Effect of Different Fertilization Practices on Soil Microbial Community in a Wheat–Maize Rotation System
Round 1
Reviewer 1 Report
This new version differs from the former ones.
Just one error; line 274 in conclusion, microorganism in plural, microorganisms
A proposition: line 273, potential of enzyme activities in state of enzyme activities. In the whole text it is important to consider enzyme activities not at actual activities, but as potential activities, that in fact reflect the actual need (except the constitutive urease)
Author Response
This new version differs from the former ones.
Just one error; line 274 in conclusion, microorganism in plural, microorganisms
A proposition: line 273, potential of enzyme activities in state of enzyme activities. In the whole text it is important to consider enzyme activities not at actual activities, but as potential activities, that in fact reflect the actual need (except the constitutive urease)
Response: Many thanks for the reviewer’s efforts. We have changed microorganism to microorganisms in line 275. We agree with the reviewer’s point that it is important to consider enzyme activities not at actual activities.
Reviewer 2 Report
Manuscript by Zhang et al. focuses on the effect of inorganic and organic fertilization on soil enzyme activities and microbial community. It is well-organized. I would recommend acknowledging the limitations of the study, but the results are valuable.
(1) Numerous studies have studied the seasonal effects of N fertilization and crop rotation. The author team should include some literature on this field in the Introduction section. Again, this study is not novel.
(2) The statistics could be improved. Repeated measurement ANOVA should be used to evaluate crop, sampling time, and N fertilization effect for soil enzymes and microbial community.
(3) Change the table 1 and 2 to figures. It is very hard to look for patterns in the tables.
(4) The discussion of crop effect is mixed with sampling time effect. We had no idea if the crop affects soil enzymes and microbial community in the literature.
Author Response
Manuscript by Zhang et al. focuses on the effect of inorganic and organic fertilization on soil enzyme activities and microbial community. It is well-organized. I would recommend acknowledging the limitations of the study, but the results are valuable.
Response: We thank the reviewer for their positive assessment of the manuscript. We carefully considered the reviewers’ comments and have revised our manuscript accordingly.
(1) Numerous studies have studied the seasonal effects of N fertilization and crop rotation. The author team should include some literature on this field in the Introduction section. Again, this study is not novel.
Response: We have added some literatures in lines 55-61 at the introduction.
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(2) The statistics could be improved. Repeated measurement ANOVA should be used to evaluate crop, sampling time, and N fertilization effect for soil enzymes and microbial community.
Response: The statistics have been improved. The repeated measurement ANOVA were used. The related information were added in line 145-148, and the results were shown in table 1 in lines 188-192.
(3) Change the table 1 and 2 to figures. It is very hard to look for patterns in the tables.
Response: Many thanks for the reviewer’s efforts. Apologized for the unclear tables 1 and 2. We have changed them to figures 1 and 2 respectively, and hope that we made it clear this time.
(4) The discussion of crop effect is mixed with sampling time effect. We had no idea if the crop affects soil enzymes and microbial community in the literature.
Response: We thank the reviewer for the thoughtful comment. Apologized for the misunderstanding and misleading for you. We double checked the data, and make sure that crop species significantly affect soil microbial community and enzyme activities. According Table 1 with the repeated measurement ANOVA, the soil microbial community were significantly affected by crop species (wheat/maize) and treatment (T) rather than year (2011, 2012 and 2013). The soil enzyme activities were significantly affected by crop species and year and to a lesser extent by treatment. We have revised the text according to the reviewer’s comment in line 243-271. We hope the discussion has improved substantially.
Round 2
Reviewer 2 Report
no
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
This study investigates how the replacement of inorganic fertilizer by compost and straw influence crop yield, soil fertility and microbial community. They found that the replacement of inorganic fertilizer by compost and straw can maintain or enhance crop productivity probably through their effects on soil fertility and microbial community. However, (1) details on the use of straw return should be added in the introduction to provide sufficient background; (2) the hypothesis should be revised based on the introduction and experimental design; (3) improve the results because there are a lot of inaccurate expression; (4) add a core sentence in each paragraph and improve the flow of ideas.
Specific comments:
Line 2-4, please revise the title and show the novelty or most important findings in this paper.
Line 24-26, please avoid the use of “affect” or influence but show the trends (eg. decrease or increase).
Line 35, “enhance” should be revised as the use of manure and straw did not always increased crop yield.
Line 43, “N deposition” is not the way by which chemical fertilizers lead to deterioration of the environment.
Line 101 -105, I think the novelty of this study is to investigate the temporal change in microbial community structure and extracellular enzyme activities, and their relationship with crop yield under different fertilization practices in a wheat-maize rotation system. Besides, Line 95-96 indicate that the objective of the present study was to monitor…. Thus, the hypothesis might be revised here other than focusing on the comparison of wheat and maize under organic manures.
Line 124, please explain “V12 stage”
Line 126-129, please explain why compost was applied at the rate of 6000 kg ha-1.
Line 129, please explain the benefits of straw return and why straw return is combined with inorganic NPK or compost in the introduction.
Line 225, replace “The Fertilization” by “Fertilization”
Line 226-227, please re-write this sentence because maize yield in the control (T1) plots were not always significantly lower than those in the fertilization (see Table 1, Maize yield in 2012 and 2013).
Line 227-229, to me, these results were interesting and important. These indicate that replace of some inorganic fertilizer by manure and straw can maintain the yield.
Line 230, please delete “Application of fertilizers increased soil nutrient contents (Table 1)” as it is an inaccurate expression.
Line 237-238, inaccurate expression. Soil pH was lower in T1 than T2 in two years (wheat in 2011 and maize 2013)?
Line 242, please add the abbreviation explanations here. For example, AP, soil alkaline phosphatase…..
Line 247 -249, this was incorrect. It seems fertilization did not significantly affect content of bacterial PLFAs and F/B ratio.
Line 270-271, inaccurate expression again. It seems fertilization did not later invertase activity at the wheat harvest in 2012. I will not talk about the inaccurate expression. Please check the expression and revise throughout the manuscript. In this sentence, please delete the first “at the wheat harvest”.
Line 294, please add the abbreviation explanations here
Line 311, please start each paragraph with a key (core) sentence in the discussion.
Line 335, please show this result at least in the supporting materials
Line 345-346, the effect of compost on soil P was discussed in this paragraph. Why did you conclude that “integration of straw amendment with compost can be a good management approach to increase crop yields and reduce N losses”? please add some discussion on straw amendment and their effects on N losses.
Line 348-349, add citations or data from the present experiment
Line 359-361, please add more details to illustrate that “manure application in combination with straw has been proposed to be the best management option….”. for instance, compare the effects of manure-straw mixture application with manure or straw application; the data on both soil fertility and yield should be included.
Line 429-430, if you did not conduct a statistic analysis on this results, please delete this conclusion.
Author Response
Line 2-4, please revise the title and show the novelty or most important findings in this paper.
Response: We have revised the title.
Line 24-26, please avoid the use of “affect” or influence but show the trends (eg. decrease or increase).
Response: We have changed.
Line 35, “enhance” should be revised as the use of manure and straw did not always increased crop yield.
Response: We have changed the word “enhance” to “maintain”.
Line 43, “N deposition” is not the way by which chemical fertilizers lead to deterioration of the environment.
Response: We agreed with you for this point and have changed“N deposition” to“N leaching”.
Line 101 -105, I think the novelty of this study is to investigate the temporal change in microbial community structure and extracellular enzyme activities, and their relationship with crop yield under different fertilization practices in a wheat-maize rotation system. Besides, Line 95-96 indicate that the objective of the present study was to monitor…. Thus, the hypothesis might be revised here other than focusing on the comparison of wheat and maize under organic manures.
Response: The hypothesis have been modified.
Line 124, please explain “V12 stage”
Response: V12 stage was that twelfth leaf emerged with ligule visible.
Line 126-129, please explain why compost was applied at the rate of 6000 kg ha-1.
Response: The application rate of compost was based on conventional farming practice.
Line 129, please explain the benefits of straw return and why straw return is combined with inorganic NPK or compost in the introduction.
Response: We have added the information about straw return in the introduction.
Line 225, replace “The Fertilization” by “Fertilization”
Response: We have changed.
Line 226-227, please re-write this sentence because maize yield in the control (T1) plots were not always significantly lower than those in the fertilization (see Table 1, Maize yield in 2012 and 2013).
Response: We have modified the sentence.
Line 227-229, to me, these results were interesting and important. These indicate that replace of some inorganic fertilizer by manure and straw can maintain the yield.
Response: Yes, we agreed with you.
Line 230, please delete “Application of fertilizers increased soil nutrient contents (Table 1)” as it is an inaccurate expression.
Response: We have deleted the sentence.
Line 237-238, inaccurate expression. Soil pH was lower in T1 than T2 in two years (wheat in 2011 and maize 2013)?
Response: Application of chemical fertilizer (T2) significantly decreased the soil pH compared with the control at wheat season in 2011 and maize season in 2013 but organic fertilization had no significant influence on soil pH value
Line 242, please add the abbreviation explanations here. For example, AP, soil alkaline phosphatase…..
Response: The abbreviation explanations have been added to the text.
Line 247 -249, this was incorrect. It seems fertilization did not significantly affect content of bacterial PLFAs and F/B ratio.
Response: Compared to T2, the application of manure (T3) showed a significantly lower content of bacterial PLFAs and higher F/B ratio at wheat season in 2012 (Table 2).
Line 270-271, inaccurate expression again. It seems fertilization did not later invertase activity at the wheat harvest in 2012. I will not talk about the inaccurate expression. Please check the expression and revise throughout the manuscript. In this sentence, please delete the first “at the wheat harvest”.
Response: Thank you for your comments. We have modified throughout the manuscript.
Line 294, please add the abbreviation explanations here
Response: We have added.
Line 311, please start each paragraph with a key (core) sentence in the discussion.
Response: The key sentences for each paragraph were added in the discussion.
Line 335, please show this result at least in the supporting materials
Response: We have changed this sentence.
Line 345-346, the effect of compost on soil P was discussed in this paragraph. Why did you conclude that “integration of straw amendment with compost can be a good management approach to increase crop yields and reduce N losses”? please add some discussion on straw amendment and their effects on N losses.
Response: The conclusion of this paragraph was that the management of P must also be ensured over the long term when applying organic fertilizer.
Line 348-349, add citations or data from the present experiment
Response: The citation was added to the text.
Line 359-361, please add more details to illustrate that “manure application in combination with straw has been proposed to be the best management option….”. for instance, compare the effects of manure-straw mixture application with manure or straw application; the data on both soil fertility and yield should be included.
Response: We have added more information.
Line 429-430, if you did not conduct a statistic analysis on this results, please delete this conclusion.
Response: We have conducted a statistic analysis on this results.
Reviewer 2 Report
You propose a short time field research on aspects with little research in the past, as you state. However, in discussion you refer about field researchs over time laps of 31 years, 30 years, 10 years. What is the special topic and new outcome of your attempt? It is well known that organic amendments support the essentials biological/microbial activities and improve consequently physical and chemical soil properties. You regional case study may be an argument to convince farmers and especially decision markers to turn toward organic manuring, a necessity for a sustainable soil use as well known by soil scientists and most agrnomists.
My questions:
Use and cropping before the start of the plot experiment, before October 2010? Give the information, and the information if the whole plot device, all plots, all treatment were established at a location with the same history. (see also line 329 in discussion)
Soil sampling at the start of the experiment, October 2010? Is of importance as you refer in line 176 about the soil pH 7,24 and give soil pH data in Table 1 far away from this value (up 8,54 in June 2011, 6 months later). Such a pH change is not possible under adequate soil use. So it would be of importance to discover and analyze the general development in the 6 starting months of the experiment, due to management changes after possibly a long time of constant management.
Field experiment: give the mode of soil management, so tillage (only rotary tillage prior to sowing? Deep of tillage?)+ pesticides amounts etc, litter management, residue management. Harvest of maize: ears or whole plants? Handling of residues?
Soil sampling time. Give the time lag between harvest and the sampling. Of importance as roots and maybe other plants residues are freshly decayed and support a new and intense microbial activity, or not so if a long time between harvest and sampling occurred. The validation of your results depends also about this aspect: short time values when residues are the support of activities, or indicators for soil statues when residues were degraded, or only the easily available components degraded.
Soil sample treatments:
Sieved 3 mm: wet soil?
Sieved 2 mm: wet soils?
Removing of roots and visible plant residues if given in the samples before or in course of sieving ? (maize residues are often visible several months after mixing into the soil, and the degradation was surely limited in your case given the dryness in wheat season, with only 100, 132, 107 mm precipitation in the 3 seasons)
Importance of presence of particulate org. matter for enzymatic activities, given the biofilm of microbes on the surface of residues, importance for SOM values, as particular OM was included in analysis by dichromate oxidation. So, your SOM values should possibly not be used as indicator for real soil OM and SOM development.
The initial SOM was 13,7 g.kg-1. In Table 1 you have still a decrease for wheat (not T5 2011, and just in 2013 higher values. I would be of importance to valuate SOM development to know and consider the values of October 2010. The validation of the fertilizing regimes became possible on that basis.
Line 159: using phospholipid fatty acid (PLFA) analysis – to be changed in “using analysis of groups of phospholipid fatty acid (PLFA)” as you use indicators for organism and analyze not at the level of all single PLFAs
Line 184: citation (Kögel-Knabner, 2006) is strange, as not in list, not numbered, and esp. as this scientist is not a specialist for enzymatic analysis, but for SOM and SOM fractions.
Line 193: “incubated at the respective temperature” and Line 196 “All temperature steps were conducted with…” Differing temperatures for differing enzymes? Temperature steps?? Please give valuable citations for these methods and the correct description
Titles table 1,2,3: complete with “in a field plot experiment”
Discussion: to be changed if soil samples contained by analysis all the particulate organic debris from harvest residues, as roots, stems, stalks…., and to be shortened with focus on the experimental region and the local agriculture.
Line 327: “Fertilizer N accumulated mostly in the top 60 cm of the soil profile and NO3--N leaching out of the root zone (top 100 cm) was negligible in wheat, but NO3--N leached out of the root zone during the summer maize season mainly due to high rainfall” Why this explanations, as you sampled 20 cm soil depth. However, line 329 specify “A previous three- year study…” Was 2010 not the start of the study?
Line 414 “not observe significant fertilization effects on urease activity…. indicating that N availability is not a key factor” – urease is a constitutive enzyme of certain microorganisms and is never an indicator for N-status of soils! Please change the statement and explanation.
Abstract: name redundancy analysis and not only the abbreviation RDA)
General remark: would it be possible to change the treatment abbreviations into No for control, M for NPK, CM for compost + NPK, MS for NPK + straw, CMS for compost + NPK + straw?
The lecture of tables and figures would be simplified, as you give no foot note for the abbreviations.
Author Response
Use and cropping before the start of the plot experiment, before October 2010? Give the information, and the information if the whole plot device, all plots, all treatment were established at a location with the same history. (see also line 329 in discussion)
Response: The winter wheat-summer maize was planted before the experiment starting in October 2010.
Soil sampling at the start of the experiment, October 2010? Is of importance as you refer in line 176 about the soil pH 7,24 and give soil pH data in Table 1 far away from this value (up 8,54 in June 2011, 6 months later). Such a pH change is not possible under adequate soil use. So it would be of importance to discover and analyze the general development in the 6 starting months of the experiment, due to management changes after possibly a long time of constant management.
Response: Thank you for your comments. As we all know, the soil nutrient show heterogeneity. And, the field management before the start of the formal experiment was realized by farmers with uneven fertilization, which leading to a larger heterogeneity.
Field experiment: give the mode of soil management, so tillage (only rotary tillage prior to sowing? Deep of tillage?)+ pesticides amounts etc, litter management, residue management. Harvest of maize: ears or whole plants? Handling of residues?
Response: We have added more detailed information.
Soil sampling time. Give the time lag between harvest and the sampling. Of importance as roots and maybe other plants residues are freshly decayed and support a new and intense microbial activity, or not so if a long time between harvest and sampling occurred. The validation of your results depends also about this aspect: short time values when residues are the support of activities, or indicators for soil statues when residues were degraded, or only the easily available components degraded.
Response: We have completed the collection of the harvest and sampling at the same time in our experiment.
Soil sample treatments:
Sieved 3 mm: wet soil?
Sieved 2 mm: wet soils?
Response: The fresh soil samples were sieved (3 mm) for soil Nmin. And the soil was sieved 2 mm after dried.
Removing of roots and visible plant residues if given in the samples before or in course of sieving ? (maize residues are often visible several months after mixing into the soil, and the degradation was surely limited in your case given the dryness in wheat season, with only 100, 132, 107 mm precipitation in the 3 seasons)
Response: All the visible roots in each soil sample were picked out by hand before sieving.
Importance of presence of particulate org. matter for enzymatic activities, given the biofilm of microbes on the surface of residues, importance for SOM values, as particular OM was included in analysis by dichromate oxidation. So, your SOM values should possibly not be used as indicator for real soil OM and SOM development.
Response: Thank you for your comments.
The initial SOM was 13,7 g.kg-1. In Table 1 you have still a decrease for wheat (not T5 2011, and just in 2013 higher values. I would be of importance to valuate SOM development to know and consider the values of October 2010. The validation of the fertilizing regimes became possible on that basis.
Response: Thank you for your comments. As we all know, the soil nutrient show heterogeneity. And, the field management before the start of the formal experiment was realized by farmers with uneven fertilization, which leading to a larger heterogeneity.
Line 159: using phospholipid fatty acid (PLFA) analysis – to be changed in “using analysis of groups of phospholipid fatty acid (PLFA)” as you use indicators for organism and analyze not at the level of all single PLFAs
Response: We have changed.
Line 184: citation (Kögel-Knabner, 2006) is strange, as not in list, not numbered, and esp. as this scientist is not a specialist for enzymatic analysis, but for SOM and SOM fractions.
Response: We have changed.
Line 193: “incubated at the respective temperature” and Line 196 “All temperature steps were conducted with…” Differing temperatures for differing enzymes? Temperature steps?? Please give valuable citations for these methods and the correct description
Response: We have changed.
Titles table 1,2,3: complete with “in a field plot experiment”
Response: We have added the words of “in a field plot experiment”.
Discussion: to be changed if soil samples contained by analysis all the particulate organic debris from harvest residues, as roots, stems, stalks…., and to be shortened with focus on the experimental region and the local agriculture.
Response: Thank you for your comments.
Line 327: “Fertilizer N accumulated mostly in the top 60 cm of the soil profile and NO3--N leaching out of the root zone (top 100 cm) was negligible in wheat, but NO3--N leached out of the root zone during the summer maize season mainly due to high rainfall” Why this explanations, as you sampled 20 cm soil depth. However, line 329 specify “A previous three- year study…” Was 2010 not the start of the study?
Response: Thank you for your comments. We have changed the sentences.
Line 414 “not observe significant fertilization effects on urease activity…. indicating that N availability is not a key factor” – urease is a constitutive enzyme of certain microorganisms and is never an indicator for N-status of soils! Please change the statement and explanation.
Response: We have changed the statement and explanation.
Abstract: name redundancy analysis and not only the abbreviation RDA)
Response: We have added.
General remark: would it be possible to change the treatment abbreviations into No for control, M for NPK, CM for compost + NPK, MS for NPK + straw, CMS for compost + NPK + straw?
The lecture of tables and figures would be simplified, as you give no foot note for the abbreviations.
Response: Thank you for your comments. The abbreviations for the treatments were added to all of the Tables and Figures.
Reviewer 3 Report
1. Please indicate what is new in your research. Write more precisely what was the purpose of this research, which the authors intended to prove.
2. Why the dose of nitrogen for wheat was 300 kg N per ha, and for 250 kg per ha of corn - please explain.
6. Please check the statistical calculations, e.g. in Table 2 Total PLFA for wheat was for T1 - 1.36, and for T5 10.08, and it was the same homogeneous group. If that was the case, then the experience error is disqualifying. The same is true for other markings in this table. Please also check the calculation of homogeneous groups in Fig. 2 - there are errors.1. Conclusions do not result from the experiment.
3. Tables 1 and 2 should be supplemented with the average values from years 2011-2013 crop yields, and soil Nmin, AP, AK, SOM and pH and contents of phospholipid fatty acids, extracellular enzyme activities (GMea). The average values enable correct inference.
4. Table No. 3, please delete - nothing adds to the value of work.
5. Figure 2 - please complete the average values for the activity of individual enzymes from years 2011-2013.
6. Why the dose of nitrogen for wheat was 300 kg N, and 250 kg for maize - please justify.
7. Please check the statistical calculations, eg in table 2 Total PLFA for wheat was for T1 - 1.36, and for T5 - 10.08, and it was the same homogeneous group. If that was the case, then the experience error is disqualifying. The same is true for other markings in this table. Please also check the calculation of homogeneous groups in Fig. 2 - there are errors.
Author Response
1. Please indicate what is new in your research. Write more precisely what was the purpose of this research, which the authors intended to prove.
Response: We have edited the purpose of this research.
2. Why the dose of nitrogen for wheat was 300 kg N per ha, and for 250 kg per ha of corn - please explain.
Response: The application rate of fertilizer for wheat and maize was based on conventional farming practice.
3. Tables 1 and 2 should be supplemented with the average values from years 2011-2013 crop yields, and soil Nmin, AP, AK, SOM and pH and contents of phospholipid fatty acids, extracellular enzyme activities (GMea). The average values enable correct inference.
Response: Thank you for your comments. The average values from years 2011-2013 were added to the Tables1 and 2.
4. Table No. 3, please delete - nothing adds to the value of work.
Response: We have deleted Table 3.
5. Figure 2 - please complete the average values for the activity of individual enzymes from years 2011-2013.
Response: We have added the average values in Figure 2.
6. Why the dose of nitrogen for wheat was 300 kg N, and 250 kg for maize - please justify.
Response: The application rate of fertilizer for wheat and maize was based on conventional farming practice.
7. Please check the statistical calculations, eg in table 2 Total PLFA for wheat was for T1 - 1.36, and for T5 - 10.08, and it was the same homogeneous group. If that was the case, then the experience error is disqualifying. The same is true for other markings in this table. Please also check the calculation of homogeneous groups in Fig. 2 - there are errors.
Response: We have checked the statistical calculations.
Round 2
Reviewer 1 Report
The authors have done a good job and addressed the major concerns. Overall, I think the present version of this manuscript has been improved according to reviewers' comments. It should make an important contribution to the use manure and straw for agricultural sustainability.
Author Response
Response to Reviewer 1 Comments
Point 1: The authors have done a good job and addressed the major concerns. Overall, I think the present version of this manuscript has been improved according to reviewers' comments. It should make an important contribution to the use manure and straw for agricultural sustainability
Response 1: We thank the reviewer for the positive assessment of the manuscript.
Reviewer 2 Report
Comments to Manuscript ID: sustainability-483846-p
They are several doubts in the quality of the analysis and so oft the results.
Given my remark on pH discrepancies (pH 7,24 for the site, pH 8,54 for plots) the authors expect now a larger heterogeneity at the site, without information if one field was used to establish the 20 plots, or several fields.
They gave a similar response considering nutrient heterogeneity: “uneven fertilization, which leading to a larger heterogeneity”.
The authors answer “the field management before the start of the formal experiment was realized by farmers” is strange, as the site information in the manuscript is “at the Quzhou Experimental Station of China Agricultural University”. It seems by the last paper information that scientists were involved in the management, and no single farmers”. Uneven fertilization on an experimental farm?? Extremely questionable, and so also still the pH discrepancies.
Furthermore, the authors add no exhaustive information on the history of the site, and if a preliminary field experiment was realized on that site, and on these plots - despite my request (“However, line 329 specify “A previous three- year study…” Was 2010 not the start of the study?”). The history is of elementary importance to validate changes do to new management. To precise “The winter wheat-summer maize was planted before” without management data is not enough to understand causes of changes and deduce interrelationships.
By soil analysis at the start of the experiment, it had been apparent if the plots are subject to the heterogeneity named now by the authors and are not comparable or if not. The authors added no soil data of the plots at the start of the experiment, October 2010, despite my request. Given such uncertainties in the validity of data, and the doubts about the effects of management vs. soil heterogeneity, the manuscript is not acceptable for publication. I note (fig1) p.ex. that for PLFA biomarkers all treatments were separated by crop species (wheat or maize) in 2011. However, only in the 1st year.
The requested technical changes and additions are ok and most addition give valuable information, so p-ex-for the right and unique temperature for enzymes essays (former manuscript: “incubated at the respective temperature” -“All temperature steps were conducted with…”. However, such important details are not enough to improve the manuscript but the manuscript: he is not acceptable, as explained.
Author Response
Response to Reviewer 2 Comments
Point 1: Given my remark on pH discrepancies (pH 7,24 for the site, pH 8,54 for plots) the authors expect now a larger heterogeneity at the site, without information if one field was used to establish the 20 plots, or several fields.
They gave a similar response considering nutrient heterogeneity: “uneven fertilization, which leading to a larger heterogeneity”.
The authors answer “the field management before the start of the formal experiment was realized by farmers” is strange, as the site information in the manuscript is “at the Quzhou Experimental Station of China Agricultural University”. It seems by the last paper information that scientists were involved in the management, and no single farmers”. Uneven fertilization on an experimental farm?? Extremely questionable, and so also still the pH discrepancies.
Response: We thank the reviewer for the thoughtful comment. Apologized for the misunderstanding and misleading. In fact the land was rented by the local farmers prior to the set-up of our experiment. That is why we answered “uneven fertilization, and the large heterogeneity.” That was demonstrated by the heterogeneity of the seedlings during the first growth season of wheat when we started our experiment.
Point 2: Furthermore, the authors add no exhaustive information on the history of the site, and if a preliminary field experiment was realized on that site, and on these plots - despite my request (“However, line 329 specify “A previous three- year study…” Was 2010 not the start of the study?”). The history is of elementary importance to validate changes do to new management. To precise “The winter wheat-summer maize was planted before” without management data is not enough to understand causes of changes and deduce interrelationships.
Response: Understand very well the reviewer’s comments on this. In 2010 we set up our experiment, and the land was recovered by the experimental station of China Agricultural University. Unfortunately, we did not have detailed information about farmer’s management, as the farmers who rented the land were free to grow any crops they liked, although winter wheat-summer maize was the main crops they grew. We mainly focused on the treatment effects after we set up our experiment.
Point 3: By soil analysis at the start of the experiment, it had been apparent if the plots are subject to the heterogeneity named now by the authors and are not comparable or if not. The authors added no soil data of the plots at the start of the experiment, October 2010, despite my request. Given such uncertainties in the validity of data, and the doubts about the effects of management vs. soil heterogeneity, the manuscript is not acceptable for publication. I note (fig1) p.ex. that for PLFA biomarkers all treatments were separated by crop species (wheat or maize) in 2011. However, only in the 1st year.
Response:
(1) We understood the reviewers’ concern about the heterogeneity vs treatment effects. As explained in above, the soil was “trained” for one growth season to make the soil more homogenously. We are quite confident that the soil was somehow homogenous. But as the soil itself is heterogeneous, as mentioned in many published papers (Li et al., 2018; Scanlan and Davies, 2019; Wallor et al., 2018). Fertilization for example itself could also lead to heterogeneity. The heterogeneity of soil can’t be fully avoided.
Li J, Guo C, Jian S, et al. Nitrogen fertilization elevated spatial heterogeneity of soil microbial biomass carbon and nitrogen in switchgrass and gamagrass croplands. Scientific reports, 2018, 8: 1734.
Scanlan C A, Davies S L. Soil mixing and redistribution by strategic deep tillage in a sandy soil. Soil and Tillage Research, 2019, 185: 139-145.
Wallor E, Kersebaum K C, Lorenz K, et al. A comprehensive data set demonstrating the spatial variability of soil properties and crop growth conditions at field scale. Open Data Journal for Agricultural Research, 2018, 5: 1-10.
(2) The soil data of the plots at the start of the experiment had been added in the line 207-208.
(3) In terms of the results of PLFAs, it is to get a snap-shot of the whole microbial community. That is why the effect of crop species is stronger than that of treatment effects. We also did pyro-sequencing in the field later on. In a similar fashion, we did not observe strong treatment effects, except the addition of organic matters (Bei et al., 2018). These results also surprised us, and our explanations for that were: we may need to do the functional genes not the whole microbial communities, as the soil was trained to mostly grown for wheat and maize. The microbials are adaptable to the cultivation already, if no new inputs (other than materials from wheat/maize, for example new crops etc) are added. That was why we found significance in microbial communities between organic added vs non-added treatments in Bei’s (2018) paper.
Bei S, Zhang Y, Li T, et al. Response of the soil microbial community to different fertilizer inputs in a wheat-maize rotation on a calcareous soil. Agriculture, Ecosystems & Environment, 2018, 260: 58-69.
Point 4: The requested technical changes and additions are ok and most addition give valuable information, so p-ex-for the right and unique temperature for enzymes essays (former manuscript: “incubated at the respective temperature” -“All temperature steps were conducted with…”. However, such important details are not enough to improve the manuscript but the manuscript: he is not acceptable, as explained.
Response: Many thanks for the reviewer’s efforts. We hope that we made it clear this time. The research work is indeed very important, and very instructive for the nutrient management in the wheat-maize rotation system. Our results suggest that (1) adding new crop species (or new materials) are important to modify the microbial-mediated processes. Crop diversification has great significance in nutrient cycling in the system. (2) Nutrient management should integrate the knowledge of microbiome information. If we understand better of the diversity and activities of soil microbiome, this is very likely that we will change the way of how we manage nutrients and cultivate crops in the future.
Reviewer 3 Report
The average results (Table 1) indicate that there were no significant differences between wheat yield from T2-T5 objects, maize yield - from T1-T5 objects and between Nmin, AK, SOM and pH contents in T1-T2 facilities. Also, the average results from Table 2 indicate no significant differences between T1-T2 objects in wheat and maize soil in the content of: total PLFA, PLFA bacteria, PLFA fungi and F/B and GMea activity in maize soil. At the same time, the results from PLFA show a large error in the analysis. This information has been omitted from the text of the manuscript and should be included in the results chapter. They should also be included in the abstract and conclusions.
Author Response
Response to Reviewer 3 Comments
Point 1: The average results (Table 1) indicate that there were no significant differences between wheat yield from T2-T5 objects, maize yield - from T1-T5 objects and between Nmin, AK, SOM and pH contents in T1-T2 facilities. Also, the average results from Table 2 indicate no significant differences between T1-T2 objects in wheat and maize soil in the content of: total PLFA, PLFA bacteria, PLFA fungi and F/B and GMea activity in maize soil. At the same time, the results from PLFA show a large error in the analysis. This information has been omitted from the text of the manuscript and should be included in the results chapter. They should also be included in the abstract and conclusions.
Response:
(1) We thank the reviewer for the thoughtful comment. Having considered their suggestions, we have added the information of Tables 1 and 2 into the results, abstract and conclusions.
(2) The data of the PLFA were tested again and log-transformed to achieve normality of distribution and homogeneity of variance before conducting ANOVA.
Round 3
Reviewer 2 Report
I will give some reasons for my proposition toward Sustainability after reviewing the manuscript 483846.
I am soil scientist with specialization in soil ecology, esp. microbial ecology, considering at first microbial functions and transformations, and the ability of soils to maintain and to express the functions at various scales, esp considering differing managements types and numerous weather impacts from dry to waterlogged, form hot to frozen.
I asked the authors about the discrepancies in pH values. Soils are regulated by pH-buffers, and generally have long time a stable pH-range. A change from pH 7,24 (as given in general description of the site) up to pH 8,54 in a short time lap by only the impact of a little variation in management (authors information: the same crop rotation occurred before) is questionable. The authors gave as response and as cause of the value the heterogeneity in the field. Despite my requirement, they never gave soil data from the beginning of the field experiments, an absolute prerequisite to monitor changes in time. Later, after 2nd reviewing, they tried to inform about the generality of soil heterogeneity. However, they were not busy at a field scale, with specific sampling, but they installed plots for experiments on a field, so that each plot was a unit and had to be analyzed also at time zero. They considered not their own argumentation based on field heterogeneity.
I was long time head of a research farm in a hilly landscape in Bavaria, with a high soil heterogeneity in each field given the topography and differing hydrological conditions in the landscape. This heterogeneity was analyzed by remote sensing approaches, characterized about soil conditions, and soil zones without great heterogeneity were used for plots where possible. In general, we used usual statistical methods to overcome heterogeneities in data, when due to field heterogeneity. Each of our numerous field experiments started with a field/soil sampling at time zero. It is a simple rule of experiments and a necessity toward monitoring and validation of effects of impacts. In the last response of the authors, they state (2) The soil data of the plots at the start of the experiment had been added in the line 207-208.”. However in V3 no new data in lines 207-208, or in other parts of the manuscript. I consider this style as not serious.
As senior scientist with long time of research and teaching, I has of course my own insight and concepts to understand microbial functionality in soils, being not only soil microbiologist focusing on microbes, their DNA, their enzymes, but considering the (physical) habitats, their steering role for microbial functions (and populations) by the (highly variable) habitat conditions. Soil are generally limited in energy, and soils activate only microbial populations they need for actual demands to realize the actual functionality. Each change, in moisture, in input of organics (manure or litter or root exudates…) leads to activation of specific populations. The resting part gets in dormancy and is not quantifiable p.ex. by PLFA analysis. Of relevance for estimation of soil quality by microbiota analysis is the total biodiversity in soils, so that, at each time, the needed populations may be activated. Of importance is to keep this biodiversity, to conserve the resiliency after end of temporally changed conditions, to keep the redundancy to maintain functions by changing of conditions. To conclude on negative impacts when populations (PLFA) or enzymes differ in comparison to a given stage is wrong. However, it is the usual interpretation of so-called soil microbiologists, microbiologists with a performing arsenal of new techniques and equipment, but without real knowledge on soils, on soil functioning, on functioning and role of microbial communities in soils. In my meaning, the authors understand not microbial soil ecology and try to prove that organic amendment is favorable for microbiota. It is evident since knowledge about microbes in soils, that to feed them is beneficial for their biomass development and their activities and is a simple necessity. The live in the soil-biogeosystem needs food, nutrients and energy, like other organisms. We have many examples worldwide for soil degradation by management without return of organic matter, without support of soil biota.
Do they analyses the microbiota involved in residue decomposition or root associated, and not microbiota representative for soil status? In fact, they sampled immediately after harvest (“at the time of harvest”), so that may produce just a short time picture in soil functioning during the high activity of beginning substrate decomposition (wheat : residues/roots after generative state; maize ears harvested, residues/root also poor in nutrients – despite “All the visible roots in each soil sample were picked out by hand”, soil material in contact to organic particles and attached to fine roots was sampled). Amounts and qualities of residues/root biomass steer such activities and related populations. When statement on soils should be issued, soil scientists would sample generally after decomposition of residues, possibly few months after harvest.
I note and explain also in my comments to the authors, that urease is never an indicator for protein decomposition ability. However, they state (line 279 in v3) “ The activity of the N-acquisition enzyme (urease)….”. Hereby it would be interesting to know the scientific domains of the authors.
I will also repeat, that the first description of the enzymatic methods was misleading, with errors. Hereby I had first doubts about the serious work of the authors.
In my opinion, the newly added text parts in V3 are not a main improvement of the manuscript.
I resume: strange pH change explained by soil heterogeneity, no consideration of soil heterogeneity by data analysis, missing data for time zero, no data addition despite affirmation of data addition “time zero” in response to the reviewer.
I add: in line 154, related to soil sampling, the statement: “One portion was air-dried for analysis of soil physico-chemical properties”. I see no data on soil physical analysis, beside the affirmation in site description “silt loam texture”.
Sorry for this long enumeration of personal details. I get the opportunity to work on a broad spectrum in agroecosystems from landscape scale, analyzed by improved technics, to microbiota and microcompartments.
