Measuring Pathogenic Soil Fungi That Cause Sclerotinia Rot of Panax ginseng Using Real-Time Fluorescence Quantitative PCR
Round 1
Reviewer 1 Report (New Reviewer)
Major corrections are required for the publication of this manuscript entitled "Measuring pathogenic soil fungi that cause Sclerotinia rot of Panax ginseng using real-time fluorescence quantitative PCR".
1. In the second paragraph of the introduction, the authors should emphasize the problems faced by fluorescent quantitative PCR assays such as screening of primers, optimization of PCR conditions, and problems faced in detecting Sclerotinia spp. which are also what you have studied It is recommended to be consistent. In my personal opinion, I suggest that the preamble can be concise, but the focus of the expression should be sharp.
2. In 2.1Materials, the materials part of the writing is wrong, you have to write about the materials used in the experiment such as the soil, the type of fungus, not the experimental apparatus and drugs, etc.
3. In Materials and Methods, the material and methods section is not properly organized, as there are too many paragraphs. Also, proper merging of paragraphs is necessary. For example, lines 205 to 208 can be put into line 74 and should describe how the samples were collected.
4. The second paragraph of the discussion is not very meaningful, and I personally suggest a discussion of some issues, such as the advantages of primer design and the effect of fluorescence quantification conditions on the results.
5. The format of the literature 16, 20, 23, 25, 27, 29, 30, 31, 32, etc. is not completely consistent with the others. The format of the line 370 must be improved.
6. The standard curve equation was Y = -4.675x + 28.031, Please check the value -4.675, The ideal value should be -3.5.
Author Response
Dear Prof.,
Thank you for your comments concerning our manuscript entitled” Measuring pathogenic soil fungi that cause Sclerotinia rot of Panax ginseng using real-time fluorescence quantitative PCR” (ID: agriculture-2495390). The comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the resubmitted paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.
Best wishes!
Baohui Lu
Author Response File: 
Author Response.pdf
Reviewer 2 Report (New Reviewer)
All comments and suggestions can be found in the attached file.
There is a need for improvement in the English language.
Comments for author File: Comments.pdf
There is a need for improvement in the English language.
Author Response
Dear Prof.,
Thank you for your comments concerning our manuscript entitled” Measuring pathogenic soil fungi that cause Sclerotinia rot of Panax ginseng using real-time fluorescence quantitative PCR” (ID: agriculture-2495390). The comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the resubmitted paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.
Best wishes!
Baohui Lu
Point to Point Responds (The modifications have been highlighted in red in the revised manuscript):
Responds to the reviewer’s comments
Reviewer #2:
1)“Sclerotinia sp.” Check the font!
Response: The front was checked.
2) “entity”, I recommend to use other synonym, for example structure!
Response: “entity” was changed to “structure”
3）“load”, changed to“presented”
Response: The scientific name was corrected.
4）“sclerotiniosis” ？
Response: The scientific name was corrected.
5）“on fungi” It is more clear to write from mycelium.
Response: The scientific name was corrected.
6）“2.1 Materials ”The entire paragraph is is not necessary.
Response: The entire paragraph was re-writed.
7）2.2This chapter is totally confusing. It is not clear enough is it the isolates part of collection, or the isolates are isolated from soil samples???
Response: DNA extraction from fungi that acquired from Table 1.
8）P108 “with” changed to“on”。
Response: The word was corrected.
9) 2.3 Cultivated in some liquid media? In the chapter 2.2. is mentioned that mycelium scraped off the agar media?
Response: “The chapter 2.2. is mentioned that mycelium scraped off the agar media”, We only use it for testing purposes, but in the chapter 2.3, we need to use a large amount of DNA to complete genome sequencing, and we need to collect a large amount of mycelium through liquid culture to obtain a large amount of DNA.
10) 2.6 Isolates were cultivated on liquid media? How many days, temperature of cultivation??? Sclerotia were presented?
Response: Isolates were cultivated on liquid media at 25℃ for 3 days, so that we can weigh the mycelium more accurately. Sclerotia were not presented.
11）Before this chapter, it should be mentioned results of 2.2. title from Materials and methods.
Response:The genomic DNA of strains or soil samples listed in Table 1 and Table 2 were extracted, purified and recovered successfully.
12) 3.5 “spp.”Not in italic
Response: The word was corrected.
13) 3.6 “Sclerotinia”Should be written in italic
Response: The word was corrected.
14）Table 4 “Sclerotinia”Should be Not in italic
Response: The word was corrected.
15）Reference 24 is not refer on soil-borne pathogen: Quantification of airborne spores of Monilinia fructicola in stone fruit 453 orchards of California using real-time
Response: The reference 24 was deleted.
16）P390“Sclerotinia”Should be written not in italic
Response: The word was corrected.
Author Response File: Author Response.pdf
Reviewer 3 Report (New Reviewer)
Methodology authors described in too much detail buying processes of the equipment, please shorten this part by giving the producer's name in brackets.
The authors described in detail real-time PCR and in the methodology started using the current name for this method: qPCR - please explain the name and use a unified name for the method.
line 169 and 302- is QPCR- SHOULD BE qPCR
line 314 - is "rot-inflicted"- infected???
some words are written in red - why?
Author Response
Dear Prof.,
Thank you for your comments concerning our manuscript entitled” Measuring pathogenic soil fungi that cause Sclerotinia rot of Panax ginseng using real-time fluorescence quantitative PCR” (ID: agriculture-2495390). The comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the resubmitted paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.
Best wishes!
Baohui Lu
Point to Point Responds (The modifications have been highlighted in red in the revised manuscript):
Responds to the reviewer’s comments
Reviewer #3:
1）Methodology authors described in too much detail buying processes of the equipment, please shorten this part by giving the producer's name in brackets.
Response: Methodology described was shorten.
2) The authors described in detail real-time PCR and in the methodology started using the current name for this method: qPCR - please explain the name and use a unified name for the method.
Response: Real-time PCR and qPCR were the same, we have corrected in the submission.
3) line 169 and 302- is QPCR- SHOULD BE qPCR
Response:“QPCR” was corrected to “qPCR”
4）line 314 - is "rot-inflicted"- infected???
Response: "rot-inflicted" have corrected to“infected”.
5）some words are written in red - why?
Response: We have previously submitted a manuscript and have requested to answer and mark the previous questions one by one for resubmission.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report (New Reviewer)
The author has made extensive revisions to the paper, and I personally believe that the current status of the article can be published in this journal.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
Major:
1) I recommend that the authors should use some help of a native English speaker or send the Ms to an English Editing Service that proofreads scientific writing.
2) Authors should improve section “3.1 Design and synthesis of specific primers”, e.g. include information about “common” gene used for A2001F/A2001R and q2001-F/R primes design, also include (as supplementary file) alignment of this gene of all used Sclerotinia species.
3) Why did the authors not use DNA from the plant material (roots, leaves of plants) in PCR?
4) Authors should shorten the abstract.
5) Is the absence of a signal in the 38th and 39th samples not a false negative result (Table 4)? For example, because of the quality of DNA isolation or the presence of polymerase inhibitors.
6) Authors should improve the description for Table 4, e.g. there are two explanation for “-”, but I do not understand differences between “+”, “++”, “+++”, “++++”.
7) Authors should decrease the figure number, e.g. by combination Fig. 1 and 2; 5 and 6; 7 and 8.
8) Authors should improve the Fig. 3(a,b), 5 (a,b), and 8 (a,b), e.g. quality for all and
in 3a, 5a, 8a – arrow for ddH2O, other pathogens – after 25 cycle;
in 3b, 5b, 8b – separate the lines for “ddH2O, other pathogens” and for “Sclerotinia sp.”.
Minor:
9) Line 118: “manufacturer’s instructions” – which manufacturer? In general, it is better to insert information about the manufacturers of reagents and equipment directly into those chapters where these reagents and equipment are mentioned.
10) Line 120: “S. ginseng YC5 was” – in Table 1 “YC5” is S. nivalis.
11) Line 130-131: “0.5 µL genomic DNA” – concentration?
12) Line 311: “sclerotium” correct to “mycelium”.
Author Response
Dear Prof.,
Thank you for your comments concerning our manuscript entitled” Measuring pathogenic soil fungi that cause Sclerotinia rot of Panax ginseng using real-time fluorescence quantitative PCR”(ID: agriculture-2252255). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.
Best wishes!
Baohui Lu
Author Response File: Author Response.pdf
Reviewer 2 Report
The scientific paper entitled: "Measuring pathogenic soil fungi that cause Sclerotinia rot of Panax ginseng using real-time fluorescence quantitative PCR" is a well-prepared work.
Sclerotinia ginseng is a major soil-borne pathogen that causes one of the most significant diseases of ginseng. In the case of monoculture cultivation, the soil is contaminated with hyphae of the pathogen and saturated with sclerotia. The development of a diagnostic method suitable for the quantitative determination of the pathogen and its persistent formulas is very important from the point of view of the planning of defenses and the prediction of plant protection problems.
The authors compared the real time PCR methodology with traditional PCR diagnostics. Limit values for effective detection were determined, and Sclerotinia specific primers were tested and selected.
The methodological descriptions are adequate and enable similar developments for others. The set control tests are a sufficient guarantee for the reliability of the results.
Their results are convincing and also new in the world regarding soil-infecting ginseng fungi.
In order to increase the professional value of the manuscript, I suggest a few corrections in the manuscript:
In line 54 - in the case of F. sambicinum, I recommend writing the full scientific name. In the same line, I consider the term used unnecessary.
In line 60 - In the case of F. brasiliense, I also recommend writing the full scientific name.
In lines 244 and 253, the spelling of Helianthus annuus is correct.
After making the changes suggested above, I recommend publishing the manuscript in the form of a scientific article.
Author Response
Dear Prof.,
Thank you for your comments concerning our manuscript entitled” Measuring pathogenic soil fungi that cause Sclerotinia rot of Panax ginseng using real-time fluorescence quantitative PCR”(ID: agriculture-2252255). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.
Best wishes!
Baohui Lu
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Major:
1) “2) Authors should improve section “3.1 Design and synthesis of specific primers”, e.g. include information about “common” gene used for A2001F/A2001R and q2001-F/R primes design, also include (as supplementary file) alignment of this gene of all used Sclerotinia species.
Response: Section “3.1 Design and synthesis of specific primers” was improved.”
- I have not found a significant explanation and the requested figures in the new manuscript (Ms) text.
2) “3) Why did the authors not use DNA from the plant material (roots, leaves of plants) in PCR?
Response: Sclerotinia ginseng is a soil-borne disease, which is difficult to observe in the soil, so it needs to be detected by molecular biological methods; If Sclerotinia ginseng infects plant tissue, we can easily observe the change of symptoms.”
- Are the symptoms of the “Sclerotinia rot” disease unique or similar to other diseases? The authors should explain this in detail in the Ms text.
Minor:
3) Line 45: “and C. carunculoides”, “and” not in italic.
4) Line 125-126, 138: “panax ginseng” correct to “P. ginseng”.
Author Response
Dear Prof.,
Thank you for your comments concerning our manuscript entitled” Measuring pathogenic soil fungi that cause Sclerotinia rot of Panax ginseng using real-time fluorescence quantitative PCR”(ID: agriculture-2252255). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.
Best wishes!
Baohui Lu
Responds to the reviewer’s comments (The modifications have been highlighted in red in the revised manuscript):
Reviewer #1:
1) “2) Authors should improve section “3.1 Design and synthesis of specific primers”, e.g. include information about “common” gene used for A2001F/A2001R and q2001-F/R primes design, also include (as supplementary file) alignment of this gene of all used Sclerotinia species.
Response: Section “3.1 Design and synthesis of specific primers” was improved.
2) “3) Why did the authors not use DNA from the plant material (roots, leaves of plants) in PCR?
Response: Sclerotinia ginseng is a soil-borne disease, which is difficult to observe in the soil, so it needs to be detected by molecular biological methods; If Sclerotinia ginseng infects plant tissue, we can easily observe the change of symptoms.”
- Are the symptoms of the “Sclerotinia rot” disease unique or similar to other diseases? The authors should explain this in detail in the Ms text.
Response: It was explained in detail in the “introduction” of the Ms text. “The sclerotia load in soil impacts the occurrence, development, and prevalence of Sclerotinia rot and when it reaches a certain level, the soil becomes unsuitable for planting ginseng. Thus, quantitative measurement of the fungal sclerotia that cause Sclerotinia rot of ginseng is critical for disease prediction and prevention.”
When the conditions are favorable in the early spring, the mycelium that germinates from the sclerotia causes infestation, allowing it to spread to adjacent ginseng plants. It caused ginseng root soft and rot, and it could be easily found by eyes. Soil DNA testing was done to detect pathogens before they infect ginseng plants.
3) Line 45: “and C. carunculoides”, “and” not in italic.
Response: It was modified in Ms text.
4) Line 125-126, 138: “panax ginseng” correct to “P. ginseng”.
Response: It was modified in Ms text.
