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[# PeerJ Staff Note - this decision was reviewed and approved by Valeria Souza, a PeerJ Section Editor covering this Section #]
Dear Dr. Delitte,
Thank you for the privilege of reviewing your work. Below you will find some minor comments suggested by the reviewer. I am willing to accept the manuscript if you are able to revise the manuscript as suggested.
Lines 179-185: “Bacteria expressing GFP were then cultivated in 50 mL Falcon containing 20 mL of LB for 24 h (30°C, 180 rpm) before being washed twice and resuspended in sterile MgCl2 + 1% Tween20. Twenty seeds from the Chevignon wheat cultivar were soaked in this suspension for 1 h before sowing in an autoclaved 70/30 mix of loam/perlite...” Please mention the concentration of the bacterial suspension as you did for the detached leaves protocol (108 CFU/mL)
Validity of the findings
The authors stated in their response letter that the genome sequences are available to reviewers for verification via Zenodo at the following link: https://doi.org/10.5281/zenodo.12743961. However, I was not able to verify the sequences as the provided link leads to a page where the files are listed but restricted. For transparency, I recommend that the authors provide the reviewers, if appropriate with a functional access to the deposited genome sequences.
As I stated in my previous review report: “Positive control refers to pathogen + known treatment (ex: fungicide or another known biocontrol agent). The use of positive control to refer to boxes where Z. tritici was applied alone can lead to confusion. Boxes with only the pathogen should be renamed adequately.” The authors only revised the title of figure 6 but not the text (361 and 362). Please revise.
Sincerely,
Sushanta Deb
The authors have adequately addressed the reviewer's comments and concerns.
The authors successfully addressed the issues stated in the review.
Based on the current revised version of the manuscript, the findings have been adequately and scientifically explained.
The title and abstract have been revised adequately. The protologue of the new species has been added.
No comment
Lines 179-185: “Bacteria expressing GFP were then cultivated in 50 mL Falcon containing 20 mL of LB for 24 h (30°C, 180 rpm) before being washed twice and resuspended in sterile MgCl2 + 1% Tween20. Twenty seeds from the Chevignon wheat cultivar were soaked in this suspension for 1 h before sowing in an autoclaved 70/30 mix of loam/perlite...” Please mention the concentration of the bacterial suspension as you did for the detached leaves protocol (108 CFU/mL)
The authors stated in their response letter that the genome sequences are available to reviewers for verification via Zenodo at the following link: https://doi.org/10.5281/zenodo.12743961. However, I was not able to verify the sequences as the provided link leads to a page where the files are listed but restricted. For transparency, I recommend that the authors provide the reviewers, if appropriate with a functional access to the deposited genome sequences.
As I stated in my previous review report: “Positive control refers to pathogen + known treatment (ex: fungicide or another known biocontrol agent). The use of positive control to refer to boxes where Z. tritici was applied alone can lead to confusion. Boxes with only the pathogen should be renamed adequately.” The authors only revised the title of figure 6 but not the text (361 and 362). Please revise.
The authors have addressed the comments effectively, and most of the suggested revisions have been incorporated. A few minor points, as outlined in the comments, still require attention before the manuscript can be considered ready for publication
The manuscript has received valuable feedback from the reviewers, and it seems that significant revisions are necessary. I agree with their suggestions, which should be addressed point by point, with detailed explanations provided for each revision
The study of the phyto-biome and plant probiotics is a rapidly evolving field, and the authors have made significant contributions through their work on the wheat phyto-biome. Their elucidation of Pseudomonas arvensis sp. nov. and P. sivasensis as a protector against fungal pathogen colonization is both relevant and impactful. The manuscript is well-organized, clearly written, and successfully meets the objectives of the study. I recommend it for publication with only minor revisions.
Major comments-
1. Species Description: The description of the novel species Pseudomonas arvensis sp. nov. is missing. Including this would be important for completeness.
Minor comments-
1. Introduction Section: It would enhance the introduction to include a broader overview of the members of the wheat phyto-biome. While Pseudomonas is a key bacterium within the wheat-associated microbiota, the inclusion of other predominant taxa would provide a more comprehensive context. Mentioning a few of these taxa in this section would add depth to the background information.
2. Image Quality: The orientation of Figure 1 could be adjusted for better readability. Improving the visual presentation would help clarify the data for readers.
3. Line 219-220: The statement "From the 444... F. graminearum" suggests that out of 444 isolates, 114 isolates exhibit resistance to one of the wheat pathogens, indicating their importance. It would be beneficial to provide a list of these isolates, perhaps in the supplementary materials, to offer further detail on these resistant strains.
No comment
No comment
The authors did a lot of work. The biocontrol and plant colonization work were well done but the identification of the bacterial isolates is not adequately done. The MLSA scheme used in the manuscript is incongruent from the identification based on whole-genome data for some strains with genome sequences. This suggest that some of genes used for MLSA are unreliable to taxonomically discriminate different species. For example, the authors report that the isolates (CR7PS1, DR1PS3, DS3PS4, and DS3PS5) that constitute a potential new species had 97.39% - 99.79% (Table4) to the type strain of P. sivasensis. The authors indicated (footnote Table 4) that these strains initially identified as P. sivasensis via MLSA subject to reidentification as P. arvensis sp. nov.” In other words, the authors are indicating that even though some of these strains are showing MLSA 99.7% similarity to P. sivasensis they are really a different species. Also, strains AR12PS3 and DR4PS3 identified as P. sivasensis using MLSA were found to be putative members of P. cyclaminis and P. marginalis (lines 275-277), respectively. This underscores my previous statement that the MLSA scheme is not suitable to reliably infer species-level identification. This puts in doubt the identification of all the other strains that used MLSA alone, since there is no genome sequence for validation.
The choice of genes and the fragment size make considerable difference in using MLSA to accurately identify Pseudomonas species. Also, the MLSA genes and length should have been already validated in the taxonomy of Pseudomonas species. The work of Mulet et al. (2010) is a good example of effective MLSA scheme for Pseudomonas species. It is unclear why the authors did not use 16S rRNA, rpoD, gyrB and rpoB (Mulet et al. 2010) but opted to only use rpoD. The Mulet et al. scheme, in most cases, seemed to be congruent with genome-based taxonomic identification. The authors are encouraged to use this scheme.
Another pitfall of the manuscript is that the authors are proposing a new species, named Pseudomonas arvensis sp. nov. The methods used for the description of the new species Pseudomonas arvensis sp. nov. are inadequate and incomplete and not in accordance with the rules of International Code of Nomenclature of Prokaryotes (ICNP). Additional data are required for a valid description. The etymology of the name has not been explained as there is no protologue section. The protologue is an ICNP requirement. Also, the cultures of type strain of the new species should be deposited into 2 international culture collections.
Other comments:
Title:
The title is not informative, and the new species proposal should be deleted/dropped. It does indicate that P. sivasensis are associated with cereals but so as other Pseudomonas species such as P. marginalis and P. cyclaminis? Is it possible to emphasize the biocontrol activity of P. sivasensis?
Abstract:
It does not give the information provided in the title except for introducing the new species, Pseudomonas arvensis. How about the data on Pseudomonas sivanensis?
Line 22: Is Pseudomonas arvensis plant pathogenic or antagonistic to fungi? Simply put, what is it doing in the wheat microbiome?
Methods:
Line 64 -65: How were the strains purified? Single colony purification? How were the strains preserved?
Line 72: “… a gelose …” could be “… an agar …”
Line 102: What is CMC medium?
Line 171: should “…2,5 kV..” be “… 2.5 kV …?
Line 190: could “…2,5 cm …” be “…2.5 cm …”
Line 209: What is a mycelium mound?
Results:
Lines 228-234: Based on the comments indicated earlier some of these MLSA-based identification might not be valid.
Table 3: Footnote-Please change “… inhibition measures …” to “…inhibition zones …”
Table 4: Please, rearrange the table to start with the isolates column. Delete the sampling dates. Please, only present matches to type strains. Include type strain code for all the best MLSA matches e.g. P. flurorescens DSM 5009T. Again some of these identifications to species level might not be accurate.
Line 243: NCBI BioProject reference PRJNA1141912 only shows the data of 2 strains: DS3PS4 and CF10PS3. In which BioProject number are the other strains?
Table 5: The assembled genome sequence of these entries SAMN42904002, SAMN42493717 SAMN42493716, SAMN42493718, SAMN42493719, SAMN42493721, and SAMN42493722 could not be found in NCBI. Please, make sure the sequences are publicly available. The lack of availability of some of these genome sequences made it impossible to further validate their taxonomic identity.
Figure 1. Quality is not good.
Lines 275-277: This indicates that the MLSA scheme is unreliable.
The experimental design is adequate, but the methods used were inadequate and incorrect especially for the choice of MLSA scheme and proposal of a new species.
The biocontrol and plant colonization work were well done. The MLSA identifications of some of the bacterial strains was incongruent with those of whole-genome sequence analysis. This created doubts on the reliability of the MLSA scheme to accurately identify strains that do not have genome sequence validation.
The data/information used in proposing the new species, Pseudomonas arvensis, is not in accordance with the ICNP rules.
The study is quite clear and addresses an important aspect of microbiome associated with crops and their crucial role in biocontrol of diseases. The study is also based on sufficient background from the field while providing sufficient results/data, but there is a good scope to build the context in a better way in the ‘Introduction’ section.
For example: Lines 45-55 represent content that looks more suitable for discussion. Instead they describe the results even before explaining the objective of the study in lines 56-59.
The research problem addressed has been supported by sufficient experimental design, both genetic and physiological. The description of the methods section also has been appropriate. The experiments have been relevant to demonstrate the association and identification of relatively novel Pseudomonas species as well as their biocontrol potential.
The study projects novelty in terms of exploring newer Pseudomonas species for their biocontrol potential as well as prevalence and localization of the species across wheat. Given that the study is carried out across several seasons, the statistical validation is evident. However, the conclusions as described in the manuscript, are more inclined towards long term implications in a generic way. Apparently, the concrete concluding remarks based on the study are not clearly included.
In the present study, the authors have explored microbiome associated with the wheat crop with a focus on Pseudomonas species, isolated from wheat heads, leaves, roots, and soil. Primary screening was done based on the antagonistic abilities, since biocontrol is a prominent phenotype associated with Pseudomonas species. Based on MLSA approach, the shortlisted isolates were primarily identified, followed by whole genome sequence based analysis of 9 strains of P. sivasensis. Subsequently, the genomes have been aligned, analyzed and mined to understand phylogenetic relationships, overall organization and genome divergence and functional capacities. In this process, a new wheat-associated species P. arvensis. The work also demonstrates the localization of a selected P. sivasensis isolate in the wheat plant and its efficacy in antagonizing Z. tritici infection.
Overall, the study has the following relevance/strengths (i) Contribution of the whole genome sequences of Pseudomonas species relatively less explored for plant-beneficial interactions. (ii) Genetic/genomic data supported by physiological experiments. The work is a good contribution to the existing repertoire of literature on plant-associated microbiomes and their significance in designing sustainable agriculture strategies.
However, following are the concerns that are realized.
1. In general, the manuscript lacks a flow of reading that could connect the flow of experiments with result; despite experimental layout being well planned and sufficiently executed.
2. The discussion section runs like a single long paragraph and this makes it difficult to read. This section requires a major re-writing.
3. The representation of the text content, in introduction and discussion looks similar.
4. The title apparently doesn’t reflect the type of work.
While the study is entirely carried out using wheat crop, the title is generalized to include the word ‘cereals’. It is important to note that the rhizobacterial colonization is largely influenced by the plant root exudates, the composition of which varies across plant species. Given this, the authors need to explain the basis of using the generalized ‘cereal’ instead of ‘wheat’. The title thus may be reconsidered.
5. Abstract does not bring out a strong summary of the study. Needs to be improvised.
Some specific points are as follows:
1. Lines 43-45: Around this text, the relevance of Pseudomonas species in general with respect to the wheat crop is required. At present, the direct mention of P. sivasensis looks abrupt
2. Lines 52-55:This content looks more befitting to results and discussion.
3. Line 218 and throughout in the later sections: The captions under ‘Results’ sections could be more descriptive and precise. In certain cases, the caption under Materials and Methods and Results is the same e.g. Line 200 and line 323. In fact, the captions in the results should be framed to establish continuity from the previous section, for better readability
4. Lines 299-311:The paragraph explains the genome mining of the selected isolates. Accordingly, none of the strains possessed complete operon for pyochelin production or the essential genes for pyoverdine production. In that case, how can the authors explain the siderophore production by the strains
5. Figure 5: Representation of the figure can be improved with better graphics
6. Line 488: The authors claim about species specific interactions being addressed through the study. In what way is the study demonstrating those?
7. Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas khavaziana species to name a few, have been explored with respect biocontrol of wheat pathogen. It would be interesting to comment upon the differences between these known biocontrol species and the P. arvensis/P. sivasensis, possibly in terms of mechanism of biocontrol or relevant genetic composition or even the localization. Including such a description in Discussion section could enhance the usefulness of the newer species explored in the present study.
8. Which Pseudomonas species are so far known to be associated with wheat microbiome? Such details in literature so far, if any, should be discussed.
9. A recent study (https://doi.org/10.3389/fmicb.2024.1440341) also reports novel Pseudomonas species associated with wheat. Discuss/analyze the results of this study (and other such studies) in comparison of those reported in the present study.
The paper titled “Pseudomonas arvensis sp. nov. and P. sivasensis are associated with cereals” treats a matter of current relevancy and is well aligned with the aims of sustainable crop protection strategies. The paper is well written, and the proper methods have been used; nonetheless, there are revisions that need to be addressed before it is ready for publication
Please find below comments relative to the points that need to be revised:
Line 63: From which part of the soil were Pseudomonas strains isolated?
Line 67: add a brief description of the “antibiosis test described for toxins”
Line 72: Mention the full form of the abbreviation STB at first use
Lines 72-77: add the source (reference) of the test
Lines 78-83: add the source (reference) of the test
Line 164: Mention the full form of the abbreviation GFP at first use
Lines 173-175: “Bacteria expressing GFP were then cultivated in LB for 24 h (30°C, 180 rpm) before being washed twice and resuspended in sterile MgCl2 + 1% Tween 20. Seeds from the Chevignon wheat cultivar were soaked in this suspension for 1 h before sowing in an autoclaved 70/30 mix of loam/perlite”
What is the concentration of the bacteria? How many seeds did you use, and what is the volume of the containers used for the cultivation?
Lines 177-184: add the source (reference) of the test
Please find below comments relative to the points that need to be revised:
Lines 225-227: in the in vitro characterization (methods section) several tests were performed but in the results section only Siderophore production, Cellulotic activity, and proteolytic activity were addressed. Please addresse the remaining tests.
Table 4: Please include the accession numbers of MLSA identification
Table 5: Please include NCBI accession number of DS3PS4
Table 5: Aside from the strain CF10PS3 (SAMN42904002), no results were found in the NCBI search for the following strains: BE10PS3 BE10PS1 CR7PS1 DR1PS3 DS3PS5 DS3PS4 AR12PS3 DR4PS3. The search results revealed that no record matches the LMG accessions for all nine strains. Please verify the accuracy or the submission of the accessions .
Line 313: which strains were tested for plant colonization? Was CF10PS3 the only strain expressing GFP?
Line 314: which strains were shown to reach the upper parts among the tested bacteria?
Line 324: Positive control refers to pathogen + known treatment (ex: fungicide or another known biocontrol agent). The use of positive control to refer to boxes where Z. tritici was applied alone can lead to confusion. Boxes with only Z. tritici are negative control. Boxes with only the buffer as well as those with only the pathogen should be renamed adequately. Please revise throughout the paper.
Certificate of deposit :
No result were found for LMG Number: LMG 33711 in the the BCCM/LMG bacteria catalogue strain search. Please verify.
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