Review Reports - Effect of Soybean and Maize Rotation on Soil Microbial Community Structure

Round 1

Reviewer 1 Report

This research attempts to assess the effects on soil microbial community structure as a result of different cropping practices (maize or soybean monoculture vs. maize-soybean rotation). Providing a better understanding of how different crops and rotations affect soil microbial communities is an interesting and worthwhile pursuit and should result in new information worthy of publication. However, thee are several problems with this paper as written that undermine its quality and usefulness, and render it in an unpublishable form at present. Major revision and a drastic overhaul of the statistical analyses, presentation of data, and interpretation and discussion of the research are needed before it can provide a worthwhile contribution to the literature in this field.

Most importantly, the analyses and presentation of the relevant data are badly mishandled. The experiment is set-up with 2 factors (cropping and fertilization), each with 3 levels, for a 3 x 3 factorial design in a randomized block (3 reps) format. However, it does not appear to have been analyzed that way, as there is no indication that the factors were analyzed as such, but only as 9 separate treatments. This must be analyzed by factor (with interactions). In this way, each factor can be analyzed as a whole, as well as determined if the factors interacted (such as fertility treatments respond differently depending on the cropping system). With the current analysis, it is impossible to determine these factor and interaction effects, and the entire results are unclear and hard to assess or make any sense of. This problem is then compounded by a very confusing and misguided presentation of data that does not show differences among the factors and treatments, but instead makes comparisons within each treatment (Table 3), which is completely pointless and counter-productive. The authors talk about supposed differences among treatments, but these relationships are not clearly shown due to the confusing and unhelpful the way data are presented.

Unfortunately, it gets even worse when the paper shifts to the presentation of PCA and RDA analyses of fatty acid components, as the authors do not seem to have a firm command of how to use these analyses or what they represent. First, the main point of these analyses are to show how the different factors and treatments affect the overall soil fatty acid profiles (through multi-variate analyses), yet the authors never present the relationships among the treatments or factors in the PCA or RDA graphs (have to plot the treatments to see their relationships, not the fatty acids). Instead, the fatty acids that are contributing to the PCs are plotted here, which provides little useful info, and pointless or incorrect statements are made, such as "The second principal component has the least influence on 18:0, 16:0, and a15:0". Note: the second PC is comprised of these fatty acid components, it cannot influence them, and in this case PC2 is negatively correlated with concentrations of those fatty acids, meaning that those FAs actually have a very strong influence on PC2, but negative correlation, meaning that treatments with high concentrations of these FAs would have a low PC2 score. Comments like this indicate that authors do not understand how to present or interpret PCA data, as many unhelpful, incorrect, or completely backward statements are made here. Same goes for the RDA analyses. Again, we need to see how these factors relate to the treatments, not just to each other, and that is not presented anywhere here. And again, interpretations appear muddled here, as statements are made that pH had great influence on fungi, but that is not what the RDA graph shows as pH as a factor had little on anything.

Throughout the paper, many statements are made that are shown or supported by the data (mostly because the appropriate data are not presented). In the discussion, they bring up FA ratios used as indicators (MUFA/STFA, cy/w7c), but these data were never presented for their research, and no reference is made to their findings, so why is this brought up if not used in this research? Also, why refer to the 'newly developed soil aggregate quality fractal dimension and S index' if these were not used or have any relevance to the current work? There are many unhelpful statements such as these throughout the discussion, and little actual discussion of the data collected and its meaning. The conclusions also present little helpful info ("Bacteria are the main components of soil microbial community"? and even incorrect or misleading statements (the greater influence on gram-positive was i15:0, most significant effect on fungi were 18:1...) - These are the components of these organisms, they do not influence or effect them, they are them. Then a series of statements are made that have no basis or support in the data presented, such as "the results showed that the physical and chemical properties of soil could be changed by crop rotation...thus affecting structure and function of soil microbial community" Where did you show this in this paper. No data presented in this regard.

Other comments - Intro should include some background info on how different crops cause changes to soil microbial communities. Section on cropping in ancient Rome and 1700's seems very out of place, and has no relevance to current topic or paper, replace with more relevant background info.

What was the previous cropping history of this field? Since samples taken after only 1 and half years of cropping, previous history may also be important for the composition and structure of soil microbial community

Table 1 - hectometers are a little used and outdated unit, show as 100 m, or convert to other unit.

Results should present overall factor effects first, then, if there are significant interactions, then can proceed to talking about specific fertility treatments within each cropping system.

Author Response

Response to Reviewer 1 Comments

Point 1:

English language and style

( ) Extensive editing of English language and style required
(x) Moderate English changes required
( ) 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)	( )

Point 2: Comments and Suggestions for Authors

Response 2: Thanks for your suggestion. According to your opinion, I had revised the article in detail. First, I used your suggestion to reanalyze the data using two-way ANOVA. Then, additional explanations were given for areas where the data charts were not clearly displayed. The conclusions and discussions of the study were revised in the way of further study from small aspects to large ones. The structure of soil microbial community under rotation was fully explained by combining data maps and tables, such as Table 3 and 4.

Point 3: Most importantly, the analyses and presentation of the relevant data are badly mishandled. The experiment is set-up with 2 factors (cropping and fertilization), each with 3 levels, for a 3 x 3 factorial design in a randomized block (3 reps) format. However, it does not appear to have been analyzed that way, as there is no indication that the factors were analyzed as such, but only as 9 separate treatments. This must be analyzed by factor (with interactions). In this way, each factor can be analyzed as a whole, as well as determined if the factors interacted (such as fertility treatments respond differently depending on the cropping system). With the current analysis, it is impossible to determine these factor and interaction effects, and the entire results are unclear and hard to assess or make any sense of.

Response 3: Thank you for pointing out incorrect data analysis, which was the cause of the confusion of conclusions. I used what you raised two-way ANOVA and analyzed the interaction effects in Table. All tables were re-analyzed strictly in accordance with the requirements of two-factor analysis, and the errors were corrected.

Point 4: This problem is then compounded by a very confusing and misguided presentation of data that does not show differences among the factors and treatments, but instead makes comparisons within each treatment (Table 3), which is completely pointless and counter-productive. The authors talk about supposed differences among treatments, but these relationships are not clearly shown due to the confusing and unhelpful the way data are presented.

Response 4: Thank you for pointing out incorrect data analysis, which was the cause of the confusion of conclusions. I chose the data with significant impact to re-analyze, as shown in the table below. And I re-write the result and the conclusions.

Point 5: Unfortunately, it gets even worse when the paper shifts to the presentation of PCA and RDA analyses of fatty acid components, as the authors do not seem to have a firm command of how to use these analyses or what they represent. First, the main point of these analyses are to show how the different factors and treatments affect the overall soil fatty acid profiles (through multi-variate analyses), yet the authors never present the relationships among the treatments or factors in the PCA or RDA graphs (have to plot the treatments to see their relationships, not the fatty acids).

Response 5: Thank you for your advice. I redraw the data. Besides, I added a specific component matrix to Figure 1 to explain the specific location. I added a heatmap to PCA for describing the correlation between environmental factors and microbial community structure was explained in detail. Detailed modifications are shown in the following figures. The reason why I do the principal component analysis of phospholipid fatty acids is to analyze the components of soil that have a greater specific coefficient of influence on the composition of microbial communities. Find these components in order to further analyze the changes of these components in the specific treatment. Redundancy analysis of environmental factors and microbial community structure is to find out the possible relationship between them. Like PCA analysis, the aim is to find out which microorganisms are more closely related to environmental factors. At the same time, the changes of phospholipid fatty acids were found by referring to the specific phospholipid fatty acids analyzed above. This change may be due to farming systems, fertilizers or their interactions.

Point 6: Instead, the fatty acids that are contributing to the PCs are plotted here, which provides little useful info, and pointless or incorrect statements are made, such as "The second principal component has the least influence on 18:0, 16:0, and a15:0". Note: the second PC is comprised of these fatty acid components, it cannot influence them, and in this case PC2 is negatively correlated with concentrations of those fatty acids, meaning that those FAs actually have a very strong influence on PC2, but negative correlation, meaning that treatments with high concentrations of these FAs would have a low PC2 score.

Response 6: Thank you for your suggestion. I re-write all wrong conclusion. I added a specific component matrix to Figure 1 to explain the specific location. Then the score of each principal component is clearer. I realized that principal components did not affect phospholipid fatty acids.

Point 7: Comments like this indicate that authors do not understand how to present or interpret PCA data, as many unhelpful, incorrect, or completely backward statements are made here.

Response 7: Same goes for the RDA analyses. Again, we need to see how these factors relate to the treatments, not just to each other, and that is not presented anywhere here. And again, interpretations appear muddled here, as statements are made that pH had great influence on fungi, but that is not what the RDA graph shows as pH as a factor had little on anything.

Also, why refer to the 'newly developed soil aggregate quality fractal dimension and S index' if these were not used or have any relevance to the current work? There are many unhelpful statements such as these throughout the discussion, and little actual discussion of the data collected and its meaning.

The conclusions also present little helpful info ("Bacteria are the main components of soil microbial community"? and even incorrect or misleading statements (the greater influence on gram-positive was i15:0, most significant effect on fungi were 18:1...) - These are the components of these organisms, they do not influence or effect them, they are them. Then a series of statements are made that have no basis or support in the data presented, such as "the results showed that the physical and chemical properties of soil could be changed by crop rotation...thus affecting structure and function of soil microbial community" Where did you show this in this paper. No data presented in this regard.

I chose these long-standing crop rotation models only to show that the research on crop rotation has been carried out very early, and to further illustrate the research progress on crop rotation.

Point 8: Table 1 - hectometers are a little used and outdated unit, show as 100 m, or convert to other unit.??

Response 8: I referred the title of “Improving Productivity of Managed Potato Cropping Systems in Eastern Canada: Crop Rotation and Nitrogen Source Effects” reference to modify units. I changed “hectometers” to “ha”.

Point 9: Results should present overall factor effects first, then, if there are significant interactions, then can proceed to talking about specific fertility treatments within each cropping system.

Response 9: Thanks for your advice. I had re-write results. I took main effect and interaction into consideration, then analyzed of the specific influence between treatments.

Author Response File: Author Response.pdf

Reviewer 2 Report

Effect of soybean and maize rotation on soil microbial community structure by Zhang et al.

Comments/suggestions:

This paper has a major problem with the statistics, which should be re-done and all the results should be re-written. The way authors used and described PCA/bi-plot analysis is not sufficient. Discussion and conclusion sections are not well developed. However, the topic is interesting. I suggest authors to re-write the manuscript.

L32: This is too general statement. Please start the sentence as “The quality of soil….”

L34: Better to say cereal or grain crop, rather than cash crop for maize.

L34: Delete “Driven by this economic value”

L41-45: For the people who never studied these models, this information is just useless. Please give what was demonstrated by these models in brief.

L53: Fertilization can also affect the population and yield of above-ground vegetation?? Please re-write.

L81: 2800 to 3200 h per ?

L91: 300 kg/hm2 ?? Please check all units.

L91-53: Please provide the information on how much NP were provided by this fertilizer?

L94-95: Please give the practice in brief.

L147: 7.2 – 8.2%, do not use % for both.

L142: The authors should analyze data as 3 (rotation) x 3 (fertilizer) x 2 (year) factorial. ANOVA table should be presented and mean separation should be done according to the p-values for main effect(s) and the interaction(s).

L150: Mean separation is very confusing, growth stages are not clear.

L182: For PCA analysis, the Eigen values should be provided along with vector loadings. Full form for each point should be given. Please explain the bi-plot more in detail. PCA/biplot analysis is more than what the authors are presenting. I did not see clear grouping and clear interpretation of the results.

L194: This figure is not clear at all. Discussion on this figure is very poor. Authors are suggested to use simple correlation coefficients or regression models to present this data.

L283: References are not formatted well.

L252: Please do not repeat all the results. Conclusion should be given based on the results of the study. What is the take home message of your study??

Author Response

Response to Reviewer 2 Comments

Point 1:

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)	( )

Response 1: Thank you so much, the MS has been smoothed by native English speaker. According to the comments of reviewer’s, we revised the INTRODUCTION, METHODS, RESULT and CONCLUSION deeply and corrected some expressions. In the interdiction I have further expanded on some unexplained elements, such as specific rotation patterns. I also rewrite some wrong statements and the error unit is amended. In the methods, the test contents are supplemented. For the results, according to your suggestions, I have re-analyzed the data and mapped them. The data of chart reaction are analyzed correctly. For the conclusion, I comprehensively consider the data and icons before and after the comprehensive analysis.

Point 2: Comments and Suggestions for Authors

Effect of soybean and maize rotation on soil microbial community structure by Zhang et al.

Comments/suggestions:

Response 2: Thanks for your suggestion. In the interdiction, I have further expanded on some unexplained elements, such as specific rotation patterns. I also rewrite some wrong statements and the error unit is amended. In the methods, the test contents are supplemented. For the results, according to your suggestions, I have re-analyzed the data and mapped them. To explain the PCA/bi-plot, I added the table of component matrix. Then data of chart reaction are analyzed correctly. By consulting the literature, I further determined the specific changes in microbial communities and their relationship with environmental factors. For the conclusion, I comprehensively consider the data and icons before and after the comprehensive analysis.

Point 3: L32: This is too general statement. Please start the sentence as “The quality of soil….”

Response 3: Thanks for your suggestion. I have changed the sentence.

Specific modification as following:

…The quality of soil is one of the important factors affecting crop growth because it is not only one of the major components of the environment but also is necessary for field crops’ survival….

Point 4: L34: Better to say cereal or grain crop, rather than cash crop for maize.

Response 4: Thanks for your advice. I have changed it into “grain crop”.

Specific modification as following:

…Maize is the world's largest grain crop with high economic value….

Point 5: L34: Delete “Driven by this economic value”

Response 5: Thanks for your advice. I have deleted the “Driven by this economic value”.

Point 6: L41-45: For the people who never studied these models, this information is just useless. Please give what was demonstrated by these models in brief.

Response 6: Thanks for your suggestion. I have outlined the content of the specific model.

Specific modification as following:

…The three-course cropping of ancient Greece and Rome ( fallow land, spring sowing land, autumn (winter) sowing land), the rotation model of Norfolk in Britain, and the six years rotation model mentioned in 1794 were all examples of the rotation of legumes and non-legumes in the United States; the British Norfolk 4-year rotation model（red clover-wheat/rye-feeding turnip/sugar-beet- two-rowed barley/red clover） in 1730…

Point 7: L53: Fertilization can also affect the population and yield of above-ground vegetation?? Please re-write.

Response 7: Thanks for your suggestion. I have re-write the sentence.

Specific modification as following:

…Fertilization affects soil microorganisms mainly by changing soil physical structure, nutrient content and the amount of root and aboveground litter of crops….

Point 8: L81: 2800 to 3200 h per ?

Response 8: Thank you for your correction. I have changed unit.

Specific modification as following:

…was more than 2800 to 3200 hours /year. The main crops…

Point 9: L91: 300 kg/hm2 ?? Please check all units.

Response 9: Thank you for pointing out the mistakes. I have changed “kg/hm²” to “kg/ha”.

Specific modification as following:

…local recommendation, 300 kg/ha or 150 kg/ha of chemical (NP) fertilizer diammonium phosphate (N 13%, P2O5 44%) and 900kg/ha of organic …

Point 10: L91-53: Please provide the information on how much NP were provided by this fertilizer?

Response 10: Thank you for pointing out the errors. I have fixed all the wrong units.

Specific modification n as following:

…diammonium phosphate (N 13%, P2O5 44%) and…

Point 11: L94-95: Please give the practice in brief.

Response 11: Thanks for your suggestion. I have briefly illustrated the field management measures.

Specific modification as following:

…Drip irrigation was carried out after sowing to ensure seedling emergence rate. Beginning on June 29, every 30 days, field weeds were removed until harvesting….

Point 12: L147: 7.2 – 8.2%, do not use % for both.

Response 12: Thanks. I have modified it.

Specific modification as following:

…accounting for 7.2-8.2%, 6.6-7.2%, 11.6-12.7%, 8.4-9.0%, 7.2-8.7% of the total phospholipid fatty acid content…

Point 13: L142: The authors should analyze data as 3 (rotation) x 3 (fertilizer) x 2 (year) factorial. ANOVA table should be presented and mean separation should be done according to the p-values for main effect(s) and the interaction(s).

Response 13: Thanks very much, it is our mistakes. So I have analyzed the data with Two-way ANOVA, and the results were presented in the revised table 3

Point 14: L150: Mean separation is very confusing, growth stages are not clear.

Response 14: Thank you for your suggestion, it is the wrong information about annotation, I have deleted the annotation about growth stage.

Point 15: L182: For PCA analysis, the Eigen values should be provided along with vector loadings. Full form for each point should be given. Please explain the bi-plot more in detail. PCA/biplot analysis is more than what the authors are presenting. I did not see clear grouping and clear interpretation of the results.

Response 15: Thanks for your suggestion, I have provided the Component Matrix table about PCA, and re-explain the bi-plot in more detail.

Point 16: L194: This figure is not clear at all. Discussion on this figure is very poor. Authors are suggested to use simple correlation coefficients or regression models to present this data.

Response 16: Thank you for your suggestion. I have revised the result of RDA and provided the correlation analysis about the soil microbial community structure and environmental soil properties.

Specific modification as following: Relationships among soil microbial community structure and soil properties were investigated by redundancy analysis (RDA) and correlation analysis in the Fig.3 where AP showed significant negative effect on OB, and inversely positively affected GP with R²=0.80 and 0.51 (P < 0.05), respectively. There were remarkable relationships between SA with Fug, GN as well as GP (P < 0.05) whose R² were 0.92, 0.65 and 0.95, respectively (Fig. 3). GP was also dramatically positively influenced by SCOM (R²=0.78, P < 0.05) and AN (R²=0.87, P < 0.05) except for SA and AP. There were no soil properties significantly affecting Act (P < 0.05).

Point 17: L283: References are not formatted well.

Response 17: Thanks for your suggestion. I have revised the references format carefully based on the latest articles of Agronomy.

Point 18: L252: Please do not repeat all the results. Conclusion should be given based on the results of the study. What is the take home message of your study??

Response 18: Thanks for your suggestion. I have revised the conclusion based on the results of the study.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

This paper improved a lot, but the statistics is not acceptable yet. The mean separation presented in Tables 3 and 4 is not correct. As I said before, mean separation should be done based on the p-values. For example, in Table 3, the cropping system x fertilizer interaction is significant only for i16:0, but authors did mean separation for all variables (a15:0, 16:0.....) which do not show the interaction effect. When the interaction is not significant there is no meaning of doing mean separation because means are not different statistically. More clearly, except for i16:0 in Table 3 and Gram-positive bacteria in Table 4, mean separation should be done only for the main effect (cropping system only, because there is no main effect of fertilizer).

Author Response

Response to Reviewer 2 Comments (round 2)

Point 1:

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)	( )	( )

Response 1: Thank you so much. According to the comments of reviewer’s, we have revised the INTRODUCTION,RESULT ,DISCUSION and CONCLUSION deeply and corrected some expressions.

Point 2: Comments and Suggestions for Authors

Response 2: Thank you so much. We have revised the section of Table 3 and Table 4 in 3.1 deeply and added the Table 5 Influence of fertilizer treatment on Gram-negative bacteria and Gram-positive bacteria. And added the discuss about the fertilizer treatment in the section of ‘4. Discussion’.

Author Response File: Author Response.docx