From Lab to Nursery: Novel Approaches of Seed Disinfection for Managing Pine Pitch Canker Propagation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments:

The study presented by Fernandes et al. recommend preventive measures for Pine Pitch Canker outbreaks in nurseries anud, consequently, in forests. In this study are comparing using four different treatments. The study is well designed and the experiments are thoroughly conducted, excellently explained and give cheap benefit exspecialy for nurseries and forests with hot water disinfection of seeds. There are several opportunities for significant improvement in MS before it is approved for publication in Forests MDPI. Mainly afer added part discussion.

1.     In article is not discussion. It is written together with Results, but in not discuss realy. I am recommending write results and discussion separately. You have only 12 articles (therefrom 8 is only report potential Pinus pathogens). Next 4 articles are only statements, but are not discussion.

One of statement is not correct. Line 325-326. „The hot water treatments results are consistent with previous studies [26,27], with the highest temperature of 60˚C yielding the best results.“ In study Agustí-Brisach, C. et al, 2012 recommended the best temperature 51-52 ˚C for 30 minutes. This study you have to more detaily and mainly correct discuss.

2.     This study is refer for your previously published work in conclussion. Which publication do you mean? Missing reference and discussion too. Please detaily compare in stis study.

Author Response

Dear Reviewer,

On behalf of all authors, I would like to thank you for your insightful comments and suggestions that contribute to improve the quality of the manuscript.

Below is an item-by-item response to the specific comments made in the review page:

The study presented by Fernandes et al. recommend preventive measures for Pine Pitch Canker outbreaks in nurseries anud, consequently, in forests. In this study are comparing using four different treatments. The study is well designed and the experiments are thoroughly conducted, excellently explained and give cheap benefit exspecialy for nurseries and forests with hot water disinfection of seeds. There are several opportunities for significant improvement in MS before it is approved for publication in Forests MDPI. Mainly afer added part discussion.

1. In article is not discussion. It is written together with Results, but in not discuss realy. I am recommending write results and discussion separately. You have only 12 articles (therefrom 8 is only report potential Pinus pathogens). Next 4 articles are only statements, but are not discussion.

The authors thank the reviewer for their comment. We have opted to maintain Results and Discussion as a single section. However, the discussion of our results was significantly deepened and improved, with further comparisons to other research.

One of statement is not correct. Line 325-326. „The hot water treatments results are consistent with previous studies [26,27], with the highest temperature of 60˚C yielding the best results.“ In study Agustí-Brisach, C. et al, 2012 recommended the best temperature 51-52 ˚C for 30 minutes. This study you have to more detaily and mainly correct discuss.

This statement has been rewritten to be more accurate. Further details of this study and comparisons between it and our own work were also added.

2. This study is refer for your previously published work in conclussion. Which publication do you mean? Missing reference and discussion too. Please detaily compare in stis study.

References to previously published work were added. Additionally, Conclusions section was improved to further discuss the impact of our work. However, considering that the other works focused on other potential sources of Fusarium circinatum contamination, and used different treatments, the results were not comparable.

Reviewer 2 Report

Comments and Suggestions for Authors

The current study deals with the application of different Pinus seed disinfection methods that are relevant for managing pine pitch canker. Although the methods that the authors selected are innovative and promising, and the authors gathered a large amount of data, the study is poorly written and discussed.

Introduction needs to be reorganized. There is need to deeper display the gap of knowledge that the study indents to fill. The aim of the study must be more specific, including what kind of treatments you analyzed and why did you choose these treatments. A paragraph on the treatments used in this study must be written with already existing scientific knowledge, gaps, and novelty. Hypotheses are also missing. The paragraph of the importance of Pinus species in Portugal is too long, please concentrate more on the experiments you did.

Line 45. “Pine” should be in a lowercase.

Line 97. “in 27 locations across the country”. The word “location” needs to be specified.

Materials and methods

Line 120. Do not write “which was a collaborating partner in the project”. Instead of this, please write more information about the seeds, such as parental trees’ age and health. Did the parental pines have symptoms of F. circinatum?

Lines 127-129. Although the manuscript is a part of the project, you do not need to write manifestations such as “partner in this project” or “our laboratory”. You all are in one team with all the project partners. Please correct this throughout the entire manuscript.

Lines 133-136. Specify treatments’ concentrations and temperatures of hot water boundaries with the link to table 1.

Lines 137-161. This is a theoretical not a methodological part and cannot be written in the section of Materials and Methods.

Table 1. The name of the column “Concentration” must be supplemented with “Temperature”.

Line 171. Were the seeds drying at room temperature?

186-187. Be more specific. Describe what is in the figures and separate them by a, b, and c. The word “Snapshots” is not appropriate, please change it.

Lines 203-230. The process of bioinformatics is gapping.

Lines 235-237. Explain why the seeds of P. pinea and P. radiata were unavailable for nursery germination trials.

Lines 237-239. Do not emphasize the importance of P. pinaster in the methodology section.

Line 246. Specify the parameters displayed in Table 2, instead of writing “Summary”. Why do all words start with a capital letter if in previous Table 1 they were in lowercase?

Lines 248-250. Not a methodological part.

Lines 273-274. The same comment as in Figure 1.

Line 278. Italicize P. pinaster.

The section of Results and Discussion were written as an experimental report, and deeper discussion for a scientific paper is needed. Discussions merely present few points from other researches. You need to compare your study with previous scientific knowledge in prevention of F. circinatum in forests.

Line 297. Italicize Fusarium circinatum and Pinus.

Lines 330-336. Repeated information.

Lines 338-340. It is a methodological part.

Lines 341-355. There is missing a table or a visualization for these results.

Lines 359-361. It is a methodological part.

Line 362. Italicize Pinus pinaster.

Line 364. “according to a Tukey’s pairwise test (p < 0.05)”. It is a methodological part.

Line 366. You cannot write “slightly lower” if there is not a significant difference.

Lines 368-369. "The highest" without a significance does not give us significant data.

Line 382. Italicize Pinus pinaster.

Lines 411-414. Please do not describe a germination rate as higher or lower between any variables if the results are not significant.

For the statistical review, please display all the numbers of your experiment in supplementary material. 

Comments on the Quality of English Language

Proofreading for scientific English, grammar and sentence construction, is mandatory.

Author Response

Dear Reviewer,

On behalf of all authors, I would like to thank you for your insightful comments and suggestions that contribute to improve the quality of the manuscript.

Regarding the suggestions of “Moderate editing of English language required” and “Proofreading for scientific English, grammar and sentence construction, is mandatory.” made by the reviewer, the authors had the manuscript be proofread by a native English speaker who corrected certain minor inconsistencies and ensured the comprehensibility of the manuscript.

Below is an item-by-item response to the specific comments made, both in the review page and the manuscript itself:

The current study deals with the application of different Pinus seed disinfection methods that are relevant for managing pine pitch canker. Although the methods that the authors selected are innovative and promising, and the authors gathered a large amount of data, the study is poorly written and discussed.

Introduction needs to be reorganized. There is need to deeper display the gap of knowledge that the study indents to fill. The aim of the study must be more specific, including what kind of treatments you analyzed and why did you choose these treatments. A paragraph on the treatments used in this study must be written with already existing scientific knowledge, gaps, and novelty. Hypotheses are also missing. The paragraph of the importance of Pinus species in Portugal is too long, please concentrate more on the experiments you did.

The authors appreciate the reviewer’s comment.

In the case of the organization of the Introduction section, we would  like to explain that our rationale was the following: presenting the Fusarium circinatum pathogen and the problems it causes, explaining the importance of Pinus species in Portugal and the potential threat of pine pitch canker in our country, reviewing the current efforts to mitigate the spread of this disease and finally exposing how our project came to be considering the points mentioned above and further explaining the goals of our work. Nonetheless, small adjustments were made to the introduction to make this clearer.

 The lines explaining the aim of this study were changed to make it clearer. Regarding the reviewer’s comment concerning the choice of treatments, the authors have opted to maintain this information in the Materials and Methods section. The novelty of our work was not in the treatments themselves, but their application in this context (against Fusarium circinatum and in a way that is possible for nursery workers to perform as needed).

The authors feel that the paragraph regarding the importance of Pinus forestry in Portugal is necessary to reinforce the contextualization and importance of not just this work, but the entire project where it is included.

Line 45. “Pine” should be in a lowercase.

Suggested change was made.

Line 97. “in 27 locations across the country”. The word “location” needs to be specified.

Locations were further specified in response to the reviewer’s comment, however the authors cannot further elaborate on the specifics of the locations due to privacy concerns.

Materials and methods

Line 120. Do not write “which was a collaborating partner in the project”. Instead of this, please write more information about the seeds, such as parental trees’ age and health. Did the parental pines have symptoms of F. circinatum?

Suggested change was made.

Lines 127-129. Although the manuscript is a part of the project, you do not need to write manifestations such as “partner in this project” or “our laboratory”. You all are in one team with all the project partners. Please correct this throughout the entire manuscript.

Suggested change was made and the entire manuscript was reviewed and corrected to be in line with the reviewers suggestion.

Lines 133-136. Specify treatments’ concentrations and temperatures of hot water boundaries with the link to table 1.

Suggested change was made.

Lines 137-161. This is a theoretical not a methodological part and cannot be written in the section of Materials and Methods.

The authors appreciate the reviewer’s comment. However, we decided to keep the structure as it was, considering that it concerns mostly technical information regarding the treatments and furthermore, the treatments fungicidal activity is not new, and as such, not the focus of our work, which focuses rather on their application in a real nursery context. Nonetheless, further information regarding past uses of the treatments (or their active compounds) as fungicides was added.

Table 1. The name of the column “Concentration” must be supplemented with “Temperature”.

Suggested change was made.

Line 171. Were the seeds drying at room temperature?

They were. Text was changed to make this fact clearer.

186-187. Be more specific. Describe what is in the figures and separate them by a, b, and c. The word “Snapshots” is not appropriate, please change it.

Text accompanying Figure 1 was changed to properly describe each of the displayed steps.

Lines 203-230. The process of bioinformatics is gapping.

Further details of the bioinformatics process are provided in the text.

Lines 235-237. Explain why the seeds of P. pinea and P. radiata were unavailable for nursery germination trials.

The nursery experimental period took place during the COVID-19 pandemic, which delayed several processes. This led to low seed stock of these species and the need to ensure seed availability for nurseries and private stakeholders outside of the context of the project. With that, only P. pinaster seeds were available in enough quantity to be used in the nursery trials and the in vitro germination assays.

Lines 237-239. Do not emphasize the importance of P. pinaster in the methodology section.

The importance of this species in the context of the Portuguese forest was already mentioned in the Introduction section, with this short note serving only as an explanation of why these assays were still carried out, in spite of the unavailability of other species.

Line 246. Specify the parameters displayed in Table 2, instead of writing “Summary”. Why do all words start with a capital letter if in previous Table 1 they were in lowercase?

Suggested change was made. Additionally, the capitalization of words was adjusted to be in line with the rest of the paper.

Lines 248-250. Not a methodological part.

The authors appreciate the reviewer’s comment. However, while it is not a methodological part, it gives important context for the certification process. We believe this to be useful for the readers and as such we’ve decided to maintain this paragraph. The authors hope that the reviewer understands our point of view.

Lines 273-274. The same comment as in Figure 1.

Text accompanying Figure 2 was changed to properly describe each of the displayed steps.

Line 278. Italicize P. pinaster.

Suggested change was made.

The section of Results and Discussion were written as an experimental report, and deeper discussion for a scientific paper is needed. Discussions merely present few points from other researches. You need to compare your study with previous scientific knowledge in prevention of F. circinatum in forests.

Line 297. Italicize Fusarium circinatum and Pinus.

Suggested change was made.

Lines 330-336. Repeated information.

The authors thank the reviewer’s comment. Changes were made to ensure that the required information was still presented in this section without repeating what had been previously said in previous sections of the manuscript.

Lines 338-340. It is a methodological part.

The authors appreciate the reviewer’s comment. Text in question was removed from this section.

Lines 341-355. There is missing a table or a visualization for these results.

A table containing all this data was constructed and can be consulted in the supplementary materials. Additionally, ITS sequences of the non-Fusarium circinatum isolates were deposited at GenBank, with this data being record in the aforementioned table. Furthermore, some isolates identification was updated to accurately depict their current scientific names. Current identification and synonymity are also presented in the supplementary table S2.

Lines 359-361. It is a methodological part.

The authors thank the reviewer’s comment. However, we feel that this sentence isn’t referring to the methodology itself, but rather to put the results shown in Table 3 in context. As such, we’ve decided to maintain this paragraph, with slight changes to better convey our intention. We hope the reviewer understands our point of view.

Line 362. Italicize Pinus pinaster.

Suggested change was made.

Line 364. “according to a Tukey’s pairwise test (p < 0.05)”. It is a methodological part.

The authors acknowledge the reviewer’s comment and corrected the text accordingly. Nonetheless, we have decided to maintain the information regarding the p value.

Line 366. You cannot write “slightly lower” if there is not a significant difference.

The authors appreciate the reviewer’s comment. Sentence in question was corrected.

Lines 368-369. "The highest" without a significance does not give us significant data.

The authors thank the reviewer’s comment. Sentence was rewritten as to not include comparisons when data was significantly similar.

Line 382. Italicize Pinus pinaster.

Suggested change was made.

Lines 411-414. Please do not describe a germination rate as higher or lower between any variables if the results are not significant.

The authors thank the reviewer’s comment. Sentence was rewritten as to not include comparisons when data was significantly similar.

For the statistical review, please display all the numbers of your experiment in supplementary material.

The authors thank the reviewer for this comment. Tables containing all raw data used for statistical analysis are available as supplementary material. Furthermore, thanks to the reviewer’s comment, the authors were able to detect a minor mistake in one of the graphs, which was corrected, and for that we are grateful.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors considered all the previous comments and made the necessary corrections based on them. The manuscript is scientifically arranged, easy to follow, and demonstrates scientific novelty.