The Resistance of Scots Pine (Pinus sylvestris L.) Half-sib Families to Heterobasidion annosum
Round 1
Reviewer 1 Report
General
This study addresses an interesting question: differences in genetic variability among Scots pine families in resistance to Heterobasidion annosum. It is based on results from an inoculation experiment with four different H. annosum strains. It presents interesting results that will be useful inbreeding programs of Scots pine.
In my opinion, the manuscript must be revised, and I have several suggestions for improving its quality. Several of these relate to the statistical analyses and how the results from these are presented, see below.
Specific comments
L 101: The “a” should be deleted.
L104: A reference is made to Table 1. There is no Table 1 in the manuscript. If the meaning is to refer to Table 1 in reference 54 this should be done differently.
L136: Inoculations were done with four different H. annosum strains. Are these the four with origins shown geographically in Figure 1?
L 137-138: What kind of experimental design was used for the inoculation experiment? Did you have any type of blocking or randomization? If so, that should be shown in the statistical model.
L 139: Define incidence or incidence rate.
L 163-164: What kind of deviations from the normal distribution? What are the consequences of these deviations, in particular for the F-tests? Why did you not make transformations and analyses of transformed values of these traits?
Why is Model 1 presented? Are there any statistical analyses done on the basis of this model? No statistical analyses are presented, neither by this model nor by Model 2. I suggest that results from the analyses of variance should be presented in a table. Model 2 should be used for analyses. I agree that both strains and families are regarded as random.
L 187-188: No significance tests are presented.
L 194 -194: Stem diameter in cm, should it be mm?
L 199 & Figure 2: Are the points presented in the figure means across the four strains?
L 201: Range of mortality from 14 to 78 %? In Figure 3 mortality varies between families from approximately 1 to 21 %. Correct the statement.
L 204-206: I cannot see this high correlation (r=0.72) from the figure. Does it show up in a plot between incidence rate and mortality?
L 209-210: “The variance component …. was 10.1 % and significant.” 10.1 % of what? Of the total v
L 216: Standard errors are shown only for pathogen spread.
Figure 4 & Figure 5: Family identification numbers cannot be identified. Which are the control groups, blue or green? The period 2, 4 and 10 months are not properly presented. The figures are of low technical quality.
L 285: Should “… variance within half sib families..” rather be “.. among half sib families.. ?
L 292-293: Where is this shown?”.. the P strain killed about three times more seedlings compared to the S strain.”
L 303-305: Analyses of variance are not shown.
Technical: There are differences in font sizes throughout the manuscript.
Author Response
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
Yes | Can be improved | Must be improved | Not applicable | |
Does the introduction provide sufficient background and include all relevant references? | (x) | ( ) | ( ) | ( ) |
Is the research design appropriate? | ( ) | (x) | ( ) | ( ) |
Are the methods adequately described? | ( ) | (x) | ( ) | ( ) |
Are the results clearly presented? | ( ) | (x) | ( ) | ( ) |
Are the conclusions supported by the results? | ( ) | (x) | ( ) | ( ) |
Comments and Suggestions for Authors
General
This study addresses an interesting question: differences in genetic variability among Scots pine families in resistance to Heterobasidionannosum. It is based on results from an inoculation experiment with four different H. annosum strains. It presents interesting results that will be useful inbreeding programs of Scots pine.
In my opinion, the manuscript must be revised, and I have several suggestions for improving its quality. Several of these relate to the statistical analyses and how the results from these are presented, see below.
Specific comments
L 101: The “a” should be deleted.
We reviewed and corrected that place of the manuscript.
L104: A reference is made to Table 1. There is no Table 1 in the manuscript. If the meaning is to refer to Table 1 in reference 54 this should be done differently.
We reviewed and corrected that place of the manuscript.
L136: Inoculations were done with four different H. annosum strains. Are these the four with origins shown geographically in Figure 1?
Yes, four different H. annosum strains are shown geographically in Figure 1. We reviewed that place of the manuscript and added link to Figure 1.
L 137-138: What kind of experimental design was used for the inoculation experiment? Did you have any type of blocking or randomization? If so, that should be shown in the statistical model.
No, there were no blocks, just the treatments and control.
L 139: Define incidence or incidence rate.
We reviewed and corrected that place of the manuscript.
L 163-164: What kind of deviations from the normal distribution? What are the consequences of these deviations, in particular for the F-tests? Why did you not make transformations and analyses of transformed values of these traits?
Why is Model 1 presented? Are there any statistical analyses done on the basis of this model? No statistical analyses are presented, neither by this model nor by Model 2. I suggest that results from the analyses of variance should be presented in a table. Model 2 should be used for analyses. I agree that both strains and families are regarded as random.
Deviations from the normal distribution, except for growth traits, were significant and due to not large sample size we decided to apply non parametric analysis approach, instead of trying to get the distribution closer to normal one.
Two statistical models were used in order to get the answer not only on pure effects of families and strains on trait variation, but also on these two effects interaction. The results on variance components from these models are presented in lines 251-255. Standard errors for variance components were added and the ratio to residuals.
L 187-188: No significance tests are presented.
Additional information was added on pairwise comparisons of H. annosum strains in between and with control.
L 194 -194: Stem diameter in cm, should it be mm?
We reviewed and corrected that place of the manuscript.
L 199 & Figure 2: Are the points presented in the figure means across the four strains?
Yes, the points represent the four strains averages in 12 Scots pine half-sib families.
L 201: Range of mortality from 14 to 78 %? In Figure 3 mortality varies between families from approximately 1 to 21 %. Correct the statement.
In our study seedlings mortality varying from 1 to 21 %. We reviewed and corrected that place of the manuscript.
L 204-206: I cannot see this high correlation (r=0.72) from the figure. Does it show up in a plot between incidence rate and mortality?
We found that there was a mistake (true value is r=0.52).
L 209-210: “The variance component …. was 10.1 % and significant.” 10.1 % of what? Of the total v
It is shown percentage from total variance. The ratio to residuals was added also.
L 216: Standard errors are shown only for pathogen spread.
Mortality and incidence rate were assessed as binomial data (0 or 1), therefore SE presented only for pathogen spread.
Figure 4 & Figure 5: Family identification numbers cannot be identified. Which are the control groups, blue or green? The period 2, 4 and 10 months are not properly presented. The figures are of low technical quality.
Sorry, but the quality of all these Figures has changed with submission the manuscript. Now Figures 4a, b, c and 5a, b, c. are corrected according changed and better technical quality.All Figures descriptions are also changed in manuscript.
L 285: Should “… variance within half sib families..” rather be “.. among half sib families.. ?
We reviewed and corrected that place of the manuscript.
L 292-293: Where is this shown?”.. the P strain killed about three times more seedlings compared to the S strain.”
In our investigation pathogen intersterility group has not been identified (lines 271-272) and in this part of manuscript lines (292-293) were the error. We reviewed and corrected that place of the manuscript.
L 303-305: Analyses of variance are not shown.
The results are described in the text. The standard errors of variance components and ratios to residuals were added.
Technical: There are differences in font sizes throughout the manuscript.
All changes were done in the manuscript.
Author Response File: 
Author Response.docx
Reviewer 2 Report
In "The resistance of Scots pine (Pinus sylvestris) half-sib families to Heterobasidion annosum," the authors examined the impact of genotype on susceptibility to H. annosum and the impact of both genotype and inoculation on total phenolic content (TPC). A better understanding of genotypic effects on TPC content and its relation to variation in susceptibility to H. annosum has the potential to positively contribute to the management of this economically important disease.
Overall, the paper was well-written and the justification of the study as well as its experimental design were clearly described. However, the paper would benefit from inclusion of additional results and some improvements to figures to increase ease of interpretation. The inclusion of this additional information in the results section (e.g. results of GLIMMIX tests are not clear and should be reported in a table or in text), which would also aid the reader in assessing the significance of the research results. In addition, figures would benefit from more complete descriptions in legends and several figures are missing y-axis labels (e.g. concentration levels of TPC's). See below for more specific comments.
Specific comments:
Line 66: Remove secondary from description of phenolic metabolite.
Line 80: Instead of "synergy", I propose you use the word "synergistic." Also, please clarify what you mean by synergy effects.
Line 135: Specify number of trees inoculated per strain overall and per genotype.
Line 157: Na 2 CO 3 needs to be reformatted.
Line 160: Please include the concentration range of chlorogenic acid used for the standard curve to quantitate TPC content, and the number of levels in the standard curve.
Line 160, 174: TPC acronym can be used here (and elsewhere, where applicable), since you've already defined it.
Line 162: Why did you use GLIMMIX, to examine the impact of genotype x strain on susceptibility outcomes and/or TPC content? Please specify.
Line 165: Please define what DDFM=KR option is for readers who are unfamiliar with SAS GLIMMIX. Is this a non-parametric approach?
Line 179 (and results in general): Please include sample sizes so that a reader can more easily interpret the presented results. This information could be included in a table, and referenced where applicable.
Line 187: Can you explain why this might have occured? How was infection incidence rate measured? Does seedling death impact pathogen growth?
Lines 190-197: Can you show results of a statistical test supporting a difference or lack there of between control and inoculated trees? Were there differences between pathogen isolates? Are results presented for all isolates together? Also, why might have there been differences between inoculated and control seedlings at the end of the study? Can you mention this briefly in the discussion. Moreover, if there are significant differences in height/stem diameter, shouldn't this be included as a variable in your GLIMMIX model?
Figure 2: Sample sizes for % should be included. From which inspection are the dead seedlings collected from (all of them together)? Lines labeled as inspection I, II, and III but in other figures are labeled by time post-inoculation (2, 6, and 10 months). I suggest making the labeling consistent throughout the manuscript.
Lines 205, 207: Include p-values for correlations.
Figure 3: Is there a reason for the specific order of the families? Are results presented across strains or just one strain? Are these results across all time periods? Please specify.
Lines 212-213: Was there a significant effect of strain on mortality, spread, or incidence? Please clarify what you mean by family x strain comparison largest for spread.
Figure 4: Specify which bars correspond to which families. Y-axis labels for TPC concentration levels are needed. Are levels (increments) consistent across plots in this figure? If not, this would aid in comparing the plots to one another. Figure 4c: X-axis labels are incomplete for 10 month time point data.
Figure 5: Y-axis labels for TPC concentration levels are needed. X-axis labels for families are needed, as well as sample sizes (this could also be included in a table). Please specify in the legend why some bars are open and others are shaded blue. Can you include standard error? Why are the time points in a different order than displayed in previous figures?
Lines 238-240: Since the study was observational in terms of TPC levels (i.e. you did not manipulate these levels directly), you can't really conclude that accumulation of TPC's in needles determined incidence of H. annosum, although you can say they are associated and that higher TPC levels likely contributed to the observed results.
Line 266: How do S-group and P-group isolates compare to the strains used in the present study? If intersterility groups were not identified in the present study (lines 271-272), how can you say that there were differences between S and P strains (lines 292-293)? Can you present the statistical results (test statistic, p-value) showing that there was a statistical difference?
Line 303-304: Can you please show the results of this analysis (test statistic, p-value)?
Lines 322-326: Terpenes are another type of plant secondary metabolites. I suggest revising the sentence order of this paragraph, since you discuss phenolics, then terpenes, and then phenolics again.
Line 338-340: Which time points corresponded to dormancy and growth? I suggest being consistent with how you describe the different time points, and/or define which time points correspond to dormancy and growth periods.
Line 444: Check reference title: "non.mycorrizal" should be "non-mycorrizal."
Author Response
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
Yes | Can be improved | Must be improved | Not applicable | |
Does the introduction provide sufficient background and include all relevant references? | (x) | ( ) | ( ) | ( ) |
Is the research design appropriate? | (x) | ( ) | ( ) | ( ) |
Are the methods adequately described? | (x) | ( ) | ( ) | ( ) |
Are the results clearly presented? | ( ) | ( ) | (x) | ( ) |
Are the conclusions supported by the results? | ( ) | ( ) | (x) | ( ) |
Comments and Suggestions for Authors
In "The resistance of Scots pine (Pinus sylvestris) half-sib families to Heterobasidion annosum," the authors examined the impact of genotype on susceptibility to H. annosum and the impact of both genotype and inoculation on total phenolic content (TPC). A better understanding of genotypic effects on TPC content and its relation to variation in susceptibility to H. annosum has the potential to positively contribute to the management of this economically important disease.
Overall, the paper was well-written and the justification of the study as well as its experimental design were clearly described. However, the paper would benefit from inclusion of additional results and some improvements to figures to increase ease of interpretation. The inclusion of this additional information in the results section (e.g. results of GLIMMIX tests are not clear and should be reported in a table or in text), which would also aid the reader in assessing the significance of the research results. In addition, figures would benefit from more complete descriptions in legends and several figures are missing y-axis labels (e.g. concentration levels of TPC's). See below for more specific comments.
Specific comments:
Line 66: Remove secondary from description of phenolic metabolite.
All changes were done in the manuscript and it can be seen with Track changes.
Line 80: Instead of "synergy", I propose you use the word "synergistic." Also, please clarify what you mean by synergy effects.
All changes were done in the manuscript and it can be seen with Track changes.
Line 135: Specify number of trees inoculated per strain overall and per genotype.
We reviewed and corrected that place of the manuscript. In our study each isolate were inoculated 240 pine seedlings; 20 seedlings in one Scots pine half-sib family.
Line 157: Na 2 CO 3 needs to be reformatted.
All changes were done in the manuscript and it can be seen with Track changes.
Line 160: Please include the concentration range of chlorogenic acid used for the standard curve to quantitate TPC content, and the number of levels in the standard curve.
Additional information was provided according to reviewer request.
Line 160, 174: TPC acronym can be used here (and elsewhere, where applicable), since you've already defined it.
All changes were done in the manuscript and it can be seen with Track changes.
Line 162: Why did you use GLIMMIX, to examine the impact of genotype x strain on susceptibility outcomes and/or TPC content? Please specify.
GLIMMIX was used because residuals showed significant deviations from the normal distribution.
Line 165: Please define what DDFM=KR option is for readers who are unfamiliar with SAS GLIMMIX. Is this a non-parametric approach?
The definition was added to M&M. The NPAR1WAY procedure was used for non-parametric analysis.
Line 179 (and results in general): Please include sample sizes so that a reader can more easily interpret the presented results. This information could be included in a table, and referenced where applicable.
Sample size in treatments was added in the Table 1.
Line 187: Can you explain why this might have occured? How was infection incidence rate measured? Does seedling death impact pathogen growth?
Incidence rate of H. annosum (fungal growth) infection was examined at three time periods over the course of 2018. For each inspection period, three visually healthy and all dead seedlings were taken from each variant (more than 180 seedlings in total). For each of these samples, a 20 cm length of stem was cut into 5 cm pieces, split in half, and placed in separate sealed bags. After three weeks incubation at room temperature, the samples were checked for mycelia, conidiophores, and conidia formation characteristic of H. annosum. If was found for mycelium, conidiophores, and conidia formation on one of the four part of stem, we attributed this plant to infected ones. As can be seen from the results, the longer the time elapses from infection, the spread of H. annosum on the stem is higher. We are thinking, that seedling death has impact for pathogen growth, because the living tissue of the plant is needed for the spread of H. annosum. After the inoculation, plant begins to die and the pathogen spreads to the rest of the plant, which it still lively. Seedlings that remain viable after inoculation prevent the spread of the pathogen to the higher parts of stem.
Lines 190-197: Can you show results of a statistical test supporting a difference or lack there of between control and inoculated trees? Were there differences between pathogen isolates? Are results presented for all isolates together? Also, why might have there been differences between inoculated and control seedlings at the end of the study? Can you mention this briefly in the discussion. Moreover, if there are significant differences in height/stem diameter, shouldn't this be included as a variable in your GLIMMIX model?
The Table with additional information on pairwise comparisons of H. annosum strains in between and with control was added. Diameter was used as a covariate (fixed effect) in the statistical model (1) for those traits which correlated with diameter, but the output was pretty similar to initial one without a covariate.
Figure 2: Sample sizes for % should be included. From which inspection are the dead seedlings collected from (all of them together)? Lines labeled as inspection I, II, and III but in other figures are labeled by time post-inoculation (2, 6, and 10 months). I suggest making the labeling consistent throughout the manuscript.
The mortality of each half-sib family seedlings is given 10 months after inoculation. We reviewed and corrected that place of the manuscriptand Figure 2, now the inspection periods of H. annosum throughout the text are represented consistently.
Lines 205, 207: Include p-values for correlations.
We reviewed and corrected that place of the manuscript.
Figure 3: Is there a reason for the specific order of the families? Are results presented across strains or just one strain? Are these results across all time periods? Please specify.
Figure 3 shows the results for the average pathogen spread and incidence rate across all strains and in all time periods. The mortality of each half-sib family seedlings is given 10 months after inoculation, because the number of dead seedlings in other inspections was very low (0-2 seedlings in one half-sib family). In Figure 3 half-sib families are presented according to seedlings mortality rates.
Lines 212-213: Was there a significant effect of strain on mortality, spread, or incidence? Please clarify what you mean by family x strain comparison largest for spread.
There was a significant effect of strain on infection incidence rate and H. annosum mycelium spread after six months and the data now are presented in the Table 1.
Figure 4: Specify which bars correspond to which families. Y-axis labels for TPC concentration levels are needed. Are levels (increments) consistent across plots in this figure? If not, this would aid in comparing the plots to one another. Figure 4c: X-axis labels are incomplete for 10 month time point data.
Sorry, but the quality of all these Figures has changed with submission the manuscript. Now Figures 4a, b, c and 5a, b, c are changed and better technical quality. Figures descriptions are also changed in manuscript.
Figure 5: Y-axis labels for TPC concentration levels are needed. X-axis labels for families are needed, as well as sample sizes (this could also be included in a table). Please specify in the legend why some bars are open and others are shaded blue. Can you include standard error? Why are the time points in a different order than displayed in previous figures?
Sorry, but the quality of all these Figures has changed with submission the manuscript. Now Figures 4a, b, c and 5a, b, c are changed and better technical quality. Figures descriptions are also changed in manuscript. The calculation of the sample size included in the Materials and Methods section of the manuscript.
Lines 238-240: Since the study was observational in terms of TPC levels (i.e. you did not manipulate these levels directly), you can't really conclude that accumulation of TPC's in needles determined incidence of H. annosum, although you can say they are associated and that higher TPC levels likely contributed to the observed results.
The main idea was paraphrased to avoid categorical conclusions.
Line 266: How do S-group and P-group isolates compare to the strains used in the present study? If intersterility groups were not identified in the present study (lines 271-272), how can you say that there were differences between S and P strains (lines 292-293)? Can you present the statistical results (test statistic, p-value) showing that there was a statistical difference?
In our investigation pathogen intersterility group has not been identified (lines 271-272) and in this part of manuscript lines (292-293) were the error. We reviewed and corrected that place of the manuscript.
Line 303-304: Can you please show the results of this analysis (test statistic, p-value)?
These results are given in the manuscript in the Table 1.
Lines 322-326: Terpenes are another type of plant secondary metabolites. I suggest revising the sentence order of this paragraph, since you discuss phenolics, then terpenes, and then phenolics again.
The order of the sentence was revised according to reviewer suggestions.
Line 338-340: Which time points corresponded to dormancy and growth? I suggest being consistent with how you describe the different time points, and/or define which time points correspond to dormancy and growth periods.
We described which time points correspond to dormancy and growth periods and the changes are included in the manuscript.
Line 444: Check reference title: "non.mycorrizal" should be "non-mycorrizal."
We reviewed and corrected that place of the manuscript.
Author Response File: Author Response.docx
Reviewer 3 Report
The resistance of Scots pine (Pinus sylvestris) half-sib families to Heterobasidion annosum General Comments This is an interesting study, which seems to be based on a well designed and implemented experiment. I enjoyed my reading as the ms is well-written with conciseness and clarity. The findings are interesting, which may provide useful inference for selecting resistant Scots pine genotypes, potentially using phenolic compounds as markers. Introduction Well-written and is very informative. Congratulations! -L38, “do” or “does”? Material and Methods Were seedling tissue samples taken from each individual seedlings or groups of seedlings. The analytical models implied that the sampling was based on individual seedlings. Please confirm. Statistical analysis Although the analytical models seem to be appropriate, the authors may, however, want to treat the pathogen types and the 12 half-sib families as fixed effects since, in the subsequent presentation of the results, the authors frequently used family means. Using a fixed model would enable the authors to test the difference between treatment and control, as well as comparisons among the families. Saying that an estimate of variance component is significantly different from zero is the same as saying that there are significant differences. Result - Figure 2. I thought that the infection incidence rate would increase from inspection I to III as fungus mycelium may continue to grow over time. The authors may want to explain why the order was reversed at the 0-5 cm stem segment. Did some initial infection die out? - Figure 3. suggest that the authors either split Figure 3 into two figures (one shows the incidence rate and the other shows the spread distance) or use two vertical axes on both sides of the figure (one shows the incidence rate and one show the spread distance). It is a bit confusing to interpret the current Figure 3. - Question: the authors stated in text that “The results of the seedlings’ mortality show large differences between half-sib families, varying from 14% to 78% (Figure 3)”. The Figure 3 however shows that mortality was less than 25% for all families. Please check the discrepancy. - L208-213 “The variance component of strain for pathogen spread was 10.1% and significant, while the family component was negligible. The opposite tendency was seen for plant mortality, where the family variance component was 6.7% and significant. If family x strain interaction was added to the statistical model, the family x strain variance component was the largest for pathogen spread (14.9%), but the results for mortality did not change”. These above statements are a little hard to interpret. It is more meaningful to show the ratio of a variance component relative to the whole phenotypic variance than show the absolute value of a single variance component, which is normally associated with “scale effect”. It is certainly good to know whether an estimate of variance component is significantly different from zero when using a random model. In fact, if the authors use a fixed model, they can actually test the differences among families or among the pathogen types. -L224, Figure 2 does not seem to indicate susceptibility that can be used as indicators as cited in subsequent sections. -Figure 4 could be replaced with a table, which will provide the exact values and take less space. Besides, Figure 4 does not seem to be drawn in good quality and with appropriate labels. -L228-246. A table or figure is needed here to better indicate the corresponding changes among families in TPC and susceptibility indicators. For example, in a figure, the vertical axis can be a susceptibility indicator and horizontal axis can be the TPC, grouped by family. -Figure 5 shows the effects of pathogen inoculation on the TPCs, which is good but does not show well the relationship between TPC and susceptibility indicators. Discussion -Explain what the S-group isolates and the P isolates are.
Author Response
Open Review
(x) I would not like to sign my review report
( ) I would like to sign my review report
English language and style
( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style
Yes | Can be improved | Must be improved | Not applicable | |
Does the introduction provide sufficient background and include all relevant references? | (x) | ( ) | ( ) | ( ) |
Is the research design appropriate? | (x) | ( ) | ( ) | ( ) |
Are the methods adequately described? | (x) | ( ) | ( ) | ( ) |
Are the results clearly presented? | ( ) | (x) | ( ) | ( ) |
Are the conclusions supported by the results? | (x) | ( ) | ( ) | ( ) |
Comments and Suggestions for Authors
The resistance of Scots pine (Pinus sylvestris) half-sib families to Heterobasidion annosum General Comments This is an interesting study, which seems to be based on a well designed and implemented experiment. I enjoyed my reading as the ms is well-written with conciseness and clarity. The findings are interesting, which may provide useful inference for selecting resistant Scots pine genotypes, potentially using phenolic compounds as markers. Introduction Well-written and is very informative. Congratulations!
-L38, “do” or “does”?
We reviewed and corrected that place of the manuscript.
Material and Methods Were seedling tissue samples taken from each individual seedlings or groups of seedlings. The analytical models implied that the sampling was based on individual seedlings. Please confirm.
In our investigation seedling tissue samples taken from each individual seedlings. We were checked for mycelia, conidiophores, and conidia formation characteristic of H. annosum in 2612 individual parts of stem in total.
Statistical analysis Although the analytical models seem to be appropriate, the authors may, however, want to treat the pathogen types and the 12 half-sib families as fixed effects since, in the subsequent presentation of the results, the authors frequently used family means.
Using a fixed model would enable the authors to test the difference between treatment and control, as well as comparisons among the families. Saying that an estimate of variance component is significantly different from zero is the same as saying that there are significant differences.
F-test were done by using the same statistical models just with fixed effects instead of random. The Table with additional information on pairwise comparisons of H. annosum strains in between and with control was added.
Result - Figure 2. I thought that the infection incidence rate would increase from inspection I to III as fungus mycelium may continue to grow over time. The authors may want to explain why the order was reversed at the 0-5 cm stem segment. Did some initial infection die out?
We are thinking, that seedling death has impact for pathogen growth, because the living tissue of the plant is needed for the spread of H. annosum. After the inoculation, plant begins to die and the pathogen spreads to the rest of the plant, which it still lively. Seedlings that remain viable after inoculation prevent the spread of the pathogen to the higher parts of stem.
- Figure 3. suggest that the authors either split Figure 3 into two figures (one shows the incidence rate and the other shows the spread distance) or use two vertical axes on both sides of the figure (one shows the incidence rate and one show the spread distance). It is a bit confusing to interpret the current
We split Figure 3 into two figures. Now, one shows the pathogen incidence rate and seedlings mortality the other shows the spread distance
Figure 3. - Question: the authors stated in text that “The results of the seedlings’ mortality show large differences between half-sib families, varying from 14% to 78% (Figure 3)”. The Figure 3 however shows that mortality was less than 25% for all families. Please check the discrepancy.
We reviewed and corrected that place of the manuscript.
L208-213 “The variance component of strain for pathogen spread was 10.1% and significant, while the family component was negligible. The opposite tendency was seen for plant mortality, where the family variance component was 6.7% and significant. If family x strain interaction was added to the statistical model, the family x strain variance component was the largest for pathogen spread (14.9%), but the results for mortality did not change”. These above statements are a little hard to interpret. It is more meaningful to show the ratio of a variance component relative to the whole phenotypic variance than show the absolute value of a single variance component, which is normally associated with “scale effect”. It is certainly good to know whether an estimate of variance component is significantly different from zero when using a random model. In fact, if the authors use a fixed model, they can actually test the differences among families or among the pathogen types.
The mentioned text was revised.
-L224, Figure 2 does not seem to indicate susceptibility that can be used as indicators as cited in subsequent sections.
We reviewed and corrected that place of the manuscript.
Figure 4 could be replaced with a table, which will provide the exact values and take less space. Besides, Figure 4 does not seem to be drawn in good quality and with appropriate labels.
Sorry, but the quality of all these Figures has changed with submission the manuscript. Now Figures 4a, b, c and 5a, b, c. are changed and better technical quality. All Figures descriptions are also changed in manuscript.We wouldn't want to replaced Figure 4 with a table so that a reader can more easily interpret the presented results.
-L228-246. A table or figure is needed here to better indicate the corresponding changes among families in TPC and susceptibility indicators. For example, in a figure, the vertical axis can be a susceptibility indicator and horizontal axis can be the TPC, grouped by family.
We selected correlation analyses instead graphical visualization. The significant correlation between TPC and susceptibility indicators are presented in the Results part to avoid large and non-informative table.
-Figure 5 shows the effects of pathogen inoculation on the TPCs, which is good but does not show well the relationship between TPC and susceptibility indicators.
We selected correlation analyses instead graphical visualization.
Discussion -Explain what the S-group isolates and the P isolates are.
Heterobasidion annosum (Fr.) Bref., found in Europe as three different intersterile groups – S, P and F. In general, the S group is specialized on Norway spruce, while P group exhibits less host specialization, primarily attacking Scots pine but also spruce and other woody species (Korhonen et al., 1992).
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
The revised version of "The resistance of Scots pine (Pinus sylvestris) half-sib families to Heterobasidion annosum" presents a more clear and comprehensive results section. I have some additional minor edits (see below). Primarily, I suggest listing families in the same order in all figures (figure 3, 4, and 5). This would make it easier for a reader to compare susceptibility measures and TPC concentrations across times, tissues, and families.
line 24: TPC-delta not previously defined
line 58: ‘annosums’ > ‘annosum’
line 66: missing space after period
line 136-137: “240 pine seedlings were inoculated per isolate”
line 149: add “from” before “three biological replicates”
line 151: missing ’s’ from “families”
line 200: distance > distant
Figure 3 legend: Indicate for figure 3A which color bar corresponds to mortality or incidence rate, e.g. mortality (%) (blue bar), incidence rate (%) (green bar).
Figures 3, 4 and 5: families should be in same order across all time points and tissues, and figures so that a reader can easily compare susceptibility measures and TPC content in individual families across tissues and times.
Figure 4: Make the y-axis minimum consistent across all tissues. Needles start at 7, stems at 6, roots at 5.
Line 307: Confirm unit is cm. mm is used previously.
Line 310: missing space after period.
Author Response
Comments and Suggestions for Authors
The revised version of "The resistance of Scots pine (Pinus sylvestris) half-sib families to Heterobasidion annosum" presents a more clear and comprehensive results section. I have some additional minor edits (see below). Primarily, I suggest listing families in the same order in all figures (figure 3, 4, and 5). This would make it easier for a reader to compare susceptibility measures and TPC concentrations across times, tissues, and families.
p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Helvetica Neue'; color: #454545}
line 24: TPC-delta not previously defined
We reviewed and corrected that place of the manuscript.
line 58: ‘annosums’ > ‘annosum’
We reviewed and corrected that place of the manuscript.
line 66: missing space after period
We reviewed and corrected that place of the manuscript.
line 136-137: “240 pine seedlings were inoculated per isolate”
We reviewed and corrected that place of the manuscript.
line 149: add “from” before “three biological replicates”
We reviewed and corrected that place of the manuscript.
line 151: missing ’s’ from “families”
We reviewed and corrected that place of the manuscript.
line 200: distance > distant
We reviewed and corrected that place of the manuscript.
Figure 3 legend: Indicate for figure 3A which color bar corresponds to mortality or incidence rate, e.g. mortality (%) (blue bar), incidence rate (%) (green bar).
We reviewed and corrected that place of the Figure 3A.
Figures 3, 4 and 5: families should be in same order across all time points and tissues, and figures so that a reader can easily compare susceptibility measures and TPC content in individual families across tissues and times.
We corrected all figures. Now families are in same order across all time points and tissues in Figures 3, 4 and 5.
Figure 4: Make the y-axis minimum consistent across all tissues. Needles start at 7, stems at 6, roots at 5.
We reviewed and corrected y-axis of the Figure 4.
Line 307: Confirm unit is cm. mm is used previously.
We reviewed and corrected that place of the manuscript.
Line 310: missing space after period.
We reviewed and corrected that place of the manuscript.
Author Response File: Author Response.docx
