Overwintering and Yield Responses of Two Late-Summer Seeded Alfalfa Cultivars to Phosphate Supply

Round 1

Reviewer 1 Report

A. General issues

1. This is only a one-year field test. The principle of the field study is a series of yearly tests. This research should be repeated in 2020/2021 season. Only two-year studies can be published in scientific journals such as Agronomy.
2. The basic condition for obtaining reliable results and conclusions is the repetition of the field experiment.

B. Main issues

1. The title of the scientific articles requires an adjustment related to the rate of the applied phosphorus and response of the tested alfalfa variety.
2. The information on the effects of phosphorus on plant growth are too general. In fact purely academic. This part should be shortened.
3. Some of the terms discussed in the Introduction are too general and even contradictory.
4. The objective of the research require clarification. The purpose(s) should be the basis for the title of the scientific article.
5. Figure 1 requires a substantive correction.
6. The dates of the consecutive alfalfa sampling (harvest) should be considered as an experimental factor. Look for the appropriate ANOVA statistical package.
7. The results are a good, overly detailed report of the one-year study.
8. The discussion is, in fact, a literature review.
9. One-year results do not allow to conducts a real scientific discussion.
10. The lack of results from two series of studies (two-seasons) leads to scientific speculation.

C. Detailed issuses are inserted directly in the text.

Comments for author File: Comments.pdf

Author Response

We appreciate your valuable comments and suggestions for our manuscript. Please check the attached file for the point-by-point response letter. 

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

I rate the work highly, but it still requires corrections. I have included all my comments in the pdf.file as the reviewer comments.

The following statements are to be considered:

- Line 2. The title is "Application rates of phosphorus fertilizer for maximum over-wintering and yield of late-summer seeded alfalfa".
Not NP-fertilizers, only P-fertilizers!
In the previous version of the manuscript, the authors wrote that as the fertilizer they applied calcium phosphate. Now, in line 131 they wrote: “Fertilization treatment was carried out on 10 September 2019, the P fertilizer was diammonium phosphate (the content of P was 23.14%)”
Diammonium phosphate - NP fertilizer ((NH₄)H₂PO₄). This fertilizer contains about 20% of P and 18% of Nitrogen! This completely changes the form of the work. The researchers did not determine the effect of phosphorus fertilization on the maximum over-wintering and yield of late-summer seeded alfalfa. They determined the effect of nitrogen-phosphorus fertilization (why nitrogen ?!). After all, the Fabaceae alfalfa does not need nitrogen fertilization? The authors must check that the name of the fertilizer is correct!

- Line 104. We still do not know the methods with which the authors determined the content of organic matter, total and available nitrogen content, and available phosphorus and potassium content. Please specify the equipment used.

- Line 106. According to the review made earlier, please explain what is the cause of such a high soil pH. Please include this explanation here and not just in response to a review. This explanation is intended for the reader, not only for the reviewer.

- Line 131. See comment to line 2

- All tables. Which may explain the change in original values compared to the manuscript "agronomy-1449247"? Why were all standard error values (from tables and figures) removed? After all, I sent a file with an example of SE calculation.

The rest of my suggestions you can find in the manuscript.pdf file

I hope, my comments will be understandable and will help the authors to properly prepare the manuscript, making it more readable.

Thank you

Reviewer

Comments for author File: Comments.pdf

Author Response

We appreciate your valuable comments and suggestions for our manuscript. Please check the attached file for the point-by-point response letter. 

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript agronomy-1545615, entitled “Application rates of phosphorus fertilizer for maximum overwintering and yield of late-summer seeded alfalfa” submitted by Wang et al. report and discussed the results related to a field experiment where three different P fertilizers doses were applied to two alfalfa varieties characterized by different dormancy behaviour. Several plant parameters were measured in order to assess the effect of fertilisation on plant growth.

Considering the importance to optimise phosphate fertilisation in order to make judicious use of this finite resource and reduce eutrophication risks due to its overuse, I believe that the manuscript is of potential interest for readers of “Agronomy” and fall within its scope.

At first reading, the experimental activity seems that was carried out following a strict scientific logic and according to widely used methods which have made it possible to obtain reliable results. However, three aspects perplex me: 1) in this experiment the fertilizer used was DAP (diammonium phosphate) that contains N besides P. This aspect was never considered in the experiment nor in the manuscript. It is necessary to consider that nitrogen fertilisation has a significant effect on plant growth, even in the case of leguminous species, where it can have a starter effect by promoting growth or, on the contrary, can have a negative effect by inhibiting symbiosis with soil microorganisms and biological nitrogen fixation. Authors must necessarily consider this throughout the manuscript. They cannot focus the manuscript only on phosphorus. Therefore, I suggest revising the entire manuscript considering both elements by establishing the dose of DAP to be administered and changing the introduction and discussion accordingly. 2) In order to assess the differences among cutting times, related to all the parameters measured during the three cutting, repeated measure statistical analysis must be applied. 3) The manuscript lacks consistency in the indication of the two varieties used and this causes confusion and tires the reader.

My assessment of the individual parts of the manuscript is:

Writing and style: The quality of the writing is good and only small changes are needed.

Introduction: it is fine and only small changes are needed.

Materials and Methods: Small changes are needed but I suggest improving the statistical analysis part adding the repeated measures analysis and the description of the performed correlation, that is missing.

Results: are clear but I suggest adding more data and statistical indications in the text. Please, avoid to resume the results related to the first two cuts and comparing them with the third cut. Follow a simple and chronological description of the effect of the treatment. Implement statistics also for the time comparisons.

Discussion: it is fine, but, I suggest to the authors to discuss also the role of N.

Conclusion: it is simple and easy to read, based on the observed results, giving a piece of useful information for the farmers.

My specific comments, which I hope will help the authors to improve the manuscript, are reported in the attached files.

Comments for author File: Comments.pdf

Author Response

We appreciate your valuable comments and suggestions for our manuscript. Please check the attached file for the point-by-point response letter. 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The reviewed manuscript does not meet the basic publication criteria. The results presented in the manuscript cover only one growing season.

Two years of research are required in a scientific journal presenting the results of a field study.

On this basis, I am obliged to present a negative opinion.

Reviewer 2 Report

Dear Authors,

I accept your manuscript entitled: Application rates of phosphorus fertilizer for maximum over-wintering and yield of late-summer seeded alfalfa in the present form and recommend it to publication in Agronomy MDPI Journal

with regards

Reviewer

Reviewer 3 Report

The authors applied all suggested changes.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Reviewer 2 Report

Dear Authors,

The manuscript requires a lot of corrections. I have included all my comments in the pdf.file as the reviewer comments. You have to recalculate standard errors

The following statements are to be considered:

- Line 16. The authors in the manuscript use the term 50, 100, 150 kg P, which is not true, as they used 21.82 or 43.64 or 65.46 kg P ha^-1, therefore the description should be unified. In this case, I suggest two ways to solve this problem.
1. using a capital P with a subscript 0, 1, 2, 3 which will be P treatment (P₀, P₁, P₂, P₃).
2. use 21.82 or 43.64 or 65.46 kg P ha^-1 or approximately 22, 44, and 66 kg P ha^-1.
All descriptions (50, 100, 150 kg P ha^-1, and P1, P2, P3, P4) in the manuscript text, tables, and figures should be changed.

- Line 100. Please provide particle size distribution of soil (percentages of sand, silt, and clay).

- Line 101. Please specify the methods were used to determine the chemical composition of soils and what equipment was used for this purpose, including the producer, city (state), and country of the producer)?

- Line 103. Could you explain why pH is so high? What is the cause of such a high soil pH?

- Line 112. Please enter the breeder's name (Seed Company) and the company location - city, USA.

- Line 120. See comment line 116.

- Line 121. Please provide the trade name of the fertilizer, give the name of the producer, city, and country. Please indicate the percentage as % P.

- 122. Herbage harvest is more associated with the harvesting of herbs than with the harvesting of the green mass of forage crops.

- Line 129. Incomprehensible description: 1-m-wide and ?-m-long? Wouldn't it be easier to tell how many square meters from each plot were assessed for determining fresh mass weight?

- 130. Word “herbage” is better to change with “samples of green mass” or “samples of above grounds parts of plants”

- 131. It's hard to understand the authors’ explanation regarding replications!
The experiment was set up in 3 replications, exactly 3 plots for one experimental treatment.
1 plot is 1 repetition.
The authors took samples: one sample from each plot = 3 repetitions of each combination.
Where did 10 replications come from ???
1st plot 3 replications + 2nd plot 3 replications + 3rd plot 4 replications?
How to understand?
Did the authors consider the actual number of repetitions when calculating the standard error (SE)?

- Line 141. 1-m-wide and ?-m-long?
Wouldn't it be easier to tell us how many square meters from each plot?

- Line 142. [total number of surviving plants/total 142 number of plants] × 100.
Can authors’ insert an equation?

- Line 148. See comment line 116.

- Line 154. See comment line 116.

- Line 155. Fig. 2 authors have to change to Figure 2A. and then see comment line 202.

- Line 166. Multiple use of notation (P <0.05) is not necessary when differences between assessed treatments or cultivars are significant, and elsewhere in the manuscript.

- Line 175. See comment line 116.

- Line 176. Fig. 2 authors have to change to Figure 2B. and then see comment line 202.

- Line 203. could you explain why standard errors have the same value? How do authors calculate SE? You should recalculate SE again for correction. Please see the comment to line 268.
Caption Figure 2. Plant density,plant height,shoots per plant and shoot mass under different P fertilization treatments in spring after autumn seeding of two alfalfa cultivars, ZC NO.3 and Knight T. P0 is 0 kg ha^-1, P1 is 50 kg ha^-1, P2 is 100 kg ha^-1, and P3 is 150 kg ha^-1 applied after autumn sowing. Error bars indicate standard error of the mean. Different capital letters indicate significant differences from the P treatment within a cultivar (P<0.05). Different lower case letters indicate significant differences from the P treatment between cultivars (P<0.05).
should be changed to:
Figure 2. Plants density, plants height, shoots per plant, and shoot mass of ZC NO.3 and Knight T alfalfa cultivars. Error bars indicate the standard error of the means. Different capital letters indicate significant differences between P treatments. Different lowercase letters indicate significant differences between cultivars (P<0.05); n=3.

- Line 226 and 253. The first column of the table should contain doses of P.
P0 is 0 kg ha-1, P1 is 50 kg ha-1, P2 is 100 kg ha-1, and P3 is 150 kg ha-1 applied after autumn sowing. For each trait the mean and standard error of the mean are reported. Different capital letters in a column indicate significant differences from the P treatment (P<0.05). Different lower case letters indicate significant differences from the P treatment between cultivars (P<0.05).
A caption should be changed to:
For each trait, the means and standard error of the means are given. Different capital letters indicate significant differences between P treatments. Different lowercase letters indicate significant differences between cultivars (P<0.05); n=3.

- Line 268. Figure 3 should be corrected as in figure 2. Could you explain why standard errors have the same value? How do authors calculate SE? You need to check how to mark error bars on the chart in MS Excel. See attached file: SE calculation - peer-review-15711099.v1.xlsx. You should recalculate SE again for correction.
- Line 281. See comments attached in pdf.file

- Line 301. Figure 5 should be corrected as in Figures 2 and 4.
- Line 442. All literature items cited by the authors must be verified again, follow the instructions for the authors. Please correct the lack of spaces, abbreviations of journal names, bolding of the year of publication, publisher names in italics, issue number in italics, periods, commas, etc.

After revising corrections and verifying the calculations, it will be possible to review the sections: Results, Discussion, and Conclusions.

The rest of my suggestions you can find in the manuscript.pdf file

I hope that my comments will be understandable and will help the authors to properly prepare the manuscript, making it more readable.

Thank you

Reviewer

Comments for author File: Comments.pdf

Reviewer 3 Report

Comments to Authors: MDPI Agronomy 1449247, Application rates of phosphorus fertilizer for maximum over-2 wintering and yield of autumn seeded alfalfa.

The manuscript is fairly well written and the results of the research potentially provide valuable information for scientists and end users regarding P application to improve winter survival and yield of dormant and semidormant alfalfa. The research appears to be well designed; however, it does not appear that sufficient replication in space or time has been done to draw definitive conclusions since the data only includes a single sampling date in a single year from what appears to be only a single field for each cropping pattern. If that is correct, it should be considered as a fatal flaw to the manuscript, but not the research. Repeating the study using the same treatments and techniques and combining that with the current data would overcome the issue, although results may be different based on the new statistical analysis of the combined data. This is the single-most important concern for the manuscript in its present state. Coincidentally, for a perennial crop, like alfalfa, collecting data from the same seeding over multiple years counts as replication over time, whether the treatment was a one time application (e.g., seed treatment) or applications made each year of the study (phosphorus applied after planting and every production year). Consequently, if the study described in this manuscript is still intact and phosphorus has been continually applied on an annual basis at the same treatment levels, that could count for continued data collection. On the other hand, you could replant the entire study and report it as evaluating the effects of phosphorus fertilization effects on establishment and first production year performance of dormant and semidormant alfalfa. The point is that to substantiate results and have power to draw conclusions, agronomic field research has to be replicated across environments, either by space or time.

Throughout the manuscript, use terms such as ‘greater’ and ‘lesser’ as opposed to ‘higher’ and ‘lower’ when describing significant differences among treatments.

Autumn actually does  not begin until about September 22^nd in the northern hemisphere. Hence, this should be termed a ‘late-summer seeding’. Avoid the use of ‘autumn’ throughout the manuscript unless an activity took place between September 22^nd and December 21^st. Start with the title.

Abstract:

Line 14: The sentence describes the importance of alfalfa in ‘northern climates’. The 40^th N latitude would not be considered that far north in the northern hemisphere. It is correctly described as ‘temperate’ at line 25 and in the body of the manuscript.

Line 25: ‘Gray-cinnamon soil,’ Unless soil types are compared, limiting the application of results to a specific soil type greatly limits the scope of application. The information presented in this manuscript is very broadly applicable to alfalfa production wherever slightly dormant and semidormant cultivars are adapted.

Keywords: I propose adding winter survival and establishment as keywords.

Introduction:

Lines 44-45: Avoid beginning a sentence with an abbreviation, acronym, or numeral. If the sentence cannot be revised to not use such terms first, spell out the term. In this particular case, the two sentences can be combined as: ‘Phosphorus (P) is one of the essential mineral elements for plant growth, but P deficiency in soil is a common phenomenon [6].’

Line 50: See previous comment. Revise to something like: ‘Application of P increased . . .’

Line 63: ‘. . . depleted soils, erosion, and long, cold winters.’

Line 67: ‘. . . soil fertility, but it also can . . .’

Line 73 and elsewhere: Avoid using terms such as higher/lower to describe significant differences. Rather use ‘greater/lesser’.

Lines 73-74: As a separate sentence, this statement requires a citation, even if it is still [18].

Lines 76-77: ‘In particular, alfalfa stubble . . . in winter [19].’ (citation required for the stand along sentence.

Materials and Methods:

Line 96: Abbreviate ‘m above sea level’ as ‘masl,’ with is a standard abbreviation in scientific literature that should not need defining.

Line 101: 6% organic matter seems rather high for semiarid, continental soils of pH 8.5.

Tables and Figures: In every figure caption and table title, provide the location of the study.

Figure 1: In the caption, give the year represented by the data. Since the study ran from August 2019 through September 2020, it would be appropriate to include weather data for both years. Move the x-axis to cross at -10C. Report the average temperatures instead of the max and min. Add the long-term average temperature and state the timespan represented by the long-term. Add precipitation to the figure as a bar graph using the secondary y-axis.

Lines 107-114: Because the two cultivars are selected based on fall dormancy, it should be assumed that they are adapted representatives of the two fall dormancy classes. Hence, the treatment should not be cultivar, but fall dormancy. Revise the manuscript to reflect the treatment categories as dormant and semidormant alfalfa. This will greatly aid with interpreting 3^rd cut height and yield differences.

Line 117-119: The study is described as a split treatment arrangement, but the whole and subplot treatments are not defined. This must be clarified.

Lines120-121: First, I recommend against using treatment numbers instead of just using the actual P₂O₅ application rates in the text where you seldom use the numbers anyway and it tables/figures where it really does not take up that much space to use the application rates. Second, the fertilizer was applied 32 days after the alfalfa was planted (by which time it should have emerged) and incorporated at a deeper depth than the alfalfa was planted (see line 117). That should have destroyed the stand. Clarify the fertilizer application process to avoid confusion.

More detail is necessary about field design and experiment management, namely: (1) Provide more detail about irrigation technique, application rate/amounts, and frequency. You could convert Figure 1 to a table to include monthly temperature and precipitation and long-term averages as well as monthly irrigation amounts, but still need to describe the technique and application process in the text. (2) Describe insect and weed problems and name the products used for control, along with application dates. This information could have influenced winter survival and stand density.

Lines 131-134: I assumed that the number of replications mentioned was within each plot. That should be stated, at least in the first instance [e.g., ‘. . .: plant density (three replications per plot) at first cut, . . .’]. Then state that the within plot data were averaged for statistical analyses.

Line 136: Singularize ‘buds’. Should be ‘bud’.

Line 141: ‘each 1-m-wide plot’. This is confusing. Do you mean that plants were counted before and after winter in a 1 m² area of each plot? Otherwise, we know how wide the plant count plot is, which is well within the 3 x 5 m whole plot size dimensions. How long was it?

Statistical analyses: Describe the model more clearly. You had a split plot with three randomized complete blocks. Thus, your model should state which are main and subplots as fixed effects and the main and subplot error terms as random effects. Additionally, you collected data from three cuttings of the same plots. Therefore, cutting is a repeated factor that can be included either as a sub-subplot or using a repeated statement in SAS GLM. Additionally, since this is a P rate study, polynomial regression to compare differences across rates would be appropriate to show linear or quadratic effects. You have to describe how response curves are generated and

Results: I recommend combining Results and Discussion sections so that discuss is directly related to results without having to repeat the results. Otherwise, I did not thoroughly review the Result, Discussion, Conclusions sections. This being said, here are some suggestions:

Line 154 and elsewhere: It is not necessary to repeat the P rates in the text. Also, do not repeat data from tables or figures in the text. Use table/figure callouts to refer the reader to the tables and figures and use callouts often as a reminder.

Table 1: First, since the plant density effects are not significant, you could delete that column from Table 1 and simply state the lack of significance and put the mean and standard deviation in the text preceding the p-value on Line 156 [e.g., (150 ± 5 plants m-2; P > 0.10). Otherwise, you need not repeat p-values in the text, refer to the table.

The tables are difficult to read as constructed. I recommend listing variables in a column (you could do that with by cutting data, but remember, this is a repeated measurements study) and having treatment effects (CV, P, CV x P) as column headings. Alternatively, prepare a separate table for each type of variable (e.g., three cuttings of plant height) with cutting and annual as column headings (as you did) and including the main effect means for CV and P above the p-values. That would be a much better presentation than Figure 2, which is poorly constructed (stacking yield data is acceptable, but not the best presentation, but stacking non-yield data like height and shoots per plant is not at all a good presentation) and not good quality for viewing (too faint and too small).

For Tables 3 & 4, including the SEs merely clutters the table. I suggest deleting the SEs

Regarding figures, they present the data as if the interactions were significant, which few are. Present the main effect means in tables. Your data is better suited for tables, except for P rates, that should be presented in figures if significant polynomial regression differences are observed.

What is the difference in shoot mass presented in Figure 1 and annual yield presented in Figure 4 besides the calculation?

Remember to discuss fall dormancy differences instead of cultivar differences.

When you have a significant interaction, like the one for the 2^nd cutting of shoot mass, that may disappear with repeated measurements analysis, but it should probably be explained away as an anomaly of some sort. Otherwise, the interactions for root traits are of considerable interest for adding to the scientific knowledge as are the interaction for forage yield. But, with only a single environment, the data does not have the reliability of an appropriately replicated study.

Since there is no CV x P interaction for winter survival, Figure 3 should be deleted and the main effects of CV and P reported only. Main effect means should be reported even when the effect is not significant.

References: There are very many missing spaces where they are appropriate. Check the manuscript template for formatting references in all aspects. Many references are almost formatted correctly, but not a single reference actually is.

The information collected during this study has potential to be of great value for the scientific community as well as end users; however, there appears to be insufficient replication in space or time for the data to support conclusions. Please follow the recommendations of the reviewers that are supported by the editors to revise and resubmit this manuscript for publication.