophytes isolated from fruits, flower petal, and stem tissue of plants from various environments. Therefore, it would be highly beneficial to include an analysis of the environmental factors and conditions influencing the growth of these plants in the various habitats from which the samples are collected. Understanding their impact on the distribution and adaptive mechanisms of Curtobacterium species within the plant could provide valuable insights into plant-microbe interactions and their potential applications in agriculture.

Thank you for all your critical feedback! We have explained the microbial habitats at the end of discussion section 4.4.

-Please provide a reference for the method used for surface sterilization of plant parts, as it is crucial to ensure the effectiveness and reliability of the sterilization process.

Surface sterilization procedure was adapted from a previous study (Burgdorf et al., 2014). We initially used a combination of ethanol and bleach but determined that omission of bleach did not impact the efficacy of surface sterilization and this was confirmed by imprinting sterilized flower petals on TSA plates as previously described (Qin et al., 2009). For the fruit samples, the surface sterilization was less critical since we used the internal pulp tissues for bacterial isolation; nevertheless, they were surface sterilized as described in the methods. We provided a more detailed explanation of the sterilization of various samples and also included references (Methods 2.2). 

-Please include a reference for Sections “2.5, 2.6, and 2.7”.

References have been added to all these sections. Routine microbiological tests (methods 2.6) were performed based on media manufacturer’s instructions.

- In heat and cold tolerance tests, are the selected temperatures of 42°C, 25°C, and 6°C adequate to accurately assess the isolates' ability to withstand heat and cold stress?

25°C was the standard growth temperature used for the Curtobacterium isolates in this study. Since psychrotrophs are recognized as microbes that grow below 7°C (Marshall 2005; Cortez et al., 2022), we believe growth at 6°C reliably classifies the bacteria as cold tolerant. While we typically use the criterion of growth at 48°C for thermotolerance in our lab, in this case, we would like to highlight the unusual heat tolerance of the isolate, RMB2, that could grow up to 45°C in liquid culture, while none of the other Curtobacterium isolates could grow at 42°C or 45°C. Interestingly, this isolate lacks the cold tolerance displayed by the other isolates. This information has been added to the discussion section (Discussion 4.4).

What was the rationale for selecting these six specific bacterial isolates from the overall collection of isolated bacteria? “As you reported in line 154”

We have provided more information in the text as to how these isolates were selected (Results 3.1). We included the supplementary table 1 describing all the isolates and explained in the results (3.1) why these six specific isolates were selected.

-The laccase test is not included in Materials and Methods Section “2.6: Metabolic Tests of Bacterial Isolates”?

This is a mistake we have fixed- thank you! A description and references for laccase assay have been added to section 2.6 in Methods.

-In Figure 5B (Lower panel), the left chart is designated for 25°C, but the Y-axis title refers to 6/25°C. Please verify and correct if necessary.

The growth in cold (6°C) relative to 25°C is expressed in the graph to show the relative cold tolerance of the isolates. For instance, based on the graph, the growth of ST1.1 at 6°C was nearly 50% that of the growth at 25°C, while most other isolates at 6C showed nearly 20% of the growth at 25°C. In contrast, RMB2 showed very little growth (less than 5% of the growth at 25°C) at 6°C.  

Typo errors:

-Line 347: “[10],[42],[43], [44],[45],[46]”. Adjust font size

-Lines 351, 352: “polysaccharides or phosphorylated polysaccharides that may be phosphorylated”. It is repetitive phrasing. Check it

-Line 356: “(Kim et al., 2008, Saranya et al., 2013)”. The authors have chosen the numbering system for citations; therefore, reference names should be removed.

-Lines 364, 365: “ [14], 364 [38],[50],[51]”. Adjust font size

-Line 371: “°C[14, 38]”. Insert space

-Line 384: “C[10]”. Insert space

-In the reference list, all organism names should be written in italics.

- The attached file may be useful.

We appreciate your time in pointing these out and apologies for the careless mistakes! All these changes have been made, thank you! We did change all scientific names in the references to italics, but as the references were created using endnote, it is possible the italics format may be lost when the document reloads on a new computer.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. There is too little information about Curtobacterium sp. endophytes in the introduction.
2. In Section 3.1, it should be mentioned how many bacteria were isolated in total, and how many Curtobacterium sp. were finally identified through 16S rRNA.
3. Figure 1 does not provide the relevant software and parameters for the construction of the phylogenetic tree.
4. The supplementary document should provide the sequences of 16S rRNA after sequencing.
5. The magnification of the Gram stains images provided in Figure 2 is not sufficient to see the specific staining details clearly.
6. It would be better if the article could conduct a basic functional analysis of the isolated Curtobacterium sp. endophytes.

Author Response

Reviewer 2

  There is too little information about Curtobacterium sp. endophytes in the introduction.

Thank you for all your critical feedback! We had ‘saved’ the information for the Discussion section, but we see now it has to be more substantial in the Introduction as well, so we have added this information to Introduction.

  In Section 3.1, it should be mentioned how many bacteria were isolated in total, and how many Curtobacterium sp. were finally identified through 16S rRNA.

We have added information about the original collection of bacteria in the beginning of results section (Results 3.1). as well added the supplementary table 1 with a list of the original isolates and how the Curtobacterium were selected.

  Figure 1 does not provide the relevant software and parameters for the construction of the phylogenetic tree.

We have added this information to the Figure 1 legend as well enhanced the description in the methods section (Methods 2.4).

  The supplementary document should provide the sequences of 16S rRNA after sequencing.

The sequences are included as Supplementary Figure 1.

  The magnification of the Gram stains images provided in Figure 2 is not sufficient to see the specific staining details clearly.

Thanks for your suggestion, we have repeated Gram stains and obtained clearer pictures that we used to remake Figure 2.

  It would be better if the article could conduct a basic functional analysis of the isolated Curtobacterium sp. endophytes.

We have a follow-up study that focuses on functional analysis, specifically with regards to the plant growth promotion capabilities of the isolates and our preliminary results suggest that some of the isolates (RMB2, C. luteum isolate) may have PGPR effect. This can be our next project.

(Note: Attachment includes a graph for your reference)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The present manuscript entitled "Comparative Analysis of Endophytic Curtobacterium Species Reveals Commonalities and Adaptations", demonstrates the the significance of endophytes in floral and fruit tissues and their application in plant growth and protection. The work is important for the development of novel ways of plant protection or plant growth promotion ; however, the present manuscript reports only the preliminary data of six endophytic Curtobacterium species. The manuscript needs to be improve by addressing the following points

1. Please introduce the significance of endophytes in floral and fruit tissues and their possible seed-borne transmission in the introduction section.
2. The present study found nearly all isolates are psychrotolerant while previous reports indicates Curtobacterium species are generally mesophilic.  Please explain the possible reason for this adaptation.  Discuss how  psychrotolerance features impact their ecological niches by comparing with other well studied psychrotolerant strains.
3. Curtobacterium is a common plant pathogen. What is the genetic distance or similarities of the isolated Endophytic Curtobacterium species with the well known pathogenic Curtobacterium species. 
 Why same bacterial genus benefits or negatively affects plant. Please discuss the possible genomic adaptations based on the existing literature. 
4. Is there any antagnostic or plant growth promoting activities (other than starch-degrading and phosphate solubilizing) of the isolated Endophytic Curtobacterium species?
5. What is the significance of "Detection of Bacterial Pigments" in this study? Please write its role in plant defence system.

Author Response

Reviewer 3

The present manuscript entitled "Comparative Analysis of Endophytic Curtobacterium Species Reveals Commonalities and Adaptations", demonstrates the the significance of endophytes in floral and fruit tissues and their application in plant growth and protection. The work is important for the development of novel ways of plant protection or plant growth promotion; however, the present manuscript reports only the preliminary data of six endophytic Curtobacterium species. The manuscript needs to be improve by addressing the following points.

1. Please introduce the significance of endophytes in floral and fruit tissues and their possible seed-borne transmission in the introduction section.

Thank you for all your critical feedback! We have included this information in the introduction section.

2. The present study found nearly all isolates are psychrotolerant while previous reports indicates Curtobacterium species are generally mesophilic.  Please explain the possible reason for this adaptation.  

We are unsure if the psychrotolerance is only limited to our Curtobacterium endophytes or if it is a more widespread ancestral characteristic of Curtobacterium species. As we mentioned in the discussion section (Discussion 4.4), most reported Curtobacterium strains have not been tested for growth in the cold (6°C) and future research may incorporate this assay in Curtobacterium characterization. 

Discuss how psychrotolerance features impact their ecological niches by comparing with other well studied psychrotolerant strains.

This is a good point and we have now addressed this in the discussion section (4.4).

3. 
   Curtobacterium is a common plant pathogen. What is the genetic distance or similarities of the isolated Endophytic Curtobacterium species with the well known pathogenic Curtobacterium species. 

We found two of the isolates (ST1.1 & WBB) clustered very closely to C. flaccumfaciens. (Fig.1, phylogenetic tree). Whether these are pathogenic requires further characterization and this would involve stem or leaf infiltration since C. flaccumfaciens can cause wilt. In a preliminary experiment, root treatment of WBB- C. flaccumfaciens and RMB2-C. luteum did not have any detrimental impact on plant growth (please see graph below in point 4).

Why same bacterial genus benefits or negatively affects plant. Please discuss the possible genomic adaptations based on the existing literature. 

This is a good point to discuss and we have addressed this in the discussion section (Discussion 4.1).

4. 
   Is there any antagnostic or plant growth promoting activities (other than starch-degrading and phosphate solubilizing) of the isolated Endophytic Curtobacterium species?

We are working on this as part of a follow-up study and preliminary results suggest that some of the isolates (C. luteum- RMB2) may be plant growth promoting.

5. What is the significance of "Detection of Bacterial Pigments" in this study? Please write its role in plant defence system.

This is quite an insightful suggestion and, indeed, upon literature research, we found that carotenoid pigments could aid plant defense and we have added these points in the discussion section (Discussion 4.2). Thank you!

(Note: Attachment includes graph for your reference)

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The paper entitled 'Comparative analysis of endophytic Curtobacterium species reveals commonalities and adaptations' is devoted to isolation and identification of endophytic Curtobacterium from fruits, flower petal and stem tissue. The authors demonstrated that Curtobacterium endophytes belong to three major clusters; phylogenetic relationships, pigmentation, and substrate specificity of tested isolates were estimated. 

I beleive the paper could be interested for the readers, but before being accepted, some serious improvements should be done.

1. Lines 27-28 (Keywords): I believe 'phylogenetics', 'pigmentation' should be added to the Keywords; 'Curtobacterium' should be in Italics.
2. Lines 100-101: The authors have to clarify what method was used for DNA extraction? Were bacteria heated or treated by a special extraction kit?
3. Whether the primers were developed in this study or taken from another work (reference?)
4. Complete amplification profile should be described.
5. I think more information about identification techniques should be provided. As I can see, identification was performed by morphological analysis and DNA sequencing. It would be useful if the authors add the pictures demonstrating the morphology of reference strains of species mentioned. These pictures can be made using any collection strains or taken from atlas or another original source. 
6. Despite the fact that obtained sequences demonstrated a high percent of identity to GenBank sequences, I can see low bootstrap support values on the dendrogram. Probably, additional sequences form the databade should be added or another algorithm of phylogenetic tree construction can be applied. Moreover, the authors could have used more than one marker to identify cultures (but I agree that 16S rDNA has been the most appropriate one). 

Author Response

Reviewer 4

The paper entitled 'Comparative analysis of endophytic Curtobacterium species reveals commonalities and adaptations' is devoted to isolation and identification of endophytic Curtobacterium from fruits, flower petal and stem tissue. The authors demonstrated that Curtobacterium endophytes belong to three major clusters; phylogenetic relationships, pigmentation, and substrate specificity of tested isolates were estimated. 

I beleive the paper could be interested for the readers, but before being accepted, some serious improvements should be done.

1. Lines 27-28 (Keywords): I believe 'phylogenetics', 'pigmentation' should be added to the Keywords; 'Curtobacterium' should be in Italics.

Thank you for all your critical feedback! These have been added.

1. Lines 100-101: The authors have to clarify what method was used for DNA extraction? Were bacteria heated or treated by a special extraction kit?

We did not need to extract DNA for the PCR. We set up colony PCR by directly adding liquid culture into the PCR reaction (as described in the methods 2.4).

1. Whether the primers were developed in this study or taken from another work (reference?)

The primers were previously described, and the references have been added (methods 2.4).

1. Complete amplification profile should be described.

This has been included in methods 2.4.

1. I think more information about identification techniques should be provided. As I can see, identification was performed by morphological analysis and DNA sequencing. It would be useful if the authors add the pictures demonstrating the morphology of reference strains of species mentioned. These pictures can be made using any collection strains or taken from atlas or another original source. 

Identification of the isolates was primarily based on 16S rRNA sequencing and confirmed using several tests recommended by EPPO for Curtobacterium identification (https://gd.eppo.int/taxon/CORBFL/documents):  yellow/orange colonies, short Gram-positive rods, catalase positive, oxidase negative and caseinase positive. Here is a reference strain for comparison (Kim et al., 2021); the colony morphology and Gram stains in our study are comparable to the images from this study (shown below).

Kim et al., 2021: https://pesquisa.bvsalud.org/gim/resource/en,au:%22Martins%20Neto,%20Viviana%22/wpr-893416

 

1. Despite the fact that obtained sequences demonstrated a high percent of identity to GenBank sequences, I can see low bootstrap support values on the dendrogram. Probably, additional sequences form the databade should be added or another algorithm of phylogenetic tree construction can be applied. Moreover, the authors could have used more than one marker to identify cultures (but I agree that 16S rDNA has been the most appropriate one). 

Thank you for this suggestion! We reconstructed the phylogenetic tree using a different strategy. We used M-Coffee for multiple sequence alignment and constructed a maximum likelihood tree with 500 bootstrap replicates and the values are much better.

(Note: Attachment includes image for your reference)

 

Author Response File: Author Response.pdf

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

In the revised version of the paper, the authors improved the text according to the comments made earlier. I believe the manuscript can be accepted for publication after some minor improvements done:

Line 10: The authors declare that '...but their function as plant endophytes is less clear...'. Probably, '...their function as endophytes of 'above-ground organs' or reproductive organs' would be more appropriate. (optional)

Lines 33-37: 'Curtobacterium' repeats in three sentences in row. 

Section 2.4. It should be noted that R. iranicus was used as an outgroup

Author Response

Line 10: The authors declare that '...but their function as plant endophytes is less clear...'. Probably, '...their function as endophytes of 'above-ground organs' or reproductive organs' would be more appropriate. (optional)

Thanks for the suggestion. This has been changed to ‘function as plant endophytes in aerial organs’.

Lines 33-37: 'Curtobacterium' repeats in three sentences in row. 

This has been fixed.

Section 2.4. It should be noted that R. iranicus was used as an outgroup

This is done. Thanks for all the constructive input through the review process.