The Immense Functional Attributes of Maize Rhizosphere Microbiome: A Shotgun Sequencing Approach
Round 1
Reviewer 1 Report
Akinola et al. have submitted a manuscript entitled “The immense functional attributes of maize rhizosphere microbiome: a shotgun sequencing approach”. The authors analyze maize rhizosphere and bulk soil microbial communities in two semi-arid locations of the North West province of South Africa, using shotgun metagenomics. This works aims at expanding the knowledge regarding the function of microorganisms in semi-arid soils, which is an important endeavor in light of global climate change. Nevertheless, I have many issues with this work, which I will detail below. I would also recommend that you revise extensively the English language throughout the manuscript.
Major comments
One of my main points of concern is regarding the results presented in the figures 3 and 5. In the material and methods you mention that three replicates were taken per site x rhizosphere/bulk soil combination. Therefore I was expecting to have 12 points on the PCA and PCoA. Why is this not the case? You also provide no justification for this. In any case, in this manner we cannot judge if all the samples from one site are similar or if the variation between replicates is smaller than the variation between sites. Please provide this data.
Another point of concern is the erroneous interpretation of PCA. In lines 259-260 you wrote: “PCA showed that there was a significant difference between the rhizosphere and surrounding soil samples (c. 96.15%).” A PCA alone cannot prove significant differences between samples or groups, for this you can employ other methods such as PERMANOVA. Also, the percentage of variability explained by both axis does not translate the differences between samples! For this you can calculate a distance measure, e.g. Bay-Curtis distances and compare them between samples.
In Figure S1 you provide a description of the taxonomical composition of the samples, but this is neither much explored (you do not even present alpha diversity measure on this) nor well linked to the functional profiling. For example, fungi are not really mentioned, although they are major players driving ecosystem processes
In the material and methods you do not explain how you collected the rhizosphere soil and what exactly what you considered rhizosphere soil. Please provide this detailed information. Rhizosphere typically spans only a few millimeters around plant roots (can be a little larger for big tree roots) and therefore it is important that you characterize how you make a distinction between this environment and the bulk soil.
I noticed that many of your references are not appropriate to fundament your statements. In lines 248-249 you wrote: “Because microorganisms inhabiting Lc are numerous, it is expected to witness a sizable reduction in the functional attributes of the community [21,22].” First you imply here that there are more microorganism in Lc than on the other locations, but I see no evidence from this. Moreover, I couldn’t find any information that supported this in the provided references. Another example is in lines 268-271: “This assertion can be supported by the abundance of microbial classes such as Deltaproteobacteria, Gammaproteobacteria, Bacilli and Planctomycetacia in Rs-site (Figure S1). Several studies have reported the tremendous contributions of these microorganisms to the improvement of plant growth [24,25].” One of the provided references focuses on the actinobacterial genus Frankia, which is not one of the mentioned bacteria. The other work referenced is focused on lake biofilms, which are very distinct environments and are not directly linked to plants. Another case is found in lines 282-283: “This is highly expected because soils with little or no plants harbor more microbes (Figure S1) [17].” The reference focuses on soil physicochemical parameters and not on microbial communities. On lines 302-304 you wrote: “It has been confirmed that environmental factors, most particularly those related to physicochemical properties drives soil microbiota diversity and structure [27].” The reference provided deals with influence of soil properties on termite mound stability and again, not with microbial communities. There are plenty of references that indeed show this, such as: Fierer, N. 2017. Embracing the unknown: disentangling the complexities of the soil microbiome. Nature Reviews Microbiology, doi: 10.1038/nrmicro.2017.87.
I also found some contradictions in your reasoning, especially in line 276-280. Here you start by saying that there was no difference on metabolic categories between areas, but right after this you claim that there is a clear separation of the sites in the PCA. Please clarify this. Also, please add why this is relevant for the environment and if this is in accordance with your expectations. This is merely a result, which you do not fundament or discuss. I also found lacking an overview on the similarities between the rhizosphere samples. Since this is about the same plant species growing in similar conditions, I would expect that the rhizosphere samples would have many similarities, but this was not a focus on this manuscript.
On lines 263-266 you mention that several functional hits were distinctive for Rs maize, but I from Figure 2 and Table S1, I see this only being true for Secondary metabolism, and even this seems to not be statistically different from Rc.
At the beginning of the Discussion you state: “Here we describe the diverse functional attributes of major taxonomic groups contributing to maize rhizosphere community performance.” This is not the case as you do not link taxonomic groups to functions.
One final aspect was that I found only limited comparison with other studies. How does this work compare to others in arid or semi-arid soils? How does this compare to other plant species?
Minor comments
Lines 11-12: “However, a lot is still needed from enormous functions possessed by rhizosphere microbiome…” This sentence is very unspecific. What is needed, in which context? Please clarify/rephrase.
Line 32: Substitute “Rhizosphere microorganisms occupy area” by “Rhizosphere microorganisms occupy the area”
Line 38: “Soil microbiota increases as a result of organic compound secretion…” What increases, number, diversity? Please be more specific.
Line 45: What you mean with “frequent disturbances”?
Line 47: Substitute “exhausting reserved soil nutrient.” by “exhausting soil nutrient reserves.”
Line 48: Substitute “mainly” by “are” and “Since” by “Therefore”
Line 50: What do you mean by likes? This is not clear.
Line 51: Please clarify what you mean with “plant soil” (do you mean rhizosphere soil?) and “potency of agricultural soils” (do you mean productivity?).
Line 55: Substitute “at” by “on the”
Line 58: Substitute “system” by “systems”
Lines 62-63: Substitute “Therefore, to maintain the level of maize production in NW and South Africa as a whole, frequent calls for sustainable land-use practices are needed.” by “Therefore, to maintain the level of maize production in NW and South Africa there is a need for sustainable land-use practices.”
Material and Methods: When were the samples taken? As mentioned above, please give more details on how the soil was collected.
Lines 86-87: Please rephrase the following: “and average yearly rainfall for both towns is between April and November with approximately 365 mm in depth.”
Line 89: What you mean by “relatively distinct plant communities”? How different are they? Was this an observation from when you sampled or is this based on vegetation records for this area?
Line 95: You didn’t determine any physical soil properties, only chemical. Please remove the mention on “physical” here and throughout the text.
Line 132: Substitute “conical” by “canonical”
Figure 1: Please add to the legend what the whiskers on the bars mean. Is it standard error, standard deviation, confidence intervals? Also, please explain what the different star symbols are.
Line 155: Substitute “mainly” by “the main”
Line 167: Substitute “insignificantly” by “not”
Line 168: You mention “The most prominent hits in the samples…” Which samples are these, all the samples?
Lines 171-172: Substitute “…activities were tremendously high…” by “…activities were higher…”
Figure 2: Please mention in the legend that these results were obtained from MG-RAST. It would also be beneficial to add the number of genes that belong to each category.
Line 186: Substitute “was higher” by “was significantly higher”
Lines 186-187: Please rephrase the following sentence: “In the case of cold shock, the rhizosphere soils were 500% higher than the surrounding samples.”
Figure 4: Figure 4a comes only after Table 2 and it is not together with 4b. Also, please add to the legend what the whiskers on the bars mean. Is it standard error, standard deviation, confidence intervals?
Line 194: In table 2 I see only alpha diversity measures described and not beta diversity, as it shows in the text. Please check this.
Figure 5: Is this based on the functional categories SEED 1 or SEED 2? If the input data is the same as for the PCA of Figure 3, then this figure and analysis are redundant and should be removed from the manuscript. Please add in the legend of the figure on which distance measure the PCoA was based.
Figure 6: You mention in the legend: “…and functional categories of both samples.” This was only for two samples? If so, which ones? This was not clear to me.
Table 3: Please remove either the “Explains %” or “Contribution %” column, as they contain the same information.
Line 219: You refer to “Ecogenomics”, a term that is not addressed in the Introduction.
Line 226: Please clarify what you mean by: “…was below the standard (6–7.2) that indicates the balance in the available soil nutrients.” From where did you derive these values?
Line 228: Please clarify what you mean by: “…were within the acceptable level…”. What is an acceptable level? Why is that the case.
Line 237-239: Please rephrase this sentence.
Line 224: Please clarify what you mean by: “…attained its theoretical limit of 2.81…” Where does this value come from?
Line 245: You mention that it was expected that Lc would have lower functional diversity. Why would this be the case? I did not see a reasoning for this anywhere in the manuscript.
Line 252: Substitute “was too low” by “was low”
Lines 255-262: The first two sentences of this paragraph are concerning plant hormones and how they help the plant. This information is disconnected from the rest of the paragraph. Please rephrase/restructure this paragraph.
Lines 274-275: Please rephrase this sentence, I could not understand what it was meant by it.
Line 252: Substitute “infestation” by “colonization”
Author Response
Title: The immense functional attributes of maize rhizosphere microbiome: a shotgun sequencing approach
Article ID: Agriculture-103307
Accompanying letter
Here is the response to Reviewers’ comments
Referee: 1
Comments to Author
Comments and Suggestions for Authors
Akinola et al. have submitted a manuscript entitled “The immense functional attributes of maize rhizosphere microbiome: a shotgun sequencing approach”. The authors analyze maize rhizosphere and bulk soil microbial communities in two semi-arid locations of the North West province of South Africa, using shotgun metagenomics. This works aims at expanding the knowledge regarding the function of microorganisms in semi-arid soils, which is an important endeavor in light of global climate change. Nevertheless, I have many issues with this work, which I will detail below. I would also recommend that you revise extensively the English language throughout the manuscript.
Major comments
One of my main points of concern is regarding the results presented in the figures 3 and 5. In the material and methods you mention that three replicates were taken per site x rhizosphere/bulk soil combination. Therefore I was expecting to have 12 points on the PCA and PCoA. Why is this not the case? You also provide no justification for this. In any case, in this manner we cannot judge if all the samples from one site are similar or if the variation between replicates is smaller than the variation between sites. Please provide this data.
Response: The replicates have been shown on the figures and they are denoted by different colors in the figures.
Another point of concern is the erroneous interpretation of PCA. In lines 259-260 you wrote: “PCA showed that there was a significant difference between the rhizosphere and surrounding soil samples (c. 96.15%).” A PCA alone cannot prove significant differences between samples or groups, for this you can employ other methods such as PERMANOVA. Also, the percentage of variability explained by both axis does not translate the differences between samples! For this you can calculate a distance measure, e.g. Bay-Curtis distances and compare them between samples.
Response: The Statement has been corrected. Likewise, Bay-Curtis distances was used for the analysis of similarities (ANOSIM) as used to confirm differences showed by PCoA. The changes made is shown in line 265-268.
In Figure S1 you provide a description of the taxonomical composition of the samples, but this is neither much explored (you do not even present alpha diversity measure on this) nor well linked to the functional profiling. For example, fungi are not really mentioned, although they are major players driving ecosystem processes.
Response: Microorganisms presented were sorted based on their hits using the SEED subsystem identification. However, only the most abundant organisms were presented in the figure.
In the material and methods you do not explain how you collected the rhizosphere soil and what exactly what you considered rhizosphere soil. Please provide this detailed information. Rhizosphere typically spans only a few millimeters around plant roots (can be a little larger for big tree roots) and therefore it is important that you characterize how you make a distinction between tis environment and the bulk soil.
Response: A comprehensive description of sampling method has been added in line 92-94 of the revised manuscript. Soil samples that were tightly bound to the roots were denoted as rhizosphere soil.
I noticed that many of your references are not appropriate to fundament your statements. In lines 248-249 you wrote: “Because microorganisms inhabiting Lc are numerous, it is expected to witness a sizable reduction in the functional attributes of the community [21,22].” First you imply here that there are more microorganism in Lc than on the other locations, but I see no evidence from this.
Response: This can be viewed on Figure S1. For easy identification, red colors on z-score denotes the least abundance and only few classes can be seen under Lc.
Moreover, I couldn’t find any information that supported this in the provided references. Another example is in lines 268-271: “This assertion can be supported by the abundance of microbial classes such as Deltaproteobacteria, Gammaproteobacteria, Bacilli and Planctomycetacia in Rs-site (Figure S1). Several studies have reported the tremendous contributions of these microorganisms to the improvement of plant growth [24,25].” One of the provided references focuses on the actinobacterial genus Frankia, which is not one of the mentioned bacteria. The other work referenced is focused on lake biofilms, which are very distinct environments and are not directly linked to plants.
Response: Relevant references have been used to replace those highlighted in red.
Another case is found in lines 282-283: “This is highly expected because soils with little or no plants harbor more microbes (Figure S1) [17].” The reference focuses on soil physicochemical parameters and not on microbial communities.
Response: The reference has been replaced.
On lines 302-304 you wrote: “It has been confirmed that environmental factors, most particularly those related to physicochemical properties drives soil microbiota diversity and structure [27].” The reference provided deals with influence of soil properties on termite mound stability and again, not with microbial communities. There are plenty of references that indeed show this, such as: Fierer, N. 2017. Embracing the unknown: disentangling the complexities of the soil microbiome. Nature Reviews Microbiology, doi: 10.1038/nrmicro.2017.87.
Response: Suggested reference has been adopted.
I also found some contradictions in your reasoning, especially in line 276-280. Here you start by saying that there was no difference on metabolic categories between areas,
Response: The first statement revealed that there was no significant difference WITHIN samples (i.e. replicates) using Pielou evenness and Shannon indices (α-diversity)
but right after this you claim that there is a clear separation of the sites in the PCoA.
Response: The PCoA was used to depict the differences BETWEEN samples and controls which was confirmed using analysis of similarities (ANOSIM) with p = 0.01 and R = 0.58 showing the differences between samples (β-diversity).
Please clarify this. Also, please add why this is relevant for the environment and if this is in accordance with your expectations. This is merely a result, which you do not fundament or discuss. I also found lacking an overview on the similarities between the rhizosphere samples. Since this is about the same plant species growing in similar conditions, I would expect that the rhizosphere samples would have many similarities, but this was not a focus on this manuscript.
Response: The relevance of the study to the environment has been added and the result discussed. The similarities between rhizosphere samples have also been discussed.
On lines 263-266 you mention that several functional hits were distinctive for Rs maize, but I from Figure 2 and Table S1, I see this only being true for Secondary metabolism, and even this seems to not be statistically different from Rc.
Response: Statement has been reconstructed and all insignificant metabolic attributes removed.
At the beginning of the Discussion you state: “Here we describe the diverse functional attributes of major taxonomic groups contributing to maize rhizosphere community performance.” This is not the case as you do not link taxonomic groups to functions.
Response: The statement has been modified.
One final aspect was that I found only limited comparison with other studies. How does this work compare to others in arid or semi-arid soils? How does this compare to other plant species?
Response: A better improvement has been done on the literature review and results compared with studies such as Chukwuneme, et al. [30], García‐Salamanca, et al. [21], and Fierer, N. (2017).
Minor comments
Lines 11-12: “However, a lot is still needed from enormous functions possessed by rhizosphere microbiome…” This sentence is very unspecific. What is needed, in which context? Please clarify/rephrase.
Response: Statement rephrased.
Line 32: Substitute “Rhizosphere microorganisms occupy area” by “Rhizosphere microorganisms occupy the area”
Response: Statement corrected
Line 38: “Soil microbiota increases as a result of organic compound secretion…” What increases, number, diversity? Please be more specific.
Response: Statement changed to ‘’the number of important soil microbiota increases’’
Line 45: What you mean with “frequent disturbances”?
Response: phrase deleted
Line 47: Substitute “exhausting reserved soil nutrient.” by “exhausting soil nutrient reserves.”
Response: Statement changed
Line 48: Substitute “mainly” by “are” and “Since” by “Therefore”
Response: Correction adopted
Line 50: What do you mean by likes? This is not clear.
Response: Other plant growth-promoting functions have been added
Line 51: Please clarify what you mean with “plant soil” (do you mean rhizosphere soil?) and “potency of agricultural soils” (do you mean productivity?).
Response: The statement has been corrected
Line 55: Substitute “at” by “on the”
Response: Correction adopted
Line 58: Substitute “system” by “systems”
Response: Corrected
Lines 62-63: Substitute “Therefore, to maintain the level of maize production in NW and South Africa as a whole, frequent calls for sustainable land-use practices are needed.” by “Therefore, to maintain the level of maize production in NW and South Africa there is a need for sustainable land-use practices.”
Response: Correction adopted
Material and Methods: When were the samples taken? As mentioned above, please give more details on how the soil was collected.
Response: A comprehensive method of sample collection has been explained.
Lines 86-87: Please rephrase the following: “and average yearly rainfall for both towns is between April and November with approximately 365 mm in depth.”
Response: Statement reconstructed as ‘’and a seasonal rainfall for both towns is between April and November with approximately 365 mm depth’’.
Line 89: What you mean by “relatively distinct plant communities”? How different are they? Was this an observation from when you sampled or is this based on vegetation records for this area?
Response: It was based on vegetational records and reference given
Line 95: You didn’t determine any physical soil properties, only chemical. Please remove the mention on “physical” here and throughout the text.
Response: The word has been removed throughout the manuscript
Line 132: Substitute “conical” by “canonical”
Response: conical has been changed to canonical
Figure 1: Please add to the legend what the whiskers on the bars mean. Is it standard error, standard deviation, confidence intervals? Also, please explain what the different star symbols are.
Response: Standard deviation bars and significance levels have been added
Line 155: Substitute “mainly” by “the main”
Response: mainly changed to the main
Line 167: Substitute “insignificantly” by “not”
Response: Insignificantly changed to not significantly
Line 168: You mention “The most prominent hits in the samples…” Which samples are these, all the samples?
Response: Changed to ‘’the most prominent hits in the rhizosphere and bulk samples’’
Lines 171-172: Substitute “…activities were tremendously high…” by “…activities were higher…”
Response: Activities were tremendously high changed to activities were higher.
Figure 2: Please mention in the legend that these results were obtained from MG-RAST. It would also be beneficial to add the number of genes that belong to each category.
Response: MG-RAST added to the legend
Line 186: Substitute “was higher” by “was significantly higher”
Response: was higher has been changed to was significantly higher
Lines 186-187: Please rephrase the following sentence: “In the case of cold shock, the rhizosphere soils were 500% higher than the surrounding samples.”
Response: Statement changed
Figure 4: Figure 4a comes only after Table 2 and it is not together with 4b. Also, please add to the legend what the whiskers on the bars mean. Is it standard error, standard deviation, confidence intervals?
Response: Legends indicating standard deviation have been added
Line 194: In table 2 I see only alpha diversity measures described and not beta diversity, as it shows in the text. Please check this.
Response: Yes, only alpha diversity was shown while beta diversity was calculated using ANOSIM- analysis of similarities using Kruskal Wallis.
Figure 5: Is this based on the functional categories SEED 1 or SEED 2? If the input data is the same as for the PCA of Figure 3, then this figure and analysis are redundant and should be removed from the manuscript. Please add in the legend of the figure on which distance measure the PCoA was based.
Response: Principal coordinate analysis (PCoA) is different from PCA. PCoA depict differences between samples and within replicates while PCA reveals the components of each sampling sites. The vector length of the functional hits depicts the functions that influences activities in the sampling sites. They PCoA and PCA serve different functions.
Figure 6: You mention in the legend: “…and functional categories of both samples.” This was only for two samples? If so, which ones?
Response: This was not clear to me. Statement modified to incorporate all samples
Table 3: Please remove either the “Explains %” or “Contribution %” column, as they contain the same information.
Response: Explain % has been removed
Line 219: You refer to “Ecogenomics”, a term that is not addressed in the Introduction.
Response: The term has been addressed.
Line 226: Please clarify what you mean by: “…was below the standard (6–7.2) that indicates the balance in the available soil nutrients.” From where did you derive these values?
Response: The sentence has been rephrased and the word ‘standard’ removed.
Line 228: Please clarify what you mean by: “…were within the acceptable level…”. What is an acceptable level? Why is that the case.
Response: The sentence has been rephrased
Line 237-239: Please rephrase this sentence.
Response: The sentence has been improved
Line 224: Please clarify what you mean by: “…attained its theoretical limit of 2.81…” Where does this value come from?
Response: Theoretical limit was given by CANOCO
Line 245: You mention that it was expected that Lc would have lower functional diversity. Why would this be the case? I did not see a reasoning for this anywhere in the manuscript.
Response: References to back up the assertion have been given
Line 252: Substitute “was too low” by “was low”
Response: The phrase has been corrected
Lines 255-262: The first two sentences of this paragraph are concerning plant hormones and how they help the plant. This information is disconnected from the rest of the paragraph. Please rephrase/restructure this paragraph.
Response: The paragraph has been restructured
Lines 274-275: Please rephrase this sentence, I could not understand what it was meant by it.
Response: Sentence has been improved
Line 252: Substitute “infestation” by “colonization”
Response: Infestation has been changed to colonization
Note: All effected corrections were highlighted in red on the main manuscript.
Olubukola Oluranti Babalola.
Reviewer 2 Report
Remarks for correction:
The usefulness of the results of the work could be better revealed by emphasizing the concrete effect in terms of environmental and economic benefits.
Manuscript visualization needs to be adjusted. Image resolution parameters are of poor quality. Need to review all.
Each picture contains many characters, abbreviations. Therefore, detailed explanations of abbreviations (Lc, RNA-M etc.) symbols (* and etc.) must be provided with the names of the pictures.
It is useful to highlight in manuscript (in results or conclusion part) and specify what will be discovered and how it will serve the world from an environmental and economic point of view if you achieve your goal? (This study aims to assess a metagenomic comparison of major metabolic attributes of two bread 13rhizosphere soils and surrounding soils as their control.).
It is not entirely correct to compare the parameters of different units of measurement in one and the same histogram ... Fig.1.
FIG. 1. Poor presentation of the confidence interval, indicating only the maximum values of the mean scatter +. It needs to be corrected and clarified. It is not explained what the intervals marked with asterisks represent and what they want to prove with them?
Why are pH, Nitrate (mg kg-1), Total Carbon (%) values compared? Such a comparison is incorrect.
It is worth considering a more accurate presentation of Figure 1.
It is necessary to adjust the presentation of units. mg / kg is not written. Need to change to mg kg-1. Throughout the manuscript text, it is necessary to review and present the units properly.
Also keep the same presentation style throughout the text, as the same is now presented chaotically differently everywhere. For example, 50 g (in 81 line), Twenty (20) grams (in 96 line) and so on. Why is it different?
Also keep the same presentation style throughout the text, as the same. It is presented chaotically differently everywhere now. For example, 50 g (in 81 line), Twenty (20) grams (in 96 line) and so on...
Please explain in more detail and detail in the methodology how the assessment of the diversity of soil functional features, CANOCO determinations, etc. is performed in order to be able to repeat the intentions.
All visualizations should be explained and followed by the figures in Fig.1, Table 1, Figs.
2, FIG. 4, Table 2, FIG. 5, FIG. 6, Table 3 must be submitted your analysis and evaluation of the results obtained. There are nothing now. Overall chapters can not end with a table or figure must be a summary, conclusion, opinion.
To consider whether at all necessary tables 2 and 3, containing very little information. It can be written in one or more sentences in the text or attached to another visual material.
Figures 2 and 3 need to be made clearer and more informative. it is necessary to provide all explanations under the title of the picture for more clear understanding for the reader. Now the images are overcrowded and don’t show off what you want to show. Please mark or somehow distinguish the essential moments and places.
The abbreviated explanations given in Figure 2 are not available. Improperly reloaded in the wrong place. Explanations of symbols can be provided next to the Figure title or in the text. The left-handed gamma of the interface also shows nothing clearly. It is necessary to highlight the essence.
Figure 4. a and b. the information is incorrect because what is written is not provided. There is no picture a at all there. Fig. 4 is not given in the usual way, there is no parameter unit or detailed explanation of the exact values. When parameter values are very small, for clarity, the smallest values can be given in the second Y axis.
A technical correction is necessary because the figure without the title and numbering before Figure 5 must be moved to the right place. Also in this figure, the presentation is superficial and needs to be improved. Because there is no complete difference between the mean values or they are all equal. In this case, a detailed description and analysis with exact values or a proper graph to ensure that the values are seen or presented in two axes is required.
Figure 5 presents similarities and dissimilarities of the relative abundance of functional annotations between samples. What has been proved by that and for what purpose.
Systematicity and integrity require the same fonts and order for manuscript text and text in all figures.
Substantial deficiency, that this manuscript does not contain conclusions, work and research, evaluation of graph data, summary of results.
Author Response
Title: The immense functional attributes of maize rhizosphere microbiome: a shotgun sequencing approach
Article ID: Agriculture-103307
Accompanying letter
Here is the response to Reviewers’ comments
Reviewer 2
Comments and Suggestions for Authors
Remarks for correction:
The usefulness of the results of the work could be better revealed by emphasizing the concrete effect in terms of environmental and economic benefits.
Manuscript visualization needs to be adjusted. Image resolution parameters are of poor quality. Need to review all.
Response: The resolution of the images has been improved.
Each picture contains many characters, abbreviations. Therefore, detailed explanations of abbreviations (Lc, RNA-M etc.) symbols (* and etc.) must be provided with the names of the pictures.
Response: Explanation of parameters has been provided beneath the figure
It is useful to highlight in manuscript (in results or conclusion part) and specify what will be discovered and how it will serve the world from an environmental and economic point of view if you achieve your goal? (This study aims to assess a metagenomic comparison of major metabolic attributes of two bread 13rhizosphere soils and surrounding soils as their control.).
Response: The discovery of novel organisms (bioinoculants) capable of ameliorating menace posed by soil stressors could help improve agricultural output in arid and semi-arid soils
It is not entirely correct to compare the parameters of different units of measurement in one and the same histogram ... Fig.1.
Response: Different units of measurement were not compared. Only values of same parameters from different samples were compared
FIG. 1. Poor presentation of the confidence interval, indicating only the maximum values of the mean scatter +. It needs to be corrected and clarified. It is not explained what the intervals marked with asterisks represent and what they want to prove with them?
Response: The asterisks have used to indicate significant difference between values and has been explained in the text
Why are pH, Nitrate (mg kg-1), Total Carbon (%) values compared? Such a comparison is incorrect.
Response: Each parameter was compared between samples not compared together
It is worth considering a more accurate presentation of Figure 1. It is necessary to adjust the presentation of units. mg / kg is not written. Need to change to mg kg-1. Throughout the manuscript text, it is necessary to review and present the units properly.
Response: Units have been corrected to mgkg-1
Also keep the same presentation style throughout the text, as the same is now presented chaotically differently everywhere. For example, 50 g (in 81 line), Twenty (20) grams (in 96 line) and so on. Why is it different?
Response: All measurements have been presented same way
Also keep the same presentation style throughout the text, as the same. It is presented chaotically differently everywhere now. For example, 50 g (in 81 line), Twenty (20) grams (in 96 line) and so on...
Response: All measurements have been presented same way
Please explain in more detail and detail in the methodology how the assessment of the diversity of soil functional features, CANOCO determinations, etc. is performed in order to be able to repeat the intentions
Response: It has been stated that the MG-RAST and CANOCO default settings were used throughout the analyses.
All visualizations should be explained and followed by the figures in Fig.1, Table 1, Figs.
Response: All figures and tables have been explained serially
2, FIG. 4, Table 2, FIG. 5, FIG. 6, Table 3 must be submitted your analysis and evaluation of the results obtained. There are nothing now. Overall chapters can not end with a table or figure must be a summary, conclusion, opinion.
Response: Summary of the tables and figures have been explained during discussion session
To consider whether at all necessary tables 2 and 3, containing very little information. It can be written in one or more sentences in the text or attached to another visual material.
Response: Table 3 is the summary of CANOCO inference from fig. 6 and could be best explained as a separate table
Figures 2 and 3 need to be made clearer and more informative. it is necessary to provide all explanations under the title of the picture for more clear understanding for the reader. Now the images are overcrowded and don’t show off what you want to show. Please mark or somehow distinguish the essential moments and places.
Response: Figures have been improved as suggested and all explanations inserted
The abbreviated explanations given in Figure 2 are not available. Improperly reloaded in the wrong place. Explanations of symbols can be provided next to the Figure title or in the text. The left-handed gamma of the interface also shows nothing clearly. It is necessary to highlight the essence.
Response: Details on all symbols have been discussed under 3.3 in methodology section
Figure 4. a and b. the information is incorrect because what is written is not provided. There is no picture a at all there. Fig. 4 is not given in the usual way, there is no parameter unit or detailed explanation of the exact values. When parameter values are very small, for clarity, the smallest values can be given in the second Y axis. Figures 4a and b have been well explained.
Response: Further information on differences between functional hits can be seen in Table S2 (Supplementary section)
A technical correction is necessary because the figure without the title and numbering before Figure 5 must be moved to the right place. Also in this figure, the presentation is superficial and needs to be improved. Because there is no complete difference between the mean values or they are all equal. In this case, a detailed description and analysis with exact values or a proper graph to ensure that the values are seen or presented in two axes is required.
Response: Figures have been moved to the right place and description of result expatiated
Figure 5 presents similarities and dissimilarities of the relative abundance of functional annotations between samples. What has been proved by that and for what purpose.
Response: α-diversity is required to show the differences within samples (i.e. the replicates) while β-diversity was used to explore differences between the samples (i.e. rhizosphere and bulk samples). Wide separations in the sampling sites indicates differences between the sampling sites and was confirmed using analysis of similarity (ANOSIM) p = 0.01 and R = 0.58.
Systematicity and integrity require the same fonts and order for manuscript text and text in all figures.
Response: Manuscript text has been arranged with same fonts.
Substantial deficiency, that this manuscript does not contain conclusions, work and research, evaluation of graph data, summary of results.
Response: The conclusions, graphs and results interpretation have been improved
Note: All effected corrections were highlighted in red on the main manuscript.
Olubukola Oluranti Babalola.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
Remarks for correction:
Most comments were not taken into account and not corrected. In response to comments, amendments must be tabled after the responses. It is not enough just to answer, but it has to be corrected or fulfilled in the manuscript too. It was tried to do it a little bit, but very timidly. I ask you to thoroughly and boldly adjust, modify and fill more useful information in the missing parts in order to make your manuscript of a higher quality and scientific level. According to the comments:Point 1:
The usefulness of the results of the work could be better revealed by emphasizing the concrete effect in terms of environmental and economic benefits. It is useful to write few sentences or paragraph about practical worth in real practice and effect for environmental and economic.
Point 2:
Manuscript visualization needs to be adjusted. Image resolution parameters are of poor quality. Need to review all. You must save in the required special format so that the images first retain their proper shape and are not distorted.
Point 3:
Each picture contains many characters, abbreviations. Therefore, detailed explanations of abbreviations (Lc, RNA-M etc.) symbols (* and etc.) must be provided after the names of the pictures. Because explained just stars now.
Point 4:
It is useful to highlight in manuscript (in results or conclusion part) and specify what will be discovered and how it will serve the world from an environmental and economic point of view if you achieve your goal? (This study aims to assess a metagenomic comparison of major metabolic attributes of two bread 13rhizosphere soils and surrounding soils as their control.). Your explanation have to be in the manuscript text.
Point 5:
It is not entirely correct to compare the parameters of different units of measurement in one and the same histogram ... Fig.1. If only values of same parameters from different samples were compared. Much better it would be presented in separate graphs or at least split on two Y axes not on one as of now because of unclear values.There is not HSD - calculated significant difference limit. It is needed to show these values too. Also in figure b useful more concrete Y axis, for example it could be numbered between 20, not 100.
Point 6 :
Fig. 1. a and b incorrect confidence intervals in all columns, indicating only the plus values of the confidence interval. It needs to be corrected.
Point 7:
After every figure and table have to be explanations and analysis of one paragraph or at least several sentences about the results given in every figure and table. A chapter or subsection can never end with a table or figure.
Point 8:
It is worth considering a more accurate presentation of Figure 1. It is necessary to adjust the presentation of units. mg / kg is not written. Need to change to mg kg-1. Throughout the manuscript text, it is necessary to review and present the units properly.
Point 9:
Also keep the same presentation style throughout the text, as the same is now presented chaotically differently everywhere. For example, 50 g (in 81 line), Twenty (20) grams (in 96 line) and so on. It was not done, because in line 98 it was also written twice - Twenty grams (20g) - it was not needed. All measurements have been presented same way in all text and not needed write surplus twice.
Point 10:
Please explain in more detail and detail in the methodology how the assessment of the diversity of soil functional features, CANOCO determinations, etc. is performed in order to be able to repeat the intentions
It useful to write in the manuscript text.
Point 11:
2, FIG. 4, Table 2, FIG. 5, FIG. 6, Table 3 must be submitted your analysis and evaluation of the results obtained. There are nothing now. Overall chapters can not end with a table or figure must be a summary, conclusion, opinion.
Every figure and table must be explained in more detail in the appropriate manuscript locations. It was not enough results summary of the tables and figures in discussion session. It is not the same and it is not good for the reader. At the end could be all results and information abstraction.
Point 12:
To consider whether at all necessary tables 2 and 3, containing very little information. It can be written in one or more sentences in the text or attached to another visual material. If the Table 3 is the summary of CANOCO inference from fig. 6 and could be best explained as a separate table. So it could be combined together od for more better way.
Point 13:
Figures 2 and 3 need to be made clearer and more informative. it is necessary to provide all explanations under the title of the picture for more clear understanding for the reader. Now the images are overcrowded and don’t show off what you want to show. Please mark or somehow distinguish the essential moments and places. Figures was not changed yet and all information like big text is in the figure 2. It was not show any concrete and clear parts, which is most important. Z score is unreadable and poor quality.
Font sizes in figure 3 in some places very big and must be the same. The same problem in figure 5 too.
Point 14:
The abbreviated explanations given in Figure 2 are not available. Improperly reloaded in the wrong place. Explanations of symbols can be provided next to the Figure title or in the text. The left-handed gamma of the interface also shows nothing clearly. It is necessary to highlight the essence. If details on all symbols have been discussed under 3.3 in methodology section, so it could be easy to make more clear shortening specifically after figure title or in the figure.
Point 15:
Fig. 4 is no parameter unit or detailed explanation of the exact values. When parameter values are very small, for clarity, the smallest values can be given in the second Y axis. Figures 4a and b have been well explained. If it can be seen in Table S2 (Supplementary section), so it need to combine or explain in two places.
Point 16:
Fig 4. Also in this figure, the presentation is superficial and needs to be improved. Because there is no significant difference between the mean values or they are all equal. In this case, a detailed description and analysis with exact values or a proper graph to ensure that the values are seen or presented in two axes is required. Also like in almost all figures and tables there is no description after figure.
Point 17:
Figure 5 presents similarities and dissimilarities of the relative abundance of functional annotations between samples. What has been proved by that and for what purpose. All this information should be written in the manuscript, not just answer.
Point 18:
Systematic and integrity require the same fonts and text size for manuscript text and text in all figures.
Point 19:
Substantial deficiency, that this manuscript does not contain conclusions, work and research, evaluation of graph data, summary of results. It should be results interpretation paragraph after figures and tables, and at the end of every section and subsection (such 3.1, 3.2, 3.3, 3.4, 3.5).
Point 20:
There is no Highlights and Conclusion sections. It should be written.
Note: All effected corrections should be highlighted in the main manuscript using Track Change and in the answer letter copied too.
Author Response
Title: The immense functional attributes of maize rhizosphere microbiome: a shotgun sequencing approach
Article ID: Here is the revised manuscript of Agriculture-103307
Accompanying letter
Thanks so much for the positive criticism. Highlighted herewith is the effected corrections and responses to your comments.
Point 1:
The usefulness of the results of the work could be better revealed by emphasizing the concrete effect in terms of environmental and economic benefits. It is useful to write few sentences or paragraph about practical worth in real practice and effect for environmental and economic.
Response: The environmental impact and economic value of the research has been modified thus;
Our study also suggests a need to explore plant growth-promoting traits of microorganisms present in the sites. The discovery of novel organisms (bioinoculants) capable of reducing the menace posed by soil stressors could serve as an alternative to the use of chemical fertilizers and improve the economic value of agricultural products in arid and semi-arid soils (line 392-395).
Point 2:
Manuscript visualization needs to be adjusted. Image resolution parameters are of poor quality. Need to review all. You must save in the required special format so that the images first retain their proper shape and are not distorted.
Response: The resolution of all the figures has been increased to 800 dpi.
Point 3:
Each picture contains many characters, abbreviations. Therefore, detailed explanations of abbreviations (Lc, RNA-M etc.) symbols (* and etc.) must be provided after the names of the pictures. Because explained just stars now.
Response: Abbreviations and other features have been defined as suggested.
Point 4:
It is useful to highlight in manuscript (in results or conclusion part) and specify what will be discovered and how it will serve the world from an environmental and economic point of view if you achieve your goal? (This study aims to assess a metagenomic comparison of major metabolic attributes of two bread 13rhizosphere soils and surrounding soils as their control.). Your explanation have to be in the manuscript text.
Response: Highlights of the result, environmental and economic impact of the research have been provided thus;
Achieved goals
This study has successfully compared the functional features of maize rhizosphere and bulk soils rhizobiome and likewise identifies soil edaphic factors as important predictor of soil functioning (line 380-382).
Environmental and economic impact
Our study also suggests a need to explore plant growth-promoting traits of microorganisms present in the sites. The discovery of novel organisms (bioinoculants) capable of reducing the menace posed by soil stressors could serve as an alternative to the use of chemical fertilizers and also improve the economic value of agricultural products in arid and semi-arid soils (line 392-395).
Point 5:
It is not entirely correct to compare the parameters of different units of measurement in one and the same histogram ... Fig.1. If only values of same parameters from different samples were compared. Much better it would be presented in separate graphs or at least split on two Y axes not on one as of now because of unclear values. There is not HSD - calculated significant difference limit. It is needed to show these values too. Also in figure b useful more concrete Y axis, for example it could be numbered between 20, not 100.
Response: Figure 1a and b has been changed to a clearer Table. All parameters related to the table have been explained beneath the table.
Each value is expressed as mean ± standard deviation of chemical properties deduced from the maize rhizosphere (Ls and Rs) and bulk (Lc and Rc) soils. All statistical analyses including mean values and analysis of variance (ANOVA) were done using GraphPad Prism (v5.0). Mean bearing different superscripts <a-z> within each row indicate significant differences at p ≤ 0.05. P values given across the rows were used to compare chemical properties of the sampling sites (line 154-158).
Point 6:
Fig. 1. a and b incorrect confidence intervals in all columns, indicating only the plus values of the confidence interval. It needs to be corrected.
Response: Fig 1a and b has been presented in a table.
Point 7:
After every figure and table have to be explanations and analysis of one paragraph or at least several sentences about the results given in every figure and table. A chapter or subsection can never end with a table or figure.
Response: The explanation of the tables and figures have been provided at the end of each table and figure.
Point 8:
It is worth considering a more accurate presentation of Figure 1. It is necessary to adjust the presentation of units. mg / kg is not written. Need to change to mg kg-1. Throughout the manuscript text, it is necessary to review and present the units properly.
Response: The units have been adjusted to mgkg-1 throughout the manuscript including figures.
Point 9:
Also keep the same presentation style throughout the text, as the same is now presented chaotically differently everywhere. For example, 50 g (in 81 line), Twenty (20) grams (in 96 line) and so on. It was not done, because in line 98 it was also written twice - Twenty grams (20g) - it was not needed. All measurements have been presented same way in all text and not needed write surplus twice.
Response: The unit has been corrected throughout the manuscript (50 g adopted).
Point 10:
Please explain in more detail and detail in the methodology how the assessment of the diversity of soil functional features, CANOCO determinations, etc. is performed in order to be able to repeat the intentions
Response: A detailed methodology explaining how the diversity of soil functional features, CANOCO determination and other statistical analysis have been written as
It useful to write in the manuscript text.
The soil functional features and rhizobiome classifications were performed using SEED subsystem level 1 and 2 (Table S1 and Figure S1) with a maximum alignment length of 15 base pairs, minimum identity of 60% and an e-value of 1e–5. Annotated functional tables extracted from MG-RAST were agglomerated based on functional level and unclassified reads retained for statistical analysis. After the 12 sequences were annotated using MG-RAST, the mean relative abundances of the triplicate samples from each site (Ls, Rs, Lc and Rc) were used for further analysis (Line 119-124).
Adopting Bray-Curtis distance matrix using CANOCO 5 (Micro-computer Power, NY, USA), an excel sheet with the mean values of relative abundances of functional features was uploaded to plot the principal coordinate analysis (PCoA) and principal component analysis (PCA) of the samples (Line 134-137)
To evaluate environmental variables that best explain the diversity of soil functional attributes, CANOCO default settings adopting forward selection of pedological variables and Monte-Carlo permutation test was used (140-141).
Point 11:
2, FIG. 4, Table 2, FIG. 5, FIG. 6, Table 3 must be submitted your analysis and evaluation of the results obtained. There are nothing now. Overall chapters can not end with a table or figure must be a summary, conclusion, opinion.
Response: As suggested, summary of each table and figures have been provided and the results have been further explained.
Every figure and table must be explained in more detail in the appropriate manuscript locations. It was not enough results summary of the tables and figures in discussion session. It is not the same and it is not good for the reader. At the end could be all results and information abstraction.
Response: The results have been summarized for each of the table and figure. The explanations were provided beneath each table and figure.
Point 12:
To consider whether at all necessary tables 2 and 3, containing very little information. It can be written in one or more sentences in the text or attached to another visual material. If the Table 3 is the summary of CANOCO inference from fig. 6 and could be best explained as a separate table. So it could be combined together od for more better way.
Response: As suggested, information in Table 2 has been incorporated into figure 5, while table 3 has been changed to table 2 to reduce the number of tables.
Point 13:
Figures 2 and 3 need to be made clearer and more informative. it is necessary to provide all explanations under the title of the picture for more clear understanding for the reader. Now the images are overcrowded and don’t show off what you want to show. Please mark or somehow distinguish the essential moments and places. Figures was not changed yet and all information like big text is in the figure 2. It was not show any concrete and clear parts, which is most important. Z score is unreadable and poor quality.
Response: The quality of all figures has been improved to 800 dpi. Meanwhile, more information about the result from the figures have been provided under Figure 3.
Font sizes in figure 3 in some places very big and must be the same. The same problem in figure 5 too.
Response: The same font sizes have been used for all axis on Figures 3 and 5.
Point 14:
The abbreviated explanations given in Figure 2 are not available. Improperly reloaded in the wrong place. Explanations of symbols can be provided next to the Figure title or in the text. The left-handed gamma of the interface also shows nothing clearly. It is necessary to highlight the essence. If details on all symbols have been discussed under 3.3 in methodology section, so it could be easy to make more clear shortening specifically after figure title or in the figure.
Response: The abbreviations have been spelt out on Figure 2 and in text. There was no left-handed gamma, we only have the z-score and the resolution has been increased to 800 dpi to reveal all information. Brief explanation of the result in Fig. 2 has been provided. For better clarification on the differences in functional hits related to SEED subsystem 1 see Table S1.
Point 15:
Fig. 4 is no parameter unit or detailed explanation of the exact values. When parameter values are very small, for clarity, the smallest values can be given in the second Y axis. Figures 4a and b have been well explained. If it can be seen in Table S2 (Supplementary section), so it need to combine or explain in two places.
Response: Fig. 4 shows the mean relative abundance of functional hits in SEED subsystem level 2. They are all in percentages. Appropriate directive have been provided to readers.
Point 16:
Fig 4. Also in this figure, the presentation is superficial and needs to be improved. Because there is no significant difference between the mean values or they are all equal. In this case, a detailed description and analysis with exact values or a proper graph to ensure that the values are seen or presented in two axes is required. Also like in almost all figures and tables there is no description after figure.
Response: Table indicating the significant difference between sampling sites have been presented on Table S1 (Supplementary). For example, enormous difference between the stress responses and other parameters can be viewed on Table S1.
Point 17:
Figure 5 presents similarities and dissimilarities of the relative abundance of functional annotations between samples. What has been proved by that and for what purpose. All this information should be written in the manuscript, not just answer.
Response: These are the parameters needed to achieve the objectives of the research and to show the differences and similarities in the sampling sites. The figure shows the diversity indices between and among the organisms in each sampling site. Meanwhile, references for justification have been added.
Point 18:
Systematic and integrity require the same fonts and text size for manuscript text and text in all figures.
Response: The same text size and font size have been used throughout the text and figures.
Point 19:
Substantial deficiency, that this manuscript does not contain conclusions, work and research, evaluation of graph data, summary of results. It should be results interpretation paragraph after figures and tables, and at the end of every section and subsection (such 3.1, 3.2, 3.3, 3.4, 3.5).
Response: The summary of figures, tables and results have been improved.
Point 20:
There is no Highlights and Conclusion sections. It should be written.
Response: The highlights have been written and conclusion section provided.
Highlights
- Differences in the functional attributes was observed in the metagenomic study of maize rhizosphere and bulk soil.
- The presence of enormous functions conferring response to soil stressors in the rhizosphere samples could highlight the presence of novel organisms with biotechnological importance.
- Environmental variables viz. N-NO3, sulfate and pH had great impact on the soil rhizobiome functioning. (line 406-411).
Note: All effected corrections were highlighted in red on the main manuscript.
O.O. Babalola
