Effects of Five Growing Media and Two Fertilizer Levels on Polybag—Raised Camden Whitegum (Eucalyptus benthamii Maiden & Cambage) Seedling Morphology and Drought Hardiness
Round 1
Reviewer 1 Report
This is a nicely designed study and concisely written paper. What's missing is context...why is this study important...
· In the introduction please give context for why you chose this species to test…where is it generally planted (be more specific than “tropical and subtropical regions of the world”), why is it planted, what is it used for, how is it generally propagated, what soil mixtures are currently used for this species, etc. Or maybe it’s not planted much yet, though the fact that there are seed orchards established for this species suggests there is a history of planting it.
· You mention that the species shows potential for cold tolerance, but drought tolerance is evaluated. Explain why you are evaluating drought tolerance of these seedlings…is this species planted in droughty regions? Without explaining this, it’s hard to understand the utility of this component of the study.
· Line 150. What type of setting outside were the seedlings moved and stored? Did they receive full sunlight?
· Line 237, you state the blocking effect was not significant. I don’t see that as an issue, it means there wasn’t variation across the blocks, which is not problematic for the analysis.
· The drought tolerance aspect of the study seems quite theoretical. Eventually all plants will die if they receive no water. Again having some context of why you were interested in drought tolerance and how droughty outplanting areas could be would be helpful in interpreting the results of this part of the study. Is there any practical implication to a difference in 12 SurvDays, in regards to a non-simulated outplanting, for example? Explain how this experiment (drought tolerance component) is important and not just something interesting to test.
· In the discussion, I suggest you mention that the results from this simulated outplanting may not necessarily reflect the performance of the seedlings had they been transplanted into an actual outplanting.
Author Response
Reviewer 1
The authors thank Reviewer 1 for his/her careful reading of the manuscript and many constructive comments. Reviewer comments are in black and our responses are in blue.
Comments and Suggestions for Authors
This is a nicely designed study and concisely written paper. What's missing is context...why is this study important...
In the introduction please give context for why you chose this species to test…where is it generally planted (be more specific than “tropical and subtropical regions of the world”), why is it planted, what is it used for, how is it generally propagated, what soil mixtures are currently used for this species, etc. Or maybe it’s not planted much yet, though the fact that there are seed orchards established for this species suggests there is a history of planting it.
Agreed. This has been addressed by adding language in the Introduction to stress the importance of this species.
You mention that the species shows potential for cold tolerance, but drought tolerance is evaluated. Explain why you are evaluating drought tolerance of these seedlings…is this species planted in droughty regions? Without explaining this, it’s hard to understand the utility of this component of the study.
Agreed. This has been addressed by adding language in the Introduction that discusses the importance of drought in reforestation efforts.
Line 150. What type of setting outside were the seedlings moved and stored? Did they receive full sunlight?
Agreed. The requested details have been added in the Methods section.
Line 237, you state the blocking effect was not significant. I don’t see that as an issue, it means there wasn’t variation across the blocks, which is not problematic for the analysis.
Agreed. Given that the block effect was non-significant for the nursery data analysis, this allowed us to redo the blocking for the drought test experiment due to space and size limitations of the larger containers. An explanation for this change in blocking structure was provided in the Methods section.
The drought tolerance aspect of the study seems quite theoretical. Eventually all plants will die if they receive no water. Again having some context of why you were interested in drought tolerance and how droughty outplanting areas could be would be helpful in interpreting the results of this part of the study.
Agreed. We added language in the Introduction and Discussion to better explain the rationale for the drought test.
Is there any practical implication to a difference in 12 SurvDays, in regards to a non-simulated outplanting, for example? Explain how this experiment (drought tolerance component) is important and not just something interesting to test.
Agreed. We added language in the Discussion to better explain the importance of testing relative drought tolerance.
In the discussion, I suggest you mention that the results from this simulated outplanting may not necessarily reflect the performance of the seedlings had they been transplanted into an actual outplanting.
Agreed. We added language in the conclusion about the limitations of the simulated outplanting.
Author Response File: 
Author Response.docx
Reviewer 2 Report
This manuscript presents some potentially interesting findings that may be applicable to nursery culture in arid locations. However, significant edits and justifications related to findings need to be made. In particular the results needs to be revised and the importance of the results for the drought study needs to be better justified.
Major issues
Section 3.1 needs to be rewritten. The experimental design was a 2 x 5 factorial, yet this design is ignored and individual means of the treatment combinations are compared. As presented, it fails to leverage the advantages from the factorial arrangement that allows for more general statements about main effects. Typically, individual treatment means might be compared if there is an interaction between treatments (and often compared within treatments, not across). I recommend that the main effects and interactions be presented first (along with p values) before delving into results comparing individual means.
In addition, I found several inconsistencies within the first paragraph of Section 3.1 when I closely examined things. Throughout the results, specific statements need to be double checked. 1) Lines 282-284 are seemingly contradiction. The first sentence says those with RH produced short seedlings, but the second sentence lists RH-S-TS as being among those with tallest heights. According to Table 3, RH-S-TS was shorter than the other three it is grouped with in the text. 2) Line 289; Says C-S-TS was not significantly different than the RH-S-TS for RCD. However in Table 3, they are assigned different letters.
Root:shoot (and other ratio based variables) change with plant size regardless of treatment effects. Table 3 reports biomass that varied by two orders of magnitude among treatments. R:S and DQI need to be considered in the context of allometric differences that change due to seedling size.
Section 3.2; similar comment regarding factorial design. It appears you will have a media x fertilization interaction due to RH media responding differently with and without fertilization. You need to at least indicate where you did and did not have interactions before jumping to comparing individual treatment means.
How meaningful is the Drought experiment? Seedling were all transplanted into sandy loam soil. This mostly eliminates any effect of media differences except perhaps related to vigor at time of transplanting. Likewise, fertilization effects can expected to be marginal except if there is a significant carry-over effect. Likely 30 days of growth before imposing drought would be long enough for any effects of fertilizer to dissipate. Rather, the drought test mainly looks at survival related to tree size. Not surprisingly, small trees survive longer as it takes longer to deplete soil moisture. What does this mean for application to the field? I don’t think we want to purposefully produce small seedlings to minimize mortality. Rather, field planting includes competition with herbaceous vegetation and there are distinct advantages to get roots deep quickly (more likely with large seedlings).
Given the influence of seedling size on survival, I suggest correlating SurvDays to size. This seems more relevant than emphasizing media treatments that ended 30 days prior to imposition of drought.
Line 412; seedling size varied by two orders of magnitude. How relevant is R:S in this context (especially for the nonfertilized RH). I assume other studies listed compared seedlings of much more similar size such that R:S might have been due to shifts in partitioning rather than allometry differences associated with overall size.
Line 413-420; The differences in seedlings size were drastic in your study. What is the normal range of seedlings planted for E. benthami? Are your results relevant in that context?
Line 421-4232; Similar to a previous comment, it appears that rather than fertilization or media, it is all about tree size. If anything, it looks like fertilization and media don’t matter. Rather really small seedlings survived longer.
Line 446: I think it is wrong to imply greater survival due to RH media. Rather, just plant smaller seedlings if you want greater initial survival under no water conditions.
Lines 451-452: Or, just manipulate seedlings size which could be done with fertilization or media, or just time between sowing and outplanting?
Minor comments
The introduction is well written, provides adequate background, and makes a good case for doing the study. But it perhaps includes a bit too much detail regarding individual studies and could be shortened.
The fertilization and media study was measured in May. How does that correspond to typical timing of Eucalyptus planting? Do the measurements in May reflect the seedlings that would eventually be planted?
Line 79; add ‘s’ on end of ‘hybrid’.
Line 143; what are the approximate dimensions of the polybags?
Line 241; what were the dimensions of the container.
Line 243; more information on the plastic cover would be helpful. How high above the seedlings was it? Did it alter the microclimate? Was it clear?
Line 388; RH were smaller both with and without fertilizer.
What is the C:N of parboiled rice hulls? It is possible that if C:N is high, it might actually be reducing the availability of nutrients.
Line 406-409; Or, was it related to leaf area, i.e., transpiring surface area.
Line 449; Did you find that fertilization reduced R:S? Just for the RH media?
Author Response
Reviewer 2
The authors thank Reviewer 2 for his/her careful reading of the manuscript and many constructive comments. Reviewer comments are in black and our responses are in blue.
Comments and Suggestions for Authors
This manuscript presents some potentially interesting findings that may be applicable to nursery culture in arid locations. However, significant edits and justifications related to findings need to be made. In particular the results needs to be revised and the importance of the results for the drought study needs to be better justified.
Major issues
Section 3.1 needs to be rewritten. The experimental design was a 2 x 5 factorial, yet this design is ignored and individual means of the treatment combinations are compared. As presented, it fails to leverage the advantages from the factorial arrangement that allows for more general statements about main effects. Typically, individual treatment means might be compared if there is an interaction between treatments (and often compared within treatments, not across). I recommend that the main effects and interactions be presented first (along with p values) before delving into results comparing individual means.
Agreed. We previously had minimized the main effects as all the interactions were significant, but in response to the reviewer’s suggestion, we included the p-values of the main effects and interactions for all the variables (Table 3) and briefly described the main effect results.
In addition, I found several inconsistencies within the first paragraph of Section 3.1 when I closely examined things. Throughout the results, specific statements need to be double checked. 1) Lines 282-284 are seemingly contradiction. The first sentence says those with RH produced short seedlings, but the second sentence lists RH-S-TS as being among those with tallest heights. According to Table 3, RH-S-TS was shorter than the other three it is grouped with in the text.
Agreed. We corrected these inconsistencies.
2) Line 289; Says C-S-TS was not significantly different than the RH-S-TS for RCD. However in Table 3, they are assigned different letters.
Agreed. The table was accurate and the text has been corrected.
Root:shoot (and other ratio based variables) change with plant size regardless of treatment effects. Table 3 reports biomass that varied by two orders of magnitude among treatments. R:S and DQI need to be considered in the context of allometric differences that change due to seedling size.
Agreed. This was addressed by providing additional language in the Discussion talking about the confounding effects of seedling size with other morphological variables.
Section 3.2; similar comment regarding factorial design. It appears you will have a media x fertilization interaction due to RH media responding differently with and without fertilization. You need to at least indicate where you did and did not have interactions before jumping to comparing individual treatment means.
Agreed. Please see response above in which we stated how we addressed the main effects and interactions. That response applies to both phases of the experiments.
How meaningful is the Drought experiment? Seedling were all transplanted into sandy loam soil. This mostly eliminates any effect of media differences except perhaps related to vigor at time of transplanting. Likewise, fertilization effects can expected to be marginal except if there is a significant carry-over effect. Likely 30 days of growth before imposing drought would be long enough for any effects of fertilizer to dissipate. Rather, the drought test mainly looks at survival related to tree size. Not surprisingly, small trees survive longer as it takes longer to deplete soil moisture. What does this mean for application to the field? I don’t think we want to purposefully produce small seedlings to minimize mortality. Rather, field planting includes competition with herbaceous vegetation and there are distinct advantages to get roots deep quickly (more likely with large seedlings).
Agreed. We added more explanation on the rationale in the Introduction and the implications in the Discussion. This includes the strong effect of seedling size, as well as the potential advantages and disadvantages of planting different-sized seedlings on challenging sites.
Given the influence of seedling size on survival, I suggest correlating SurvDays to size. This seems more relevant than emphasizing media treatments that ended 30 days prior to imposition of drought.
Agreed. We correlated four important size variables with survival and added a new figure to graphically display the results (Figure 5). In addition, several sentences on this subject have been added to the Discussion.
Line 412; seedling size varied by two orders of magnitude. How relevant is R:S in this context (especially for the nonfertilized RH). I assume other studies listed compared seedlings of much more similar size such that R:S might have been due to shifts in partitioning rather than allometry differences associated with overall size.
Agreed. We correlated R:S ratio with survival (see response just above) that showed similar correlations with survival for both shoot height and R:S ratio and added explanation in the Discussion section.
Line 413-420; The differences in seedlings size were drastic in your study. What is the normal range of seedlings planted for E. benthami? Are your results relevant in that context?
Agreed. We added language in the Discussion acknowledging the large range in seedling sizes in this experiment.
Line 421-4232; Similar to a previous comment, it appears that rather than fertilization or media, it is all about tree size. If anything, it looks like fertilization and media don’t matter. Rather really small seedlings survived longer.
Agreed. We have clarified the language in the Discussion that fertilization and media (i.e., nursery cultural practices) are the driving factors influencing seedling size in this study. Thus, we agree it is mainly about tree size as determined by nursery practices. We clarified this more in the Discussion and Conclusion sections.
Line 446: I think it is wrong to imply greater survival due to RH media. Rather, just plant smaller seedlings if you want greater initial survival under no water conditions.
We agree that we can’t directly connect RH to greater survival. However, as we mention in our response above, the nursery practices have an influence on seedling morphology and physiology that translates to improved growth and survival in the field or in our case simulated outplanting. We clarified this in both the Discussion and Conclusion sections.
Lines 451-452: Or, just manipulate seedlings size which could be done with fertilization or media, or just time between sowing and outplanting?
It is possible to manipulate seedling size through a large number of nursery cultural practices. However, the focus of this study was to use existing resources in developing countries to make these changes with little additional input. This is clarified more in the Conclusion section.
Minor comments
The introduction is well written, provides adequate background, and makes a good case for doing the study. But it perhaps includes a bit too much detail regarding individual studies and could be shortened.
We agree and shortened the Introduction section.
The fertilization and media study was measured in May. How does that correspond to typical timing of Eucalyptus planting? Do the measurements in May reflect the seedlings that would eventually be planted?
We did not attempt to coordinate nursery production with operational planting dates, but did grow seedlings in the nursery phase for a corresponding length of time to produce a range of seedling sizes.
Line 79; add ‘s’ on end of ‘hybrid’.
Agreed. The phrase has been changed to “….seedlings of a Eucalyptus hybrid….”
Line 143; what are the approximate dimensions of the polybags?
Agreed. This information has been added.
Line 241; what were the dimensions of the container.
Agreed. This information has been added.
Line 243; more information on the plastic cover would be helpful. How high above the seedlings was it? Did it alter the microclimate? Was it clear?
Agreed. More information on the growing environment has been added.
Line 388; RH were smaller both with and without fertilizer. What is the C:N of parboiled rice hulls? It is possible that if C:N is high, it might actually be reducing the availability of nutrients.
Agreed. We added C:N ratio of the rice hulls and discussed its possible effects in the Discussion section.
Line 406-409; Or, was it related to leaf area, i.e., transpiring surface area.
Agreed. We added language regarding leaf area.
Line 449; Did you find that fertilization reduced R:S? Just for the RH media?
Yes. We found that non-fertilized RH media had significantly higher R:S ratios compared to fertilized RH media. These were also significantly different from other fertilized and non-fertilized media. We have made this finding and its relevance more clear in the revised Discussion.
Round 2
Reviewer 2 Report
The manuscript is improved. I appreciate the work the authors took to address comments.
My only comments relate to Figure 5. This is the analysis I suggested adding. 1) I really don't think it is appropriate to fit 5th or 6th order polynomials. For instance, the curve in 5d does not likely reflect a biologically relevant relationship. To me it seems like there is a threshold response on most of these curves. I recommend fitting simpler equations. This would reduce r2, but make more sense. 2) Should Figure 5 be moved to the Results? 3) a bit more can be added to the text about the implications of size and managing size based in results of Figure 5.
Author Response
We thank Reviewer 2 for further constructive comments. We agree with both of his/her recommendations; (1) We re-ran the correlations in figure 5 using only quadratic equations, which make more biological sense. (2) We moved Figure 5 into the results section (now Figure 2) and provided explanatory text in the Methods and Results sections.
