The Dark Septate Endophytes and Ectomycorrhizal Fungi Effect on Pinus tabulaeformis Carr. Seedling Growth and their Potential Effects to Pine Wilt Disease Resistance
Round 1
Reviewer 1 Report
The authors present a series of experiments to test whether dark septate endophytic fungi and ectomycorrhizal fungi affect the susceptibility of Pinus tabulaeformis seedlings to the nematodes that cause pine wilt disease. The experiments used seemed appropriate to test the hypotheses and the study is scientifically relevant.
I have made a number of edits and suggestions on the PDF file attached. Most are related to interpretation of the text as there were multiple instances where it was difficult to understand or that need better explanation. I will look forward to your revised manuscript.
Comments for author File: 
Comments.pdf
Author Response
Responses to Reviewer 1:
The authors present a series of experiments to test whether dark septate endophytic fungi and ectomycorrhizal fungi affect the susceptibility of Pinus tabulaeformis seedlings to the nematodes that cause pine wilt disease. The experiments used seemed appropriate to test the hypotheses and the study is scientifically relevant.
Answer: Thanks very much for your approval on the scientific merit of our work and suggestions! The amendments are marked using the "Track Changes" function in Microsoft Word in the revised manuscript. The attachment is the revised manuscript.
I have made a number of edits and suggestions on the PDF file attached. Most are related to interpretation of the text as there were multiple instances where it was difficult to understand or that need better explanation. I will look forward to your revised manuscript.
Answer: Thanks for your insightful comments and kind advices. We've revised the manuscript according to your suggestions. The following are the point by point responses to your comments.
1. Wouldn't it be cumulative (mortality and morbility up to the end of the experiment)? Please revise and if necessary, change throughout the manuscript. (Page1, Line25)
Answer: Yes, we've changed it to cumulative throughout the manuscript.
2. If you have information on the scope of the economic and environmental impact, please include and cite. Also, which countries are most affected? (Page1, Line42)
Answer: We have revised it. (Page1 and 2, Line43-45)
3. This sentence is incomplete and not very clear. Is it supposed to be joined with the next sentence?
I have made some edits to these two sentences. Please revise to check whether this is the message you intend to convey. (Page2, Line43-53)
Answer: We've revised these according to your advice. (Page2, Line45-56)
4. Citation is needed to provide evidence that Pinaceae trees were obligate to ECMF. (Page2, Line54)
Answer: We've added a citation. (Page2, Line57)
5. plant health? metabolism?
It is not clear from this sentence what the tremendous effect are. Also, this sentence can be merged with the next so that the role of ECMF is better explained. (Page2, Line55-57)
Answer: We've revised it. (Page2, Line60)
6. mycorrhizal fungi?
This sentence is incomplete as it is. Please clarify. (Page2, Line61)
Answer: Yes, it is mycorrhizal fungi. We've revised it. (Page2, Line63-64)
7. It would make it easier to readers that are not familiar with these species if you indicate here which species are ECMF and which are DSE. (Page2, Line87-92)
Answer: We've revised it. (Page2, Line90-94)
8. Here it is not clear whether 5 disks of mycelium were each put in one 250 ml flask and 5 replicates were tested per strain or if one disk was put in one flask and 5 replicates were tested per strain. Please review the wording to explain this better. (Page3, Line97-99)
Answer: We've revised it. (Page3, Line101-102)
9. incubation plates or incubated plates? (Page3, Line107 and Line110)
The way it is written it seems like the plates were pre-incubated with something that is not indicated. Please clarify.
Answer: We've revised it. (Page3, Line109-110)
10. It is not clear whether the control group consisted of seedlings inoculated with a sterilized solution of all strains mixed together. This is what I was able to interpret from the text. Please clarify. (Page3, Line126-128)
Answer: The control group consisted of seedlings inoculated with a sterilized solution of all strains mixed together. (Page3, Line129)
11. What is this following experiment? Or is it the experiment you just described above? Please explain. The way the text is written it seems like there is a subsequent experiment that is not indicated. (Page3, Line129-L134)
Answer: We took 70 cups of uniform growth seedlings of each treatment out from 80 cups for the following experiment. Because from the appearance the growth seedlings of some treatments were not that uniform. These differences might be caused by the seed we used, for reducing the error caused by seed, we selected 70 cups of uniform growth seedlings of each treatment.
12. Here it would be good to indicate the P-values at which your tests were considered significant. P<0.05, P<0.01? (Page5, Line193)
Answer: We've revised it. (Page5, Line188)
13. This is not very clear. Were all comparisons significant at P<0.01? Or was there a variety of responses depending on the comparisons? Did you do all pairwise comparisons or only compared the treated vs the control? (Page5, Line207-209)
Answer: All comparisons significant at P < 0.05. We did all pairwise comparisons. We've corrected it. (Page5, Line203-204)
14. It would make it more clear for the reader if you indicate here which strains are ECMF and which are DSE. Maybe a new column to the left with ECMF, merging the rows corresponding to those species, control, and DSE with the merged rows for those species. It is well explained in the foot of the table, but it would make it much easier to read if they are indicated directly on the table. (Page6, Table 1)
Answer: Thanks for your good advice, we've revised it. (Page6, Line213-220)
15. See table 1. May incorporate additional column or indicate "see Table 1" if you do not wish to rewrite the text at the foot of the table. Since each figure and table should be self-explanatory, such information on strains, significant tests, etc, are lacking here. (P7, L16)
Answer: Thanks for your good advice, we've revised it. (Page7, Line238-243)
16. Here the legend title should be indicated for clarity. D is Diameter?
Also, it would also help to mention which species are ECMF and which are DSE. (Page8, Line18-24)
Answer: We've revised it. (Page7, Line246-252)
17. Explanatory text on the species, significance, etc. should be added at the bottom of the table. May make reference to Table 1 if you don't wish to rewrite everything, but may be a decision to consult with the editors. (Page8, Line31-32)
Answer: We've rewrote it. (Page8, Line259-266)
18. This is not clear. What is the meaning of respectively in this context? Were all the treatments combined and used for PCA?
Answer: We've corrected our expression. All the treatments experimental data sets were used for PCA. (Page8, Line268)
19. Information on the strains would be helpful to make the figure self-explanatory. (p9, L50)
Answer: We've revised it. (Page9, Line284-288)
20. substitute (day) for (days); Also, add strain information. (Page10, Line62-64)
Answer: We've substituted (day) for (days), and added strains information.
21. Explain what the asterisks mean. Please write in bold, as this is another title row.
Answer: We've explained what the asterisks mean. We've revised it. (Page10, Line313-314)
22. Please revise this last sentence. It is not very clear what you are trying to discuss. (Page11, Line95-97)
Answer: We've revised this sentence. (Page11, Line339-342)
23. Was this reduction in mortality significant with respect to the controls? (Page11, Line101)
Answer: Compared to the control, 30% reduction in mortality.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The paper covers an interesting and actual topic. Authors present interesting results. I acknowledge the tremendous and difficult work of the authors in a subject that is quite difficult to address. In this study, the authors have examined the influence of ECMF and DSE symbiosis on P. tabulaeformisinfected by PWN. The results showed that the growth seedlings and root morphology of P. tabulaeformisseedlings as well as their resistance to PWD varied according the ECMF/DSE species applied. The hypotheses and speculations are carefully identified.
Introduction
Within the introduction I think the part about the EMC is relatively short.
Some specific remarks:
Line 37: It is better to change keywords, because they are in the title. For example, ectomycorrhizal fungi – ectomycorrhizal colonization.
Line 53, 54, 63, 69, 74, 78: Please give ‘Space’.
Materials and Methods
The study is correctly designed, but need more details.
Some specific remarks:
Line 133-134: you need to provide additional details relating to your experimental design. Specifically - what do you mean be a ‘randomized complete block design’ (was this a randomized complete block design, with 4 blocks?). Please provide more details that describe the design of this experiment.
Line 165: Please clarify what you mean by saying‘colonisation rate’. You mean that roots were colonized by other fungal species and not only by mycorrhizal fungi? If yes, I believe that this should be expressed as percent of colonized roots and not percent of colonized plants.Please explain how vital ECM fungi and non-vital roots were distinguished or give a reference.
Line 175-177: Why is different number of seedlings for each group?
Results
The results provide an advance in current knowledge. The data and analyses are presented appropriately.
Some specific remarks:
Line 199: Where in section ‘Materials and Methods’ is information how did you investigate fungal colonisation? Only in line 165?
Line 1: ‘root morphological characters’ – maybe better ‘roots parameters’
Line 70 (Table 4): physiology parameters should be shown in bold.
Discussion
Nice ideas, connection of results to those of others. Please discuss future work that is needed in this particular field in order to be able to suggest applications of such findings in the future.
Line 108: Please give Space ‘pine[ ‘.
Line 110: ‘The’ should be ‘the’.
Conclusions
The conclusions are interesting for the readership of the Journal.
Author Response
Responses to Reviewer 2:
The paper covers an interesting and actual topic. Authors present interesting results. I acknowledge the tremendous and difficult work of the authors in a subject that is quite difficult to address. In this study, the authors have examined the influence of ECMF and DSE symbiosis on P. tabulaeformis infected by PWN. The results showed that the growth seedlings and root morphology of P. tabulaeformis seedlings as well as their resistance to PWD varied according the ECMF/DSE species applied. The hypotheses and speculations are carefully identified.
Answer: Thanks very much for your approval on the scientific merit of our work and suggestions!
The amendments are marked using the "Track Changes" function in Microsoft Word in the revised manuscript. In addition, the attachment is the revised manuscript.
Introduction
Within the introduction I think the part about the EMC is relatively short.
Answer: Thanks for your suggestion! We've done some revisions in the introduction part. Please see (Page 2, Line 57-80).
Some specific remarks:
Line 37: It is better to change keywords, because they are in the title. For example, ectomycorrhizal fungi – ectomycorrhizal colonization.
Answer: We've revised it. (page1, line 39-40)
Line 53, 54, 63, 69, 74, 78: Please give ‘Space’.
Answer: We've revised it.
Materials and Methods
The study is correctly designed, but need more details.
Answer: Thanks for your suggestion. We've made some revisions. (page 3, line 94-136)
Some specific remarks:
Line 133-134: you need to provide additional details relating to your experimental design. Specifically - what do you mean be a ‘randomized complete block design’ (was this a randomized complete block design, with 4 blocks?). Please provide more details that describe the design of this experiment.
Answer: We took 70 cups of uniform growth seedlings of each treatment out from 80 cups for the following experiment. Because from the appearance the growth seedlings of some treatments were not that uniform. These differences might be caused by the seed we used, for reducing the error caused by seed, we selected 70 cups of uniform growth seedlings of each treatment.
‘Randomized complete block design’ in the paper means that all the seedlings of different treatments were arranged randomized. We want to reduce interference caused by human factors.
Line 165: Please clarify what you mean by saying‘colonisation rate’. You mean that roots were colonized by other fungal species and not only by mycorrhizal fungi? If yes, I believe that this should be expressed as percent of colonized roots and not percent of colonized plants. Please explain how vital ECM fungi and non-vital roots were distinguished or give a reference.
Answer: The "colonization rate" means the percentage of the root length colonized by fungus in the root length we measured. The detailed method for measuring mycorrhizal colonization rate was in " Phillips, J.; Hayman, D. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 1970, 55, 158-IN118. and Giovannetti, M.; Mosse, B. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 1980, 84, 489-500."
Line 175-177: Why is different number of seedlings for each group?
Answer: The group with 50 seedlings of each treatment was used for measuring the cumulative mortability and mortality after PWN inoculation. The more seedlings of the group, the more accurate the result. While the left 5 seedlings were enough for contrast. Considering the tremendous total works we need to do, we just took 50 seedlings for measuring cumulative mortability and mortality and took 5 seedlings as a contrast of each treatment.
Results
The results provide an advance in current knowledge. The data and analyses are presented appropriately.
Answer: Thanks for your approval on the results.
Some specific remarks:
Line 199: Where in section ‘Materials and Methods’ is information how did you investigate fungal colonisation? Only in line 165?
Answer: We investigate the fungal colonization according to " Phillips, J.; Hayman, D. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 1970, 55, 158-IN118. and Giovannetti, M.; Mosse, B. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 1980, 84, 489-500." It's a common technique in studying mycorrhizal.
Line 1: ‘root morphological characters’ – maybe better ‘roots parameters’
Answer: Thanks for your good advice. We've corrected it. (page 6, line 222)
Line 70 (Table 4): physiology parameters should be shown in bold.
Answer: We've corrected it. (page 10, line 313-314)
Discussion
Nice ideas, connection of results to those of others. Please discuss future work that is needed in this particular field in order to be able to suggest applications of such findings in the future.
Answer: Thanks for your approval on the discussion.
Line 108: Please give Space ‘pine[ ‘.
Line 110: ‘The’ should be ‘the’.
Answer: We've corrected them. (page 11, line 353 and line 356)
Conclusions
The conclusions are interesting for the readership of the Journal.
Answer: Thanks for your approval on the conclusions.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Thank you for making the corrections and addressing the questions I had during my previous review. The manuscript is much improved and I think that there are only some minor details left to correct and it will be ready for publication.
I am attaching a file with some itemized edits.
