Localized and Moderate Phosphorus Application Improves Plant Growth and Phosphorus Accumulation in Rosa multiflora Thunb. ex Murr. via Efficient Root System Development
Round 1
Reviewer 1 Report
This manuscript describes a glasshouse pot study of Rosa multiflora examining the effects of P fertilizer application method (localized versus mixed) and application rates (low, moderate, high) on root developmental traits and P uptake efficiency over two growth periods (45 versus 92 days). For the most part, this was a well-described study and well-written manuscript that provides interesting and relevant information with respect to plant (root) growth and P uptake in response to differential P fertilizer application. I have provided editorial suggestions on the uploaded .pdf of the manuscript to help tighten up the writing.
My main concern deals with the statistical analyses applied to this three factor factorial experiment (with replication – 4). In section 2.3 (statistical analysis), the data were first subjected to a series of one-way ANOVAs to examine main effect responses? This is not correct, and would require reduction in degrees of freedom for each successive ANOVA applied to the same data set – fairly sure this was not done. Then, almost as an afterthought, the same data set was subjected to a two-way ANOVA to identify significant interactions between application method and application rate (although the significant interactions were not thoroughly described in the results). Further to this, each ANOVA was run separately for the two growth periods considered – also not correct. If I understand the experimental design correctly (probably could be explained better), 48 pots were randomly assigned to either local or mixed fertilizer application (M), one of three fertilizer rates (L), and 2 growth periods (G) with 4 replicates of each (this was not stated in the methods, but should be). This design results in a 3-way ANOVA with replication, and a GLM:
Yijkl = µ + Mi + Lj + Gk + MLij + MGik + LGjk + MLGijk + ξ(ijk)l
The interactions determining if the various measured growth attributes respond differently depending on the interacting factor (e.g., ML: does root development response to application level (L) differ depending on the application method (M)?). Critical to remember that one always reads an ANOVA from the bottom up: if there is a significant interaction between main factors, it becomes meaningless to describe the overall main effect response, since the response varies depending on the condition/level of the interacting factor. Too much time is spent on describing the effects of main effects that are interacting with other factors. The results section should start with the full 3-way ANOVA results (not just p-values – include df, MS, F statistic, and actual p-values (you can also add *, **, *** to denote the levels of significance).
My second concern is the use of a post-hoc means separation test (here Duncan’s multiple range test) to examine the effects of a quantitative variable (in this case level of fertilizer application). Use of a means separation test is fine (and what they were designed to examine) for qualitative variables (in this case application method). For quantitative variables, one should be defining response functions (and inflection point that would define the “optimal” level). The authors suggest the moderate application rate might be “optimal”, but how do we know it is not somewhere between say moderate and adequate – means separation tests are inappropriate to determine optimums. Related to the development of response functions, this study would have greatly benefitted from the inclusion of a “control” (no fertilizer application) – this control would have provided the baseline on which to compare the responses of all fertilizer application methods and rates. This should be acknowledged in the methods and discussion.
With the above said, the authors have a very nice dataset that just requires a more comprehensive analysis that includes all factors, and associated interactions, in one ANOVA and proceed with describing/illustrating the key responses. With these important (moderate) revisions, the revised manuscript would be acceptable for publication in Forests.
Comments for author File: 
Comments.pdf
Author Response
Point 1: I have provided editorial suggestions on the uploaded .pdf of the manuscript to help tighten up the writing.
Response 1: We are grateful for these suggestions. We have made a thorough revision in the revised manuscript. All the suggestions have been accepted.
Point 2: My main concern deals with the statistical analyses applied to this three factor factorial experiment (with replication – 4). In section 2.3 (statistical analysis), the data were first subjected to a series of one-way ANOVAs to examine main effect responses? The results section should start with the full 3-way ANOVA results (not just p-values – include df, MS, F statistic, and actual p-values (you can also add *, **, *** to denote the levels of significance).
Response 2: We are appreciated for the comment. Three-way analysis of variance (ANOVA) was performed to test the effect of treatment days, P supply methods and levels and their interaction on growth and nutrient accumulation. We have added relevant information in the Materials and Methods (line 156-158), Results section (line 162-171) and Abstract (line 25-26) and Table 1.
(1) Abstract (line 25-26)
There was a significant interaction effect among treatment days, P supply methods and levels (P ≤ 0.05) on shoot DW, root DW, root/shoot ratio and total P accumulation.
(2) 2.3. Statistical Analyses (line 156-158)
Three-way analysis of variance (ANOVA) was performed to test the effect of days, P supply methods and levels and their interaction on growth and nutrient accumulation.
(3) results (line 162-171)
3.1. Interactive Effects of sampling time, P fertilizer supply methods and levels
The three-way ANOVA showed a significant interaction among days, P fertilizer supply methods and levels (P≤0.05) for shoot DW, root DW, root/shoot ratio and total P accumulation, but not for total root length and root surface area (Table 1). The interaction effect between days and P supply levels was significant (P≤0.05) for all the variables, except shoot DW. The interaction effect between P supply methods and levels was significant (P ≤ 0.05) for total root surface area and total P accumulation. P supply methods and days had a significant interaction (P≤0.05) for root DW, root/shoot ratio and total root surface area. In contrast, the main effects of days, P supply methods and levels were significant (P≤0.05) for shoot DW, Root DW and root/shoot ratio, respectively, but not for the other variables.
Point 3: My second concern is the use of a post-hoc means separation test (here Duncan’s multiple range test) to examine the effects of a quantitative variable (in this case level of fertilizer application). Use of a means separation test is fine (and what they were designed to examine) for qualitative variables (in this case application method). For quantitative variables, one should be defining response functions (and inflection point that would define the “optimal” level). The authors suggest the moderate application rate might be “optimal”, but how do we know it is not somewhere between say moderate and adequate – means separation tests are inappropriate to determine optimums. Related to the development of response functions, this study would have greatly benefitted from the inclusion of a “control” (no fertilizer application) – this control would have provided the baseline on which to compare the responses of all fertilizer application methods and rates. This should be acknowledged in the methods and discussion.
Response 3: Thanks. We have added the explanation in the Methods (line 101-104, 111-112 and 112-1113), Abstract (line 27) and Discussion (line 346-350).
(1) line 101-104
We choose the soil P supply levels based on a preliminary 2-year experiment with five P supply rates (0, 40, 80, 160 and 320 mg P kg−1 soil, supplied as superphosphate). The results showed that the application rate of 80 mg P kg-1 soil was appropriate for root growth and trunk expansion of Rosa multiflora.
(2) line 111-112
P-deficient condition with 40 mg P kg−1 soil conventionally mixed application served as the control.
(The soil Olsen-P concentration of the P-deficient treatment with 40 mg P kg−1 mixed application was 12.5 mg kg-1, which belongs to the deficiency range and is usually treated as a control.)
(3) line 112-113
There were six treatments in the study, with 8 replicates per treatment.
(4) line 27
In the present study, The moderate application rate of P (80 mg N kg-1) appeared optimal, but it may between moderate and adequate. Thus, the word of optimal was been replaced with efficient.
(5) line 346-350
Localized application at the moderate P supply significantly stimulated root proliferation and improved root spatial distribution, which can be an effective management strategy for enhancing Rosa multiflora growth and nutrient uptake. A greater understanding of P accumulation by field-grown Rosa multiflora in relation to soil P properties is needed for better defining optimum P fertilizer rate recommendations.
We have made a thorough revision in the revised manuscript. We are appreciative of the constructive criticisms and comments offered by the reviewers and yourself on the manuscript. In the following experiments, we will further study this topic to reveal the underlying mechanism.
Reviewer 2 Report
The introduction should be re-organized to make each paragraphs have one topic, and the paragraphs should be distinctly separated from each paragraphs to get distinct topic.
I also suggested that the introduction should include the review of other literatures closely related to this research, i.e., the effect of P supply methods and levels. I think that higher DWs in localized application than mixing application is due to more amount addition in localized application.
Expression error: [16] (Ma et al. 2013).
Why did you use the different amounts of P fertilization for mixed and localized application? I think that the amount should be same for each level.
What is the full description of IBA? The abbreviations should be used after defining.
Please describe the process of digestion of samples for P analysis in more detail.
Why did you use capital letter for “PHOSPHATE2”??
Author Response
Point 1: The introduction should be re-organized to make each paragraphs have one topic, and the paragraphs should be distinctly separated from each paragraphs to get distinct topic.
Response 1: We are appreciated for the comment. Some literatures were added in the introduction. The whole introduction has been re-organized to make each paragraphs have one topic. The paragraphs were distinctly separated from each paragraphs to get distinct topic (line 46-72).
Point 2: I also suggested that the introduction should include the review of other literatures closely related to this research, i.e., the effect of P supply methods and levels. I think that higher DWs in localized application than mixing application is due to more amount addition in localized application.
Response 2: Good comments. Some literatures about the effect of P supply methods and levels has been added in 55-61 and line 66-68. There were six treatment combinations of three soil P supply levels (deficient, moderate and adequate) and two supply methods (mixed or localized application) in the study. The amount of P was the same for each supply level but varied in supply methods. The same amounts of P as in the mixing treatment were applied in the localized treatment, which made concentration in that zone higher than the concentration in the mixing treatment. The relevant information was added in line 104-107.
Point 3: Expression error: [16] (Ma et al. 2013).
Response 3: Thanks. The repeated reference of Ma et al. 2013 has been deleted.
Point 4: Why did you use the different amounts of P fertilization for mixed and localized application? I think that the amount should be same for each level.
Response 4: The amount of P was the same for each supply level but varied in supply methods (line 104-107). World P reserves are very limited and the availability of applied fertilizer P is very low (10–25%). And there is a paucity of knowledge about the effects of heterogeneous P fertiliser application on root growth of perennial non-herbaceous plants such as bushes and trees. Thus, Rosa multiflora Thunb. ex Murr. was chosen was the subject of our study. Two supply methods and Three P supply levels were used to characterize root morphological traits and nutrient accumulation. The results showed that localized and moderate P supply can enhance plant growth and nutrient accumulation by modifying root traits and improving root spatial. It suggested that localized and moderate P supply may be an effective P management strategy for optimization plant productivity and nutrient-use efficiency.
Point 5: What is the full description of IBA? The abbreviations should be used after defining.
Response 5: Thanks. The full description of IBA is indole-3-butyric acid and it has been added in line 119.
Point 6: Please describe the process of digestion of samples for P analysis in more detail.
Response 6: Done. We have added the relevant information in the Materials and Methods in line 142-149 as follows. Plant samples (0.5000 g) were weighted, placed in a digestion tube and added 8 ml of concentrated sulphuric acid (H2SO4). Mix gently and leaved to stand at room temperature for about 12 h. Then added 4 mL of 30% v/v hydrogen peroxide (H2O2, Guaranteed reagent), shook gently and put a small funnel in the mouth of the tube. Sample was digested at 250℃ for 30 min, then raised temperature to 370 ℃ and digested for 10 min. The tubes were took out, cooled down, added 2 mL H2O2 and digested again. Repeated 2-3 times until the solution was colorless. After cooling, dilute to a constant volume.
Point 7: Why did you use capital letter for “PHOSPHATE2”??
Response 7: Thanks. The lower letters for PHOSPHATE2 was also used in the literatures. And it has been changes to lower letters in line 314.
We have made a thorough revision in the revised manuscript. We are appreciative of the constructive criticisms and comments offered by the reviewers and yourself on the manuscript. In the following experiments, we will further study this topic and reveal the underlying mechanism.
Author Response File: 
Author Response.doc
Round 2
Reviewer 1 Report
This is a revised manuscript (2nd submission) that describes a glasshouse pot study of Rosa multiflora examining the effects of P fertilizer application method (localized versus mixed) and application rates (low, moderate, high) on root developmental traits and P uptake efficiency over two sampled growth periods (45 versus 92 days). As noted in my previous review, the previous manuscript was, for the most part, well written, but the authors have incorporated the complete set of editorial suggestions provided in that original review that has greatly improved the writing quality along with the description and interpretation of results.
My key comments/suggestions for the original submission, dealt with the statistical approach (i.e., treat the experiment as a 3-way ANOVA with replication and develop response functions for the quantitative factors). The authors have revised based on the 3-way ANOVA (Table 1), and have better described the interacting effects between the three factors. Not sure it is necessary to still run a preliminary one-ANOVA (L.154-156)?
Based on the substantive improvements to this manuscript, I now feel it is acceptable for publication in Forests.
