Novel Molecular Techniques for Identifying Agricultural Microorganisms
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments for author File: 
Comments.pdf
Extensive editing of English language required
Author Response
We greatly appreciate the time and dedication to our manuscript; your comments have been very valuable to improve the document.
1.- The manuscript provides an overview of novel techniques for identifying agricultural microbes. In total, the language of this review requires further modification.
Answer: The manuscript was proofread and edited in English.
Following are some detail suggestions:
2.- Line 15 and 58: “chapter” is not the appropriate term here. Please consider replacing “chapter” with “review” to better align with the context of the document.
Answer: The change has been made, please look at line 60
3.- Line 48 to 59: This section needs further enhancement. It only mentions PCR and ELISA methods and fails to provide a comprehensive summary of all the methods included in the paper. In the following sections of the main text, there is no detailed description of the ELISA technique. Please revise this part to include a clear and complete summary of the methodologies employed.
Answer: The other methods discussed in the manuscript have been added because it is the introductory section, they are only mentioned but not detailed because they are discussed in more detail in later sections. ELISA was not detailed because it is a routine and low-range technique for the identification of microorganisms, and the idea of the present work was to present new methodologies according to the scope of the work. Please see lines 48-61.
4.- Line 94: In section 2.3, it would be beneficial to include more specific examples or case studies to illustrate NGS applications.
Answer: This section 2.3 has been modified to include more examples of NGS applications. Please review.
5.- Line 246, 259 and 290: The sensors are used to detect the toxins which are not relevant to the discussion of detecting various microorganisms. It would be more appropriate to provide an example that aligns with the primary focus on microbial detection. Consider revising the example to better reflect the scope of detecting different types of microorganisms using these immunosensors.
Answer: This observation has been addressed, but most of the sensor examples focus on indirect detection of microorganisms based on the compounds they produce. Please review this section.
6.- Line 310 and 313: In line 310, the full name "Escherichia coli" should be written out the first time it is mentioned, followed by the abbreviation (E. coli) in parentheses. Subsequent mentions, such as in line 313, should use the abbreviation "E. coli". The same issue appears with other bacterial names, such as "P. aeruginosa" in line 310, which has not been mentioned previously and therefore needs to be written in full. Please review the entire manuscript for Latin names to ensure that they are correctly introduced.
Answer: The name of the microorganisms has been corrected as suggested by you.
Reviewer 2 Report
Comments and Suggestions for Authors
First paragraph of Introduction (Line 24 to 40) needs a lot of referencing. There is none. Where do these times come from, where does the info come from, Gram staining is a technique with a reference. Please include references in this paragraph.
Line 44: Rather use non-culturable than non-cultivable
Line 45 - 47: Provide references. Also, what is meant with it being expensive? Compared to what. Picking up colonies and doing Sanger is one of the cheaper methods when compared to some other methods newer methods.
Line 48 - 59: Needs more references to back up the statements here.
Line 58: What "Chapter" this is a paper.
Line 59: I feel you are discussing more methods than PCR and ELISA.
Line 62: Check grammar for this sentence, as is it makes almost no sense.
Line 63 to 66: These sentences are technically not correct. For example, NGS and third generation sequencing still depend on PCR steps before sequencing for targeted microbial diversity sequencing. PCR is also used for many other application than diversity. I would rethink what I'm trying to say here. Factually it makes little sense. Maybe just say that there are various methods that employ DNA to determine diversity, or something like that.
Section 2.1: References required for all the methods, how do I know you are not just making up names here. Definitely give more info on how these methods work and how you get diversity information from the results. When and how would I use these methods? What advantages do they offer?
Section 2.3: NGS is a very broad term that comprises various sequencing technologies with various applications, advantages and shortcomings. You can probably write a review paper on NGS alone (I'm sure this exists and I would probably reference it here). What is presented here is woefully inadequate to present the scope of NGS. Also, what happened to third generation sequencing that was mentioned earlier?
Section 2.4: FISH applications needs to better defined in the context of the paper. Don't just say what has been found with FISH, why are those organisms important. There is also good examples in FISH looking for nitrate reducers, for example, that is applicable to agriculture.
Section 3: Good amount of info on MALDI-TOF, but written very poorly, like how you would tell it to somebody, not scientifically written. Also, need things like advantages in agriculture and compared to other methods.
Section 4: I like this section, would like to see more depth toward the application in agriculture. Certain bacteria is mentioned that have been detected by sensors, but no reason as to why you would like to pick them up in agriculture is given.
Section 5: Also a nice section, however, also needs so depth on agriculture spesific application, ie, not just mention, expand.
General Comment:
I would personally shift the tone of the paper a bit, it begins by stating that we do not get a full overview of microbes in soil since most of then cannot be cultured using the "golden standard", but the bulk of the paper is spent on methods that only identify certain microbes and glosses over methods that can give you a more complete diversity, such as NGS and other sequencing methods.
Personally I would shift towards saying that you can get most of the sequencing, but the major issue is cost and training to analyze and interpret the results. I would put section 4 and 5 on the forefront as methods that can screen and identify agri relevant microbes at lower cost and ease of use, emphasizing why identifying them easily is important. Otherwise the nuc acid part has to be extensively rewritten to at least be of the same depth as sections 4 and 5.
Comments on the Quality of English Language
Overall the english isn't horrible but it needs input from a native speaker and there needs to be a lot of work done to write more scientifically and less conversationally.
Author Response
Reviewer 2
We greatly appreciate the time and dedication to our manuscript; your comments have been very valuable to improve the document.
1.- First paragraph of Introduction (Line 24 to 40) needs a lot of referencing. There is none. Where do these times come from, where does the info come from, Gram staining is a technique with a reference. Please include references in this paragraph.
Answer: In the current version of the manuscript, references were added to reinforce the information presented.
2.- Line 44: Rather use non-culturable than non-cultivable
Answer: This word has been changed to “unculturable”. Please review lines 44 and 49.
3.- Line 45 - 47: Provide references. Also, what is meant with it being expensive? Compared to what. Picking up colonies and doing Sanger is one of the cheaper methods when compared to some other methods newer methods.
Answer: References have been added. It was an error that what was meant “expansive” and “not expensive”, this has been corrected.
4.- Line 48 - 59: Needs more references to back up the statements here.
Answer: References have been added
5.- Line 58: What "Chapter" this is a paper.
Answer: This word has been changed to review, see line 60.
6.- Line 59: I feel you are discussing more methods than PCR and ELISA.
Answer: The other methods discussed in the review have been added.
7.- Line 62: Check grammar for this sentence, as is it makes almost no sense.
Answer: The grammar has been corrected. Check the current line 63-64.
8.- Line 63 to 66: These sentences are technically not correct. For example, NGS and third generation sequencing still depend on PCR steps before sequencing for targeted microbial diversity sequencing. PCR is also used for many other application than diversity. I would rethink what I'm trying to say here. Factually it makes little sense. Maybe just say that there are various methods that employ DNA to determine diversity, or something like that.
Answer: These sentences have been corrected, please see lines 63-68.
9.- Section 2.1: References required for all the methods, how do I know you are not just making up names here. Definitely give more info on how these methods work and how you get diversity information from the results. When and how would I use these methods? What advantages do they offer?
Answer: Section 2.1 has been modified by adding references and further expanding the information for each method.
10.- Section 2.3: NGS is a very broad term that comprises various sequencing technologies with various applications, advantages and shortcomings. You can probably write a review paper on NGS alone (I'm sure this exists and I would probably reference it here). What is presented here is woefully inadequate to present the scope of NGS. Also, what happened to third generation sequencing that was mentioned earlier?
Answer: Section 2.3 has been modified based on your suggestion, please review it.
11.- Section 2.4: FISH applications needs to better defined in the context of the paper. Don't just say what has been found with FISH, why are those organisms important. There is also good examples in FISH looking for nitrate reducers, for example, that is applicable to agriculture.
Answer: Section 2.4 was changed to contextualize the paper and to include more examples related to Agriculture.
12.- Section 3: Good amount of info on MALDI-TOF, but written very poorly, like how you would tell it to somebody, not scientifically written. Also, need things like advantages in agriculture and compared to other methods.
Answer: Section 3 has been modified with the addition of advantages of this method.
13.- Section 4: I like this section, would like to see more depth toward the application in agriculture. Certain bacteria is mentioned that have been detected by sensors, but no reason as to why you would like to pick them up in agriculture is given.
Answer: More examples of using these methods in agriculture have been added. See section.
14.- Section 5: Also a nice section, however, also needs so depth on agriculture spesific application, ie, not just mention, expand.
Answer: We also discussed agricultural applications in more detail.
15.- General Comment:
I would personally shift the tone of the paper a bit, it begins by stating that we do not get a full overview of microbes in soil since most of then cannot be cultured using the "golden standard", but the bulk of the paper is spent on methods that only identify certain microbes and glosses over methods that can give you a more complete diversity, such as NGS and other sequencing methods.
Answer: Based on your observations, the tone of the article has been improved in this new version.
16.- Personally I would shift towards saying that you can get most of the sequencing, but the major issue is cost and training to analyze and interpret the results. I would put section 4 and 5 on the forefront as methods that can screen and identify agri relevant microbes at lower cost and ease of use, emphasizing why identifying them easily is important. Otherwise the nuc acid part has to be extensively rewritten to at least be of the same depth as sections 4 and 5.
Answer: Please review the various sections as your comments have been taken into consideration.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThis revised manuscript can be accepted for publication
Author Response
Thank you again for your comments on our manuscript. They have greatly improved the document.
Reviewer 2 Report
Comments and Suggestions for AuthorsI can see the authors did a lot of work to improve the paper based on the comments, suggestions and changes I provided. The language has been drastically improved along with the overall scope of the paper. As is I don't see a reason not to accept the paper.
One small comment, for some reason the written out names have been deleted and only the abbreviation kept in the headers to the sections. I would put back the whole written out word with the abbreviation in brackets for the heading -> Line 123, 136, 249
Author Response
Thank you again for your comments on our manuscript, which have improved the document considerably.
The full names with their abbreviations in brackets have been reinserted in the section headings. They are marked in yellow for easier checking.
