the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 13 May 2026
Impact of perennial forage plants on microbial carbon use efficiency in weathered Ethiopian soils
Abstract. Aims: Ethiopian soils are highly weathered and in urgent need of remediation. Increasing the soil carbon (C) pool by perennials potentially remediates soil degradation while sequestering atmospheric CO2. The soil microbial loop is thought to play an important role in soil C sequestration as it contributes significantly to the stabilization of plant litter C. The amount of stabilized litter C has been hypothesized to depend on microbial carbon use efficiency (mCUE), which is sensitive to soil nutrients and plant inputs. Here, we investigate the effect of two perennial forage crops on mCUE in four weathered Ethiopian soils.
Methods: We cultivated the grass Urochloa cv. Cayman as single stand and in mixture with the legume Desmodium intortum in a fully factorial greenhouse experiment with four distinct farmers’ soils from the Ethiopian Rift Valley (Amhara and Sidama). Microbial CUE was assessed after 104 days of plant growth using 18O-H2O incorporation.
Results: Microbial CUE varied between the four soils, with Amhara soils showing lower mCUE than Sidama soils. Overall, mCUE was little affected by plant growth but increased significantly with plant dry weight (DW) in one of the two Sidama soils. Mass specific growth rates remained small in Amhara soils, indicating that factors other than plant C input inhibited microbial growth. Plant composition had no effect in Amhara soils while the Urochloa × Desmodium mixture tended to have lower mCUE in Sidama soils.
Conclusions: Perennials may improve mCUE in weathered soils, but this stimulation strongly depends on soil nutrient availability and suggests that there are critical nitrogen (N) and phosphorous (P) thresholds below under which mCUE does not respond to growing plants. This implies that highly degraded soils must be ameliorated before using perennials to stimulate nutrient cycling and restore soil health.
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RC1: 'Comment on egusphere-2026-2488', Anonymous Referee #1, 16 Jul 2026
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AC1: 'Reply on RC1', Niklas Wickander, 14 Oct 2026
Reviewer 1
This paper quantifies the effect of two types of perennial forage crops on microbial carbon use efficiency (CUE) in four highly weathered soils. Soils were collected from four different Farming Training Centers in Ethiopia, which allowed the authors to study the effects of perennial crops on microbial CUE across different soil physicochemical features.
This paper integrated a 104 day fully factorial greenhouse experiment to examine plant dry weight, microbial CUE, mass-specific growth rate, respiration, and microbial biomass carbon across soils planted with Urochloa cv. Cayman as single stand and in mixture with the legume Desmodium intortum. The paper uses linear regression and two-way ANOVA to examine the effect of the predictor variables and their interactions on the response variables. The results are overall robust and contribute to knowledge of microbial CUE under different soils under plant treatments. However, not all assumptions and conclusions were clear or supported by the data. Notably, the effects of soil and plant type could not be isolated, nor could the effects of soil pH and soil phosphorus, and critical nitrogen thresholds were not explicitly explained but assumptions were made in the discussion, conclusion, and abstract. After these minor revisions, and further suggestions detailed below, I recommend this article to be accepted for publication.
We thank the reviewer for the overall positive response to our manuscript. We address direct comments below one by one and believe they will improve the quality of the revised manuscript.
- Does the paper address relevant scientific questions within the scope of SOIL?
Yes, the paper addresses the important topic of soil remediation in highly weathered soils using plant remediation approaches to improve microbial CUE as microbial CUE may control the stabilization of soil litter C. The authors measured the effect of perennial plant treatment, soil, and their interactions on microbial carbon use efficiency. Additional microbial metrics were measured including mass-specific growth, respiration, and microbial biomass carbon.
- Does the paper present novel concepts, ideas, tools, or data?
Yes, this article contributes to existing work that seeks to address how plant remediation approaches can influence microbial CUE, which is hypothesized to regulate soil litter C stabilization. The research is conducted on four highly weathered agricultural Ethiopian soils that are amended with fertilizer and used for maize production. This work contributed to advancing knowledge on modern agricultural practices by utilizing four different soils from Farmers’ Training Centers in Ethiopia to explore plant remediation effects on microbial CUE.
- Does the paper address soils within a multidisciplinary context?
Yes, soils here are addressed through the lens of soil fertility, agricultural sciences, climate change science, and microbiology through examining the effects of plant remediation using two perennial crops on microbial CUE.
- Is the paper of broad international interest?
This paper would be of broad interest to farmers, microbiologist, ecologist, climate change scientist, soil scientist, and politicians as it relates to soil sustainability and crop production, and general audience as it relates to improving soil health through plant remediation approaches.
- Are clear objectives and/or hypotheses put forward?
Yes, the author's motivation for the work and aims were clearly laid out. The hypotheses are well laid out at the end of the introduction, which were 1) microbial CUE and response to plant inputs depend on soil properties, with nutrient poor soils having a limited response , 2) legume inclusion would increase microbial CUE by alleviating nitrogen limitation, 3) plant functional diversity exerts a greater influence on microbial CUE than plant biomass.
- Are the scientific methods valid and clear outlined to be reproduced?
Yes, each methodology has a clear subheader, followed by description, formulas, and citations.
- Is the soil type/classification adequately described?
Soil pH, soil textural class, CEC, and certain nutrients are well described, however there is no overarching soil classification data. Soil texture class is used interchangeably here with soil type, but these are two different things. If possible, I think it would be helpful to list the soil classification as soil type, or make it clear the authors refer to a soils textural class as the soil type. Including soil classification data may yield more insight into plant and microbial responses to plant remediation strategies since soils in the different regions actually had overlapping soil texture classes.
We will add the soil classification to the manuscript and be more consistent with the use of soil texture class and soil type in the revised manuscript.
- Are analyses and assumptions valid?
Yes, the analysis is generally valid. However, the assumptions and conclusions could use additional clarification. Since microbial CUE was not able to be measured in the unplanted soil controls, the authors could not isolate the effect of the soil from the effect of the potted plants. The soils from each site were sampled in five locations (corner and center) at depths between 0 - 25 cm, which could be mixing different soil layers with unique physicochemical properties. Additionally, the authors did not mention why the data was appropriate for linear regression and two-way ANOVA.
We agree that the results can only be interpreted in relation to each other and not as definitive effects. This is a consequence of the failed mCUE analysis of the control soils; DNA extraction was not successful in the control soils, despite measures taken to increase the DNA yield, likely because microbial biomass was very small in unplanted soil.
In theory, we may have mixed different soil layers by sampling soil down to 25 cm. However, all soils were highly weathered, having deep, homogeneous soil horizons. We therefore believe that mixing layers of soil with different properties is a minor problem in the present study. In any case, all four soils were sampled in the same way, dried and shipped and homogenized before potting. In this way, results are valid relative to each other.
We will add an explanation to justify the choice of linear regression and two-way ANOVA. We used linear regression to search for trends between increased DW biomass and mCUE and the two-way ANOVA to assess how the soil and plant simultaneously affected mCUE.
- Are the presented results sufficient to support the interpretations and associated discussion?
It is not clear how the analysis supports the conclusion that highly degraded Sub-Saharan soils must be amended with phosphorus fertilizer before plant remediation approaches occur. The soils that had higher microbial CUE did have more P, but they also had higher pH which the authors noted in the discussion. Since the effects of soil pH from soil phosphorus could not be isolated, the statement that P amendment must occur before plant remediation approaches warrants further investigation.
Overall, the conclusion is based on the difference in mCUE between the two regions the soils were sampled from. The major differences were soil pH and available P (Melich III). We deemed the difference in P availability to be the ruling factor for mCUE as it was shown to affect the stoichiometric nutrient balance and microbial nutrient demand in these soils in a companion experiment (Wickander et al., 2026). We will address the ambiguity of P availability and pH in more detail in the revised manuscript.
It is also unclear why the authors stated that there are critical nitrogen (N) thresholds below which microbial CUE does not respond to growing plants in the abstract. Can the authors state this threshold? Total nitrogen was highest in soils with the lowest microbial CUE so this statement could use revision to make it more clear.
This is out of place in the abstract conclusion and will be rephrased in the R1. We speculated about this threshold in the discussion as literature suggests that N availability is a major constraint for mCUE, not just by fertilizer input, but also by plant input. We speculate that this is an explanation to the effect observed in the mixture treatment.
- Is the discussion relevant and backed up?
The discussion is well written but could benefit from clearer sub sections. The authors note that the significant correlation between microbial CUE and plant biomass could suggest that increased biomass is a driving force for increasing microbial CUE if P is available. Yet, microbial CUE in Baricha soils that had high P was not significantly correlated with plant biomass. The authors state that the perennial mixture significantly lowered mCUE in one soil, yet there was no change in microbial respiration.
We will define clearer sub-sections.
This is a good point, the effect of plant biomass was generally limited on mCUE, with only Bilate showing a significant correlation, which is also the soil which saw a significant decrease in mCUE from the plant mixture. One reason for these inconsistent responses may be soil specific condition as the mixtures had lower DW biomass in the Bilate than in the Boricha soil, which may have affected the overall plant effect on mCUE. The statement of the plant DW biomass effect is based on the regional variation, with low-P soils (Amhara) having less biomass, and high-P soils (Sidama) having larger biomass, thus affecting the C and N input in these soils. By contrast, Boricha and Bilate soils were too similar to show measurable differences between soils. Here, differences arose from differences in plant biomass accumulation between treatments. We will clarify this in the discussion section.
Additionally, we discuss on L. 328 - 335 that the likely reason for the absence of a visible effect on soil respiration is the allocation of C input from the roots being different in the plant mixture compared to the monoculture.
Line 298 - 299 - soils from the Amhara and the Sidama region had overlapping soil textural classes (silt loam) so the statement that different soil textures may contribute to different microbial CUE should be revised to clarify that the same soil textural classes had different responses based on clay content.
We will reformulate this to highlight the clay content rather than the soil texture class.
- Are accurate conclusions reached based on the presented results and discussion?
Please see section 6 and section 7.
See the reply to section 6 and 7.
- Do the authors give proper credit to related and relevant work and clearly indicate their own original contribution?
Yes
- Does the title clearly reflect the contents of the paper and is it informative?
Yes
- Does the abstract provide a concise and complete summary, including quantitative results?
Yes, however the importance of legumes for microbial CUE is not established in the introductory section of the abstract but it is an important component of the approach and results. The abstract conclusion sentences about nitrogen thresholds and fertilizer amendment strategy should be revised as the results and discussion did not clearly state the necessary nitrogen threshold for plant remediation to be successful. Without isolating the effects of pH and phosphorous content, it's unclear if soils should be amended to increase pH or increase phosphorus.
We will revise the abstract introduction to highlight the effects of legumes and reformulate the conclusion to tone down the critical N-threshold, pH and P.
- Is the overall presentation well structured?
Yes.
- Is the paper written concisely and to the point?
Yes, the paper is well written and clear, with clearly laid out importance statements, aims, hypothesis, and approach.
- Is the language fluent, precise, and grammatically correct?
Yes
- Are the figures and tables useful and all necessary?
Yes
- Are mathematical formulae, symbols, abbreviations, and units correctly defined and used according to the author guidelines?
Yes
- Should any parts of the paper (text, formulae, figures, tables) be clarified, reduced, combined, or eliminated?
It's not clear if the authors measured respiration in the unplanted controls (methods section:). It is clear they could not measure microbial CUE or mass-specific growth, but can it be stated whether respiration was measured in unplanted controls?
It was measured for the unplanted controls, but for the same reason as mCUE, the results were omitted due to the low quality of the data. We will state this clearly in the manuscript.
The caption for Figure 3 could be more descriptive by explaining that regression equations are shown based on p-value significance and linear regression was done on a per pot basis. The light color lines and small text are somewhat difficult to see in Figure 3, especially in panel C that is crowded with the legend and panel D where the equation is in small light pink colored text. Can the color legend be shifted outside of the four panels?
We will look it over and amend the figure layout to improve the readability.
The legend for figure 4 states that different letters indicate significance, but no letters are shown.
Thank you for pointing this out. We forgot to add the letters of significance and will do that.
- Are the number and quality of references appropriate?
Yes
- Is the amount and quality of supplementary material appropriate and of added value?
There is no supplementary table. A table of all statistical analysis used to test the effect of the predictor variable and interaction of predictor variables on the response variable would help summarize the data.
We will add a supplementary table for this.
Citation: https://doi.org/10.5194/egusphere-2026-2488-AC1
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AC1: 'Reply on RC1', Niklas Wickander, 14 Oct 2026
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RC2: 'Comment on egusphere-2026-2488', Jeroen Zethof, 07 Sep 2026
Dear authors,
I read with great interest your work on microbial Carbon Use Efficiency in four Ethiopian soils and how this is affected by the native Urochloa, with and without the legume Desmodium. Overall, the manuscript is well and clearly written. However, a few points were encountered that needs to be cleared up or improved. Also the statistical analysis is limited and for instance the regressions were only tested on single treatments, no combinations of them. For example from Figure 3C and D, one could expect a significant difference between regions, but not plant species.
It is not completely clear why the four soils were (initially) chosen, i.e. in which aspect were the expected to differ? Especially as they all have similar total N and C:N ratios, it needs to be better explained why the legume (an N-fixer) was chosen in your experiment. Were there also (subjective) observations for the legume performance? If I understood it right, the Dry Weights in Figure 1 includes for both the grass and legume. Sometimes the soils are also grouped per regions and sometimes evaluated as 4 individual soils. Please ensure consistency.
Minor points for improvement:
As the legume was only tested in combination with the grass Urochloa, it is not a “fully factorial experiment” (see lines 20, 87) Please revise.
Line 94: “N co-limitation” -> Either focus on N-limitation or better explain the co-limiting factors that you expect.
Line 229: “On a single pot basis” What is meant here? For individual pots there was a correlation? Linear mixed effects models with random effect can overcome the problem of different “baselines” between individual pots.
Line 242-245: Not really clear where this is referring to as Fig. 2B clearly shows significant differences and Fig. 3B is not sufficient to claim “a significant difference between the two plant treatments in Abiyot Fana”. The significant differences could be further tested.
Figure 3: Please indicate in the figure description why only these regressions are displayed in the figures.
Line 291: “Melich III” -> This method was not mentioned in the method section, please include (“Mehlich III”)
Line 378: “must be phosphorous amended” -> This advice is a bit out of nowhere and was not (directly) tested in this study. Also other potential nutrients were not discussed, therefore it would be better to generalize your statement.
Citation: https://doi.org/10.5194/egusphere-2026-2488-RC2 -
AC2: 'Reply on RC2', Niklas Wickander, 14 Oct 2026
Dear authors,
I read with great interest your work on microbial Carbon Use Efficiency in four Ethiopian soils and how this is affected by the native Urochloa, with and without the legume Desmodium. Overall, the manuscript is well and clearly written. However, a few points were encountered that needs to be cleared up or improved. Also the statistical analysis is limited and for instance the regressions were only tested on single treatments, no combinations of them. For example from Figure 3C and D, one could expect a significant difference between regions, but not plant species.
We thank reviewer #2 for the feedback, which will help to improve our manuscript. We omitted the interaction in the linear regression as the main point was to test the specific effects in the four soils, and not variation between regions, which were already obvious. However, if the reviewer wishes, we can perform and add an interaction analysis.
It is not completely clear why the four soils were (initially) chosen, i.e. in which aspect were the expected to differ? Especially as they all have similar total N and C:N ratios, it needs to be better explained why the legume (an N-fixer) was chosen in your experiment. Were there also (subjective) observations for the legume performance? If I understood it right, the Dry Weights in Figure 1 includes for both the grass and legume. Sometimes the soils are also grouped per regions and sometimes evaluated as 4 individual soils. Please ensure consistency.
The soils and plants were selected based on careful considerations within the “Ethiopia-Grass” project, which aimed to explore the feasibility of using perennial grasses and mixtures in rotation with cereals for soil amelioration in the Great Rift valley. Field experiments were run in two pedo-climatically distinct regions (Amhara, Sidama) differing in parent soil materials. The plant varieties were selected based on their forage potential and growth habits. We will clarify this in more detail in the R1 version.
Reported DW biomasses are the sum of both grasses and legumes, but we can add a visual assessment on the plant performance and mixture composition.
We will check and ensure consistency when we group the soils regionally. Often, this was done to highlight the variation across regions, and when comparing all four soils it is to highlight that regardless of region there is variability between the soils.
Minor points for improvement:
As the legume was only tested in combination with the grass Urochloa, it is not a “fully factorial experiment” (see lines 20, 87) Please revise.
Good point, we will revise this
Line 94: “N co-limitation” -> Either focus on N-limitation or better explain the co-limiting factors that you expect.
We will revise this sentence to explain that we refer to P.
Line 229: “On a single pot basis” What is meant here? For individual pots there was a correlation? Linear mixed effects models with random effect can overcome the problem of different “baselines” between individual pots.
We mean that each dot in the regression represents one pot, and the correlation then appears from plotting each pot. We will revise this to better explain what we mean.
Line 242-245: Not really clear where this is referring to as Fig. 2B clearly shows significant differences and Fig. 3B is not sufficient to claim “a significant difference between the two plant treatments in Abiyot Fana”. The significant differences could be further tested.
Fig 2B refers to the difference according to plant treatment. Fig 3B refers to the differences according to plant DW biomass, and there we see a correlation in one of the soils. We will revise the paragraph to improve readability.
Figure 3: Please indicate in the figure description why only these regressions are displayed in the figures.
We will add a sentence explaining this.
Line 291: “Melich III” -> This method was not mentioned in the method section, please include (“Mehlich III”)
We will mention in the materials and methods that Mehlich III was used?
Line 378: “must be phosphorous amended” -> This advice is a bit out of nowhere and was not (directly) tested in this study. Also other potential nutrients were not discussed, therefore it would be better to generalize your statement.
We will revise the conclusion.
Citation: https://doi.org/10.5194/egusphere-2026-2488-AC2
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AC2: 'Reply on RC2', Niklas Wickander, 14 Oct 2026
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- 1
Principal criteria
Excellent (1)
Good (2)
Fair (3)
Poor (4)
Scientific significance:
Does the manuscript represent a substantial contribution to scientific progress within the scope of SOIL (novel concepts, theories, methods, or data; interdisciplinary)?
2
Scientific quality:
Are the scientific approach and applied methods valid and reproducible? Are the results discussed in a thorough and balanced way (consideration of related and relevant work, including appropriate references)?
2
Presentation quality:
Are the scientific results and conclusions presented in a clear, concise, and well-structured way (appropriate number and quality of figures/tables, appropriate use of English)?
1
Summary and recommendation:
This paper quantifies the effect of two types of perennial forage crops on microbial carbon use efficiency (CUE) in four highly weathered soils. Soils were collected from four different Farming Training Centers in Ethiopia, which allowed the authors to study the effects of perennial crops on microbial CUE across different soil physicochemical features.
This paper integrated a 104 day fully factorial greenhouse experiment to examine plant dry weight, microbial CUE, mass-specific growth rate, respiration, and microbial biomass carbon across soils planted with Urochloa cv. Cayman as single stand and in mixture with the legume Desmodium intortum. The paper uses linear regression and two-way ANOVA to examine the effect of the predictor variables and their interactions on the response variables. The results are overall robust and contribute to knowledge of microbial CUE under different soils under plant treatments. However, not all assumptions and conclusions were clear or supported by the data. Notably, the effects of soil and plant type could not be isolated, nor could the effects of soil pH and soil phosphorus, and critical nitrogen thresholds were not explicitly explained but assumptions were made in the discussion, conclusion, and abstract. After these minor revisions, and further suggestions detailed below, I recommend this article to be accepted for publication.
Yes, the paper addresses the important topic of soil remediation in highly weathered soils using plant remediation approaches to improve microbial CUE as microbial CUE may control the stabilization of soil litter C. The authors measured the effect of perennial plant treatment, soil, and their interactions on microbial carbon use efficiency. Additional microbial metrics were measured including mass-specific growth, respiration, and microbial biomass carbon.
2. Does the paper present novel concepts, ideas, tools, or data?
Yes, this article contributes to existing work that seeks to address how plant remediation approaches can influence microbial CUE, which is hypothesized to regulate soil litter C stabilization. The research is conducted on four highly weathered agricultural Ethiopian soils that are amended with fertilizer and used for maize production. This work contributed to advancing knowledge on modern agricultural practices by utilizing four different soils from Farmers’ Training Centers in Ethiopia to explore plant remediation effects on microbial CUE.
3. Does the paper address soils within a multidisciplinary context?
Yes, soils here are addressed through the lens of soil fertility, agricultural sciences, climate change science, and microbiology through examining the effects of plant remediation using two perennial crops on microbial CUE.
4. Is the paper of broad international interest?
This paper would be of broad interest to farmers, microbiologist, ecologist, climate change scientist, soil scientist, and politicians as it relates to soil sustainability and crop production, and general audience as it relates to improving soil health through plant remediation approaches.
5. Are clear objectives and/or hypotheses put forward?
Yes, the author's motivation for the work and aims were clearly laid out. The hypotheses are well laid out at the end of the introduction, which were 1) microbial CUE and response to plant inputs depend on soil properties, with nutrient poor soils having a limited response , 2) legume inclusion would increase microbial CUE by alleviating nitrogen limitation, 3) plant functional diversity exerts a greater influence on microbial CUE than plant biomass.
6. Are the scientific methods valid and clear outlined to be reproduced?
Yes, each methodology has a clear subheader, followed by description, formulas, and citations.
7. Is the soil type/classification adequately described?
Soil pH, soil textural class, CEC, and certain nutrients are well described, however there is no overarching soil classification data. Soil texture class is used interchangeably here with soil type, but these are two different things. If possible, I think it would be helpful to list the soil classification as soil type, or make it clear the authors refer to a soils textural class as the soil type. Including soil classification data may yield more insight into plant and microbial responses to plant remediation strategies since soils in the different regions actually had overlapping soil texture classes.
8. Are analyses and assumptions valid?
Yes, the analysis is generally valid. However, the assumptions and conclusions could use additional clarification. Since microbial CUE was not able to be measured in the unplanted soil controls, the authors could not isolate the effect of the soil from the effect of the potted plants. The soils from each site were sampled in five locations (corner and center) at depths between 0 - 25 cm, which could be mixing different soil layers with unique physicochemical properties. Additionally, the authors did not mention why the data was appropriate for linear regression and two-way ANOVA.
9. Are the presented results sufficient to support the interpretations and associated discussion?
It is not clear how the analysis supports the conclusion that highly degraded Sub-Saharan soils must be amended with phosphorus fertilizer before plant remediation approaches occur. The soils that had higher microbial CUE did have more P, but they also had higher pH which the authors noted in the discussion. Since the effects of soil pH from soil phosphorus could not be isolated, the statement that P amendment must occur before plant remediation approaches warrants further investigation.
It is also unclear why the authors stated that there are critical nitrogen (N) thresholds below which microbial CUE does not respond to growing plants in the abstract. Can the authors state this threshold? Total nitrogen was highest in soils with the lowest microbial CUE so this statement could use revision to make it more clear.
10. Is the discussion relevant and backed up?
The discussion is well written but could benefit from clearer sub sections. The authors note that the significant correlation between microbial CUE and plant biomass could suggest that increased biomass is a driving force for increasing microbial CUE if P is available. Yet, microbial CUE in Baricha soils that had high P was not significantly correlated with plant biomass. The authors state that the perennial mixture significantly lowered mCUE in one soil, yet there was no change in microbial respiration.
Line 298 - 299 - soils from the Amhara and the Sidama region had overlapping soil textural classes (silt loam) so the statement that different soil textures may contribute to different microbial CUE should be revised to clarify that the same soil textural classes had different responses based on clay content.
11. Are accurate conclusions reached based on the presented results and discussion?
Please see section 6 and section 7.
12. Do the authors give proper credit to related and relevant work and clearly indicate their own original contribution?
Yes
13. Does the title clearly reflect the contents of the paper and is it informative?
Yes
14. Does the abstract provide a concise and complete summary, including quantitative results?
Yes, however the importance of legumes for microbial CUE is not established in the introductory section of the abstract but it is an important component of the approach and results. The abstract conclusion sentences about nitrogen thresholds and fertilizer amendment strategy should be revised as the results and discussion did not clearly state the necessary nitrogen threshold for plant remediation to be successful. Without isolating the effects of pH and phosphorous content, it's unclear if soils should be amended to increase pH or increase phosphorus.
15. Is the overall presentation well structured?
Yes.
16. Is the paper written concisely and to the point?
Yes, the paper is well written and clear, with clearly laid out importance statements, aims, hypothesis, and approach.
17. Is the language fluent, precise, and grammatically correct?
Yes
18. Are the figures and tables useful and all necessary?
Yes
19. Are mathematical formulae, symbols, abbreviations, and units correctly defined and used according to the author guidelines?
Yes
20. Should any parts of the paper (text, formulae, figures, tables) be clarified, reduced, combined, or eliminated?
It's not clear if the authors measured respiration in the unplanted controls (methods section:). It is clear they could not measure microbial CUE or mass-specific growth, but can it be stated whether respiration was measured in unplanted controls?
The caption for Figure 3 could be more descriptive by explaining that regression equations are shown based on p-value significance and linear regression was done on a per pot basis. The light color lines and small text are somewhat difficult to see in Figure 3, especially in panel C that is crowded with the legend and panel D where the equation is in small light pink colored text. Can the color legend be shifted outside of the four panels?
The legend for figure 4 states that different letters indicate significance, but no letters are shown.
21. Are the number and quality of references appropriate?
Yes
22. Is the amount and quality of supplementary material appropriate and of added value?
There is no supplementary table. A table of all statistical analysis used to test the effect of the predictor variable and interaction of predictor variables on the response variable would help summarize the data.