Nematicidal Properties and Chemical Composition of Pinus rigida Mill. Resin against Pinewood Nematodes
Round 1
Reviewer 1 Report
General
The manuscript titled „Nematicidal properties and chemical composition of Pinus rigida resin against pine wood nematodes” studied the toxic effects of pine resin on nematodes in several points of view and methods. The work substantially explored the issue and it is well-written. I have only minor questions and suggestions.
Details
Abstract
Abstract should have the form of a typical article with introduction, methods, results and conclusions. Lines 10-11, 13, 16-17, 22-23 are about methods. They should be described together and then results should be written together.
Introduction
line 32: year of description and the describer.
lines 42-43: Reference?
lines 46-47: When was first reported? In 1913?
lines 86-91. This is the Introduction. There is no need to write about the results and conclusions. Here should be summarized the point of the study. What did you study, what were the questions or hypotheses?
Material and methods
line 94: Botrytis cinerea. Please refer to the study of culturing these nematodes. (If it is the article of Togashi et al. 2003, I couldn’t reach it on the internet.)
lines 99: Other sizes of the stem segments should be added (width: cm). Were these stems freshly taken?
line 140: P. rigida
line 138: only P. rigida? What about the other two pine species?
line 146. I missed here the explanation, why the control was needed. Later, in the section Discussion I found the answer in lines 323-324, but I think it would be useful to explain also here that it was important to measure the stilbene values without pathogen attack.
line 160. In triplicate and repeated in three times means that there were 9 samples for each treatment?
Results
Figures 1, 2, 3, 6: I miss the numbers of repetitions, n=3?
lines 175-181 These are parts for introduction.
lines 181, 183: I would add the SE values for the percent values.
line182 and 183: 62+39≠100%
line 185-186: Were the nematode specimens staying in the resin of P. densiflora and P. koraiensis also died?
line 183: I would make it clear that “outside of the resin” means that these nematodes moved on the surface of the tube (not in a liquid above the resin). (It appeared also later in the discussion section but it was confusing to me.)
Figure 2: Label of y axis: Is this specimen number or percent of specimens?
Figure 3: P. densiflora, P koraiensis, P. rigida
line 212. for P. rigida
Table 1: In the legend, it should be mentioned what the bold characters/numbers indicate. Are these values average values? There were three replicates, didn’t they have three values? Then they should have SE as well?
lines 242-244. These are method descriptions.
Figure 5E: what about the orange part?
Discussion
lines 281-285, 292-296: repetitions…
Section 4.3. These are only the repetitions of the results. You should interpret all these results. For instance, what about these chemicals and their effects on nematodes or other animals?
Author Response
General
The manuscript titled ”Nematicidal properties and chemical composition of Pinus rigida resin against pine wood nematodes” studied the toxic effects of pine resin on nematodes in several points of view and methods. The work substantially explored the issue and it is well-written. I have only minor questions and suggestions.
Answer: We appreciate your valuable comments for our manuscript.
Details
Abstract
Abstract should have the form of a typical article with introduction, methods, results and conclusions. Lines 10-11, 13, 16-17, 22-23 are about methods. They should be described together and then results should be written together.
Answer: We revised the format of Abstracts as suggested.
Introduction
line 32: year of description and the describer.
Answer: The description and the describer is Jones et al. (2013).
lines 42-43: Reference?
Answer: We inserted a reference in text.
lines 46-47: When was first reported? In 1913?
Answer: Yes, to avoid confusion, we deleted a word, Asia.
lines 86-91. This is the Introduction. There is no need to write about the results and conclusions. Here should be summarized the point of the study. What did you study, what were the questions or hypotheses?
Answer: We revised the last phrase of Introduction.
Material and methods
line 94: Botrytis cinerea. Please refer to the study of culturing these nematodes. (If it is the article of Togashi et al. 2003, I couldn’t reach it on the internet.)
Answer: We added new reference for the information of culturing nematodes.
lines 99: Other sizes of the stem segments should be added (width: cm). Were these stems freshly taken?
Answer: We modified the sentence.
line 140: P. rigida
Answer: We deleted the section.
line 138: only P. rigida? What about the other two pine species?
Answer: We deleted the section.
line 146. I missed here the explanation, why the control was needed. Later, in the section Discussion I found the answer in lines 323-324, but I think it would be useful to explain also here that it was important to measure the stilbene values without pathogen attack.
Answer: The stilbene values are gained without pathogen attack.
line 160. In triplicate and repeated in three times means that there were 9 samples for each treatment?
Answer: yes, all experiments were conducted in triplicate, and repeated three times
Results
Figures 1, 2, 3, 6: I miss the numbers of repetitions, n=3?
Answer: yes, all experiments were conducted in triplicate, and repeated three times
lines 175-181 These are parts for introduction.
Answer: We deleted the sentences of 176-178.
lines 181, 183: I would add the SE values for the percent values.
Answer: We added the SE values.
line182 and 183: 62+39≠100%
Answer: The mistake is corrected as 61+39.
line 185-186: Were the nematode specimens staying in the resin of P. densiflora and P. koraiensis also died?
Answer: The nematodes immersed in the P. densiflora and P. koraiensis resins showed mobility until after 3 days of culture.
line 183: I would make it clear that “outside of the resin” means that these nematodes moved on the surface of the tube (not in a liquid above the resin). (It appeared also later in the discussion section but it was confusing to me.)
Answer: In natural condition, resin is secreted with some exudates containing waters. Moreover, PWNs should be isolated from liquid medium. Thus PWN dipping in resin in tube can make high humidity condition in upper aerial part of tube, which may permit the movement in aerial part of tube.
Figure 2: Label of y axis: Is this specimen number or percent of specimens?
Answer: the label is the percent of PWNs
Figure 3: P. densiflora, P koraiensis, P. rigida
line 212. for P. rigida
Table 1: In the legend, it should be mentioned what the bold characters/numbers indicate.
Answer: The bold characters/numbers indicate P. rigida resin-specific compounds.
Are these values average values? There were three replicates, didn’t they have three values? Then they should have SE as well?
Answer: The values of peak area are mean of three replicates.
lines 242-244. These are method descriptions.
Answer: We deleted a sentence.
Figure 5E: what about the orange part?
Answer: We corrected the Figure E.
Reviewer 2 Report
The paper under review describes the effects of resins derived from three pine species (Pinus. densiflora, P. koraiensis, and P. rigida) on the pinewood nematode, Bursaphelenchus xylophilus. Pinus rigida is known to be resistant to the pinewood nematode. Therefore, it is very interesting that the resin components of P. rigida suppressed the behavior of nematodes and had a lethal effect on nematodes. However, there is a major problem with this paper that no statistical analysis has been performed in any of the experiments. Since scientific papers require discussions based on the results of statistical analysis, it is essential to carry out statistical analysis in this study as well. Results and discussion should be reconstructed after conducting statistical analysis in each experiment. Major revisions are required to be published in this journal.
Specific comments are as follows:
--------------------------------------------------------------------
(Introduction)
L57:
Sugar => Suga
L81-82:
“Although PWN migration… migrate in P. rigida (Son et al. 2014)”
The sentence is strange.
(Materials and Methods)
L93:
Pine wood nematode (PWN) => PWN
L94:
PWNs (Bursaphelenchus xylophilus) => PWNs
L96-97:
Togashi et al. (2003) is not suitable as a citation of the Baermann funnel technique. There are many papers that explain this method in English (e.g. Hooper (1986)).
Baerman => Baermann
L98-106:
Information on the branch thickness (diameter) is needed because there may be some relationship between the thickness of branches and the number (area) of resin canals. The diameter of hole made to inoculate the nematodes should also be shown here. How many repetitions in this experiment?
L107-113:
How did you collect the resins from each pine trees? A detail description is needed.
“values in all data…standard error (SE)” => the sentence should be moved to the legend of figure 2.
L114-121:
How many nematodes are used in this experiment?
““values in all data…standard error (SE)” => the sentence should be moved to the legend of figure 3.
L134-135:
“Compounds presented as … 0.05% are indicated” => the sentence should be moved to the legend of table 1.
L137-146:
Where is the result of this experiment? I could not find it in Result section.
L156:
He et al. (2017) => this paper is not listed in Reference section.
L161-163:
Statistical analysis is necessary in experiments 2.3, 2.4, 2.5, and 2.7. Statistical method used in each experiment should be explained here.
(Results)
L165-170 (Figure 1):
It is recommended to show the total number (including repetition) rather than the average ratio of nematodes in each segment in figure1. Then, it may be better to analyze by chi-square test.
L168:
“after 3 days” => after 1 day? (L104)
L175-181:
“Generally, …small PCR tube.”.
These sentences are not explanations of the results. Not necessary.
L181-192:
The result of statistical analysis should be described here. In figure 2A, it is difficult to distinguish between the area inside or outside resin. Is it better to show difference in color between a and b? In figure2B, the result of statistical analysis should be shown. Chi-square test may be effective.
L190:
“during 6 days => during 3 days? (L111)
L193-203: (Figure 3)
It should be clarified whether the difference in the progress of mortality rate of nematodes among three pinus species is statistically significant.
L205-208:
“Because the resins… pine plants by GC-MS”
This sentence isn’t result of the experiment. Not necessary.
L217:
“peak numbers 25 and 29” => peak numbers 23 and 28?
L224-225: (figure 5E)
To state that the amounts of two substances in bark and resin was higher than those in leaf and xylem, statistical analysis is needed. There is no description of the orange bar (Figure 5E).
L241-251: (figure 6)
Similar to above, to state the strength of PWN toxicity in cis-3,5-dimethoxystilbene, statistical comparison is necessary.
(Discussion)
L281-285:
“We tested… were inoculated in the resin.”
The description about the experimental method is already in M & M. These sentences are not necessary.
L292-295:
“To investigate the resin toxicity … and P. rigida trees”
The sentence is also unnecessary for the same reason as above.
L313-314:
“compared to those from P. densiflora and P. koraiensis,”
The phrase is also unnecessary.
L318-341:
It is difficult to distinguish between the findings in past studies and in this study. The paragraph should be revised so that the reader can easily understand it.
The discussion may need to be significantly modified due to the results of statistical analysis.
(References)
L382:
1980 => 1979
Author Response
The paper under review describes the effects of resins derived from three pine species (Pinus. densiflora, P. koraiensis, and P. rigida) on the pinewood nematode, Bursaphelenchus xylophilus. Pinus rigida is known to be resistant to the pinewood nematode. Therefore, it is very interesting that the resin components of P. rigida suppressed the behavior of nematodes and had a lethal effect on nematodes. However, there is a major problem with this paper that no statistical analysis has been performed in any of the experiments. Since scientific papers require discussions based on the results of statistical analysis, it is essential to carry out statistical analysis in this study as well. Results and discussion should be reconstructed after conducting statistical analysis in each experiment. Major revisions are required to be published in this journal.
Answer: We appreciate your valuable comments for our manuscript.
Specific comments are as follows:
--------------------------------------------------------------------
(Introduction)
L57:
Sugar => Suga
Answer: We corrected.
L81-82:
“Although PWN migration… migrate in P. rigida (Son et al. 2014)”
The sentence is strange.
Answer: We modified.
(Materials and Methods)
L93:
Pine wood nematode (PWN) => PWN
Answer: We corrected.
L94:
PWNs (Bursaphelenchus xylophilus) => PWNs
Answer: We corrected.
L96-97:
Togashi et al. (2003) is not suitable as a citation of the Baermann funnel technique. There are many papers that explain this method in English (e.g. Hooper (1986)).
Baerman => Baermann
Answer: We changed the citation reference to Hoopper (1986).
L98-106:
Information on the branch thickness (diameter) is needed because there may be some relationship between the thickness of branches and the number (area) of resin canals. The diameter of hole made to inoculate the nematodes should also be shown here. How many repetitions in this experiment?
Answer: We describe the details of branch sizes and hole diameter.
Repetition was described in Fig. 1 legend.
L107-113:
How did you collect the resins from each pine trees? A detail description is needed.
“values in all data…standard error (SE)” => the sentence should be moved to the legend of figure 2.
Answer: Resins were collected from surfaces of excised branches of three-year-old pine saplings (P. densiflora, P. koraiensis, and P. rigida) obtained by germination of seeds.
The sentence is moved to the figure legend.
L114-121:
How many nematodes are used in this experiment?
““values in all data…standard error (SE)” => the sentence should be moved to the legend of figure 3.
Answer: Approximately 50 nematodes were dipped into resin.
The sentence is moved to the figure legend.
L134-135:
“Compounds presented as … 0.05% are indicated” => the sentence should be moved to the legend of table 1.
Answer: The sentence is moved to the figure legend.
L137-146:
Where is the result of this experiment? I could not find it in Result section.
Answer: We forgot the deletion of this section during submission.
L156:
He et al. (2017) => this paper is not listed in Reference section.
Answer: We added the paper in references.
L161-163:
Statistical analysis is necessary in experiments 2.3, 2.4, 2.5, and 2.7. Statistical method used in each experiment should be explained here.
Answer: We revised figures as suggested. Statistical methods are described. Moreover, Figures are revised.
(Results)
L165-170 (Figure 1):
It is recommended to show the total number (including repetition) rather than the average ratio of nematodes in each segment in figure1. Then, it may be better to analyze by chi-square test.
Answer: Important finding is that spreading of PWNs is suppressed in P. rigida branch. Data are not changed. We analyzed the data statistically using one-way ANOVA.
L168:
“after 3 days” => after 1 day? (L104)
Answer: we corrected.
L175-181:
“Generally, …small PCR tube.”.
These sentences are not explanations of the results. Not necessary.
Answer: The sentences are deleted as suggestion.
L181-192:
The result of statistical analysis should be described here. In figure 2A, it is difficult to distinguish between the area inside or outside resin. Is it better to show difference in color between a and b? In figure2B, the result of statistical analysis should be shown. Chi-square test may be effective.
Answer: We modified the Figure.
L190:
“during 6 days => during 3 days? (L111)
Answer: we corrected to 3 days.
L193-203: (Figure 3)
It should be clarified whether the difference in the progress of mortality rate of nematodes among three pinus species is statistically significant.
Answer: we revised the sentences.
L205-208:
“Because the resins… pine plants by GC-MS”
This sentence isn’t result of the experiment. Not necessary.
Answer: we revised the sentences.
L217:
“peak numbers 25 and 29” => peak numbers 23 and 28?
Answer: we corrected to 23 and 28.
L224-225: (figure 5E)
To state that the amounts of two substances in bark and resin was higher than those in leaf and xylem, statistical analysis is needed. There is no description of the orange bar (Figure 5E).
Answer: we corrected the orange bar in Figure 5E.
L241-251: (figure 6)
Similar to above, to state the strength of PWN toxicity in cis-3,5-dimethoxystilbene, statistical comparison is necessary.
Answer: we revised Figure 6 as suggested.
(Discussion)
L281-285:
“We tested… were inoculated in the resin.”
The description about the experimental method is already in M & M. These sentences are not necessary.
Answers: We revised the sentences.
L292-295:
“To investigate the resin toxicity … and P. rigida trees”
The sentence is also unnecessary for the same reason as above.
Answers: We revised the sentences.
L313-314:
“compared to those from P. densiflora and P. koraiensis,”
The phrase is also unnecessary.
Answers: We deleted the phrase.
L318-341:
It is difficult to distinguish between the findings in past studies and in this study. The paragraph should be revised so that the reader can easily understand it.
The discussion may need to be significantly modified due to the results of statistical analysis.
Answers: We revised the paragraph of Discussion part.
(References)
L382:
1980 => 1979
Answers: We corrected.
Reviewer 3 Report
The Manuscript ID: forests-1793974 examines nematicidal properties and chemical composition of Pinus rigida resin against pine wood nematodes (PWNs). While Pinus rigida showed PWN resistance, P. densiflora and P. koraiensis were the most disease-affected species. The authors demonstrated that the resin from PWN-resistant P. rigida could directly affect PWN mobility and mortality via toxic phytochemicals in the resin. Generally, the subject is worth publication though it is solely based on three-year-old saplings of P. rigida obtained by germination of seeds. It would have been better if further research were conducted on how tree growth stage influences susceptibility/resistance to the PWN and on the possibility of cross-breeding among provenances as well as their reaction to the nematodes. Also, the writing style is somewhat awkward with some misprints in this manuscript. An examples is: Line 366: “toxic to PWNs, which may be create PWN resistance in P. rigida pine.” Should be “toxic to PWNs, which may create PWN resistance in P. rigida pine.”
Therefore, I would suggest a rewriting of the ms and resubmitting after minor revision.
Author Response
The Manuscript ID: forests-1793974 examines nematicidal properties and chemical composition of Pinus rigida resin against pine wood nematodes (PWNs). While Pinus rigida showed PWN resistance, P. densiflora and P. koraiensis were the most disease-affected species. The authors demonstrated that the resin from PWN-resistant P. rigida could directly affect PWN mobility and mortality via toxic phytochemicals in the resin. Generally, the subject is worth publication though it is solely based on three-year-old saplings of P. rigida obtained by germination of seeds. It would have been better if further research were conducted on how tree growth stage influences susceptibility/resistance to the PWN and on the possibility of cross-breeding among provenances as well as their reaction to the nematodes. Also, the writing style is somewhat awkward with some misprints in this manuscript. An examples is: Line 366: “toxic to PWNs, which may be create PWN resistance in P. rigida pine.” Should be “toxic to PWNs, which may create PWN resistance in P. rigida pine.”
Therefore, I would suggest a rewriting of the ms and resubmitting after minor revision.
Answer: We appreciate your valuable comments for our manuscript.
Round 2
Reviewer 2 Report
The manuscript has been properly revised by authors, but there are still some inadequacies. Specific comments are below.
P3:
“PWN migration in PWN-inoculated branches failed to migrate in P. rigida (Son et al. 2014).”
As mentioned in the original paper, the sentence is strange. => PWNs inoculated on branches failed to migrate in P. rigida (Son et al. 2014).
P4:
Pinus densiflora => P. densiflora
P5-6: Statistics
“All experiments were conducted in triplicate, and repeated three times.” => All experiments were repeated in triplicate.
You must explain here not only the statistical method used in this study, but also the purpose of comparison using the method. To compare the ability of nematodes to migrate in cut branches among three species (Figure 1), to compare the activity of nematodes soaked in resin among three species (Figure 2), to compare the mortality of nematodes in resin among three species (Figure 3),….
P6-8: Results
“Inhibition of PWN migration in pine stems”
“PWN inoculation in resin”
“Resin toxicity to PWNs”
“Chemical composition of resins from P. densiflora, P. koraiensis, and P. rigida”
“Nematicidal activity of 3,5-dimethoxystilbene against PWNs”
The results of the statistical analysis obtained in each experiment should be shown in each section (F=, df=, p=), including an explanation of whether it was statistically significant or not.
P17, 18, 19, and 22: legends for figures 1, 2, 3, and 6
“All experiments were conducted in triplicate, and repeated three times.” => Experiment was repeated in triplicate.
Author Response
The manuscript has been properly revised by authors, but there are still some inadequacies. Specific comments are below.
Answer: We deeply appreciate your kind and valuable suggestions and comments for our article.
P3:
“PWN migration in PWN-inoculated branches failed to migrate in P. rigida (Son et al. 2014).”
As mentioned in the original paper, the sentence is strange. => PWNs inoculated on branches failed to migrate in P. rigida (Son et al. 2014).
Answer: We revised the sentence as suggested.
P4:
Pinus densiflora => P. densiflora
Answer: We corrected.
P5-6: Statistics
“All experiments were conducted in triplicate, and repeated three times.” => All experiments were repeated in triplicate.
Answer: We corrected as suggested.
You must explain here not only the statistical method used in this study, but also the purpose of comparison using the method. To compare the ability of nematodes to migrate in cut branches among three species (Figure 1), to compare the activity of nematodes soaked in resin among three species (Figure 2), to compare the mortality of nematodes in resin
among three species (Figure 3),….
Answer: We modified the sentences as suggested.
P6-8: Results
“Inhibition of PWN migration in pine stems”
“PWN inoculation in resin”
“Resin toxicity to PWNs”
“Chemical composition of resins from P. densiflora, P. koraiensis, and P. rigida”
“Nematicidal activity of 3,5-dimethoxystilbene against PWNs”
The results of the statistical analysis obtained in each experiment should be shown in each section (F=, df=, p=), including an explanation of whether it was statistically significant or not.
Answer: We tried to revised the sentences based on statistical data analysis.
P17, 18, 19, and 22: legends for figures 1, 2, 3, and 6
“All experiments were conducted in triplicate, and repeated three times.” => Experiment was repeated in triplicate.
Answer: We corrected as suggested
Author Response File: 
Author Response.docx
