Trichoderma harzianum and Bacillus subtilis in Association with Rock Powder for the Initial Development of Maize Plants

Round 1

Reviewer 1 Report (Previous Reviewer 1)

The revised manuscript presented here (Trichoderma harzianum and Bacillus subtilis in association with rock powder for the initial development of maize plants) shows noticeable improvement over previously submitted versions. I note some relatively minor concerns that, if addressed, may improve the overall readability of the manuscript in the future:

·       Lines 31-32: us of the phrase “increase the analyzed analysis” is awkward and hard to follow (especially “analyzed analysis”

·       Line 33: I would suggest replacing “….when applied to the bacterium….” with something more like “…when coupled with inoculation of…” since the rock powder was not actually applied directly to the microorganisms but, rather, to the soil.

·       Please clarify the number of replicate plants used for each experiment as this remains unclear (e.g., how many plants were used for Experiment 1, T1 and all of the other treatments?)

·       Line 62 suggests that global food production is decreasing. If this is true, a reference could be provided.

·       Line 353 – please define “SDM” when it is first used.

·       Line 357: please define “RDM” when it is first used.

·       The tables need to be renumbered as the draft currently has tables 1, 2, 6 and 8.

Author Response

We thank the reviewer for the opportunity to improve our manuscript

Reviewer 1 - The revised manuscript presented here (Trichoderma harzianum and Bacillus subtilis in association with rock powder for the initial development of maize plants) shows noticeable improvement over previously submitted versions. I note some relatively minor concerns that, if addressed, may improve the overall readability of the manuscript in the future:

Answer: We authors want to thank the reviewer for the opportunity given to us to improve our manuscript.

• Reviewer 1- Lines 31-32: us of the phrase “increase the analyzed analysis” is awkward and hard to follow (especially “analyzed analysis”

Answer: It has been removed.

• Line 33: I would suggest replacing “….when applied to the bacterium….” with something more like “…when coupled with inoculation of…” since the rock powder was not actually applied directly to the microorganisms but, rather, to the soil.

Answer: I agree with you. It has been changed.

• Please clarify the number of replicate plants used for each experiment as this remains unclear (e.g., how many plants were used for Experiment 1, T1 and all of the other treatments?)

Answer: Each treatment had four repetitions.

• Line 62 suggests that global food production is decreasing. If this is true, a reference could be provided.

Answer: The word decreasing has been changed for issues.

• Line 353 – please define “SDM” when it is first used.

Answer: SDM – shoot dry matter

• Line 357: please define “RDM” when it is first used.

Answer: RDM  root dry matter

• The tables need to be renumbered as the draft currently has tables 1, 2, 6 and 8.

Answer: The numbers were wrong. Now is right.

 

Author Response File: Author Response.docx

Reviewer 2 Report (New Reviewer)

As general comment the work is well written and designed with relevant results.

In general terms the topic of the article is interesting, the methodology is explicitly presented and the results reported are interesting.

The abstract need to added some value of the results.

The introduction chapter should end with a paragraph indicating the purposefulness of the conducted research. Authors should clearly define the purpose of the work and formulate research hypotheses. They wrote to paragraph about the objective it should be modified

Some table and Fig need to improve Fig 1 what the litter mean on the culman and what the bar is this SD or SE?

Discussion part should be discussed some part

Make sure that all scientific names in the References list are italics.

The paper needs some editorial corrections.

I recommend the publication of this manuscript in the Journal of Agronomy  journal after minor revisions.

 

Author Response

As general comment the work is well written and designed with relevant results.

In general terms the topic of the article is interesting, the methodology is explicitly presented and the results reported are interesting.

Answer: Thank you for your support.

The abstract need to added some value of the results.

Answer: It has been added.

The introduction chapter should end with a paragraph indicating the purposefulness of the conducted research. Authors should clearly define the purpose of the work and formulate research hypotheses. They wrote to paragraph about the objective it should be modified

Some table and Fig need to improve Fig 1 what the litter mean on the culman and what the bar is this SD or SE?

Answer: It has been added.

Discussion part should be discussed some part

Make sure that all scientific names in the References list are italics.

It has been done

The paper needs some editorial corrections.

It has been done

I recommend the publication of this manuscript in the Journal of Agronomy  journal after minor revisions.

 

Author Response File: Author Response.docx

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

The manuscript presented here (Trichoderma harzianum and Bacillus subtilis in association with rock powder for the initial development of maize plants) describes an interesting experiment in an interest research area. However, portions of the manuscript are challenging to follow, there are some potential concerns with some of the data presented, and the data analysis itself does not appear complete. I will list below some of the concerns I developed looking through the manuscript:

·       Lines 18-20: It is extremely challenging to understand what the authors are trying to convey in this sentence which states that the application of two microbes did not improve growth over the “application” alone. The application of what? Do the authors mean this to be a comparison to the fertilizer application? Or perhaps they are trying to compare the application of single microbes versus both together? The use of “application” and “application alone” appear to be referring to different things but it is challenging to decipher this.

·       The manuscript never actually provides a description of the rock powder being tested. There is a reference to “basalt” in two of the table captions but that is it. Since the rock powder is a central facet of the work it seems reasonable to describe what it is, where it came from, etc. Is it locally sourced? Why did the authors pick this particular powder versus any others? Was it chosen to intentionally augment the local soil conditions?

·       Further, there is very little nutrient information provided on the rock powder itself. Lines 110-111 list some details but leave out important components like phosphorus and organic carbon data which would be helpful for later data interpretation and discussion.

·       There is a recurring issue with abbreviations not being defined before use:

o   Line 123 refers to “DIC” – what is “DIC” as it is presumably not dissolved inorganic carbon which is typically abbreviated as DIC.

o   Lines 147 and 156 refer to “BOD” – what is BOD as it is presumably not biological oxygen demand as is often abbreviated in environmental literature

o   Line 161 defines CFU but not in the first use of that abbreviation in the manuscript

o   Line 173 – What is IAFE?

·        I do not understand the distinction between sections 3.5.3 and 3.5.6. Both seem to be measuring root biomass but section 3.5.6 has a specific storage at 20 deg C for two days prior to drying. What is the purpose of this hold time? And, there is reference to the 20 deg C being in a “cold chamber” whereas it is typically assumed that room temperature is roughly 20 deg C. Did the authors consider biological degradation of a wet sample “stored” at 20 deg C?

·       Lines 205-206: There is some text here that makes no sense: “initial temperature of 100 °C, left for 20 min and gradually increased from 50 °C to 50 °C until reaching 350 °C.” I am guessing that the second mention of “50 °C” was meant to be a time unit and this is a typo but it is unclear.

·       Table 1 has many issues:

o   Why is colony length reported in units of area (cm²)? Is not length typically reported in cm, m, or comparable unit?

o   What does “No Conidia mL^-1” refer to? As stated, it says “no conidia” while I am guessing the authors intend for this to be number of conidia.

o   What is “UFC” as reported in the table? Is this supposed to be “CFU”?

o   In both this table and subsequent tables it is unclear what “F” and “CV (%)” are referring to at the bottom of the table. My apologies if I missed this in the text but it is very unclear to me.

·       Tables 2 and 3

o   These tables could likely be combined to make comparisons between the before and after soils more apparent.

o   Why is the units associated with pH listed as “CaCl₂”?

o   As listed, all of the concentrations appears to be provided in units of volume (e.g., mg dm^-3). Is this intentional? How was the volume of soil used for each analysis measured as this is not described in the methods.  

o   What is “CTC”?

o   Why is there not mention at all of variability in the measurements? Were replicates run for these analyses and how much variability was observed?

o   The text associated with these tables suggests that both OM and phosphorus increases with addition of rock powder but it seems challenging to believe that a small addition of powdered rock is going to actually increase soil OM by roughly 15%. If the rock powder was basalt I can’t imagine there being a large amount of OM inherent to the powder itself. Further, why would adding a small amount of rock powder decrease Cu, Mn, and Zn concentrations? It seems more likely that variability in the analytical results could be the cause.

·       Lines 296-298: This section highlights why the data from these experiments need to be reported. For instance, this section suggests that there is no statistical difference between values of 0.60 and 1.13. This is likely only explainable by having very large variability in the replicates of each treatment type and showing that variability would be very helpful to the reader.

·       Tables 5, 7, and 9 all list results / data for “No.” and “Q”. What are these? Is this a repeated typo in which the authors are actually referring to nitrogen (N) and phosphorus (P)?

·       Lines 384-385: This is a very confusing sentence as it appears to say that treatments 3, 4, and 5 achieved better results than treatments 3, 4, and 5 which does not make sense.

·       Tables 4, 6, 8 – all of these tables list the stem diameter for maize grown to 40 days post germination as being on the order of roughly 0.6 to 1.2 mm. I want to question the authors on this as 0.6mm stem diameter for a 40 day old maize plant is extremely small and I would argue that many maize plants exceed this diameter at emergence. Further, I do not believe that a maize plant over 1m tall would have a stem diameter less than a mm. Care needs to be taken and the tables thoroughly reviewed for accuracy.

·       Tables 4, 5, 6, 7, 8 – There is a lot of repeated data in these table as the same controls are used in each case. This results in the tables taking up a lot more room than needed and may be confusing to the reader. I would recommend condensing Tables 4, 6, and 8 into one, combined table and likewise condensing Tables 5, 7, and 9 into one, condensed table to improve overall readability.

 

I presented here some of the more glaring issues I had with the manuscript. Again, I believe the scientific motivation behind this experiment is sound. However, a large number of potential errors in the data presentation, lack of data on measurement variability, sparce information on the motivation and nature of the added rock powder, and related issues make the current version of the manuscript challenging to follow and raises questions regarding the stated outcomes. It is also challenging to evaluate some of the conclusions presented without having a better understanding of the data owing to what appear to be errors in the data (as reported) and not being able to evaluate the variability in the presumed results from experimental replicates.

Author Response

Reviewer - The manuscript presented here (Trichoderma harzianum and Bacillus subtilis in association with rock powder for the initial development of maize plants) describes an interesting experiment in an interest research area. However, portions of the manuscript are challenging to follow, there are some potential concerns with some of the data presented, and the data analysis itself does not appear complete. I will list below some of the concerns I developed looking through the manuscript:

Answer: We want to thank the reviewer for the opportunity to improve our manuscript. We appreciate your help.

• Reviewer- Lines 18-20: It is extremely challenging to understand what the authors are trying to convey in this sentence which states that the application of two microbes did not improve growth over the “application” alone. The application of what? Do the authors mean this to be a comparison to the fertilizer application? Or perhaps they are trying to compare the application of single microbes versus both together? The use of “application” and “application alone” appear to be referring to different things but it is challenging to decipher this.

Answer: The word “application” has been changed to “inoculation”. The results also show that inoculation with two microbes, B. subtilis and T. harzianum, in the mixture form did not promote a greater increase in the analysed parameters compared to inoculation with either microbe alone.

• Reviewer - The manuscript never actually provides a description of the rock powder being tested. There is a reference to “basalt” in two of the table captions but that is it. Since the rock powder is a central facet of the work it seems reasonable to describe what it is, where it came from, etc. Is it locally sourced? Why did the authors pick this particular powder versus any others? Was it chosen to intentionally augment the local soil conditions?

Answer – This description has been added. The rock powder was acquired directly from its manufacturer. The brand is Coplan, produced in the Embaúba city Sao Paulo State Brazil. Rock powder is a residue of basalt rock extraction and has the ability to remineralize soil. Their physical and chemical attributes are shown in Table 2. This rock powder was chosen due to its proximity to the manufacture, and it was kindly donated.

• Reviewer - Furthermore, there is very little nutrient information provided on the rock powder itself. Lines 110-111 list some details but leave out important components like phosphorus and organic carbon data which would be helpful for later data interpretation and discussion.

Answer – Very good question. There is no phosphorus, organic matter or other kind of nutrients in the rock powder. Rock powder is poor in nutrient content. The potential of the rock powder is its capacity to remineralize the soil.

 

Reviewer-   There is a recurring issue with abbreviations not being defined before use:

         Reviewer-   Line 123 refers to “DIC” – what is “DIC” as it is presumably not dissolved inorganic carbon which is typically abbreviated as DIC.

Answer: DIC means completely randomized design. It has been changed to CRD.

 

Reviewer-  Lines 147 and 156 refer to “BOD” – what is BOD as it is presumably not biological oxygen demand as is often abbreviated in environmental literature.

Answer: BOD has been changed to Microbiological greenhouse.

Reviewer-  Line 161 defines CFU but not in the first use of that abbreviation in the manuscript.

Answer: In the section Microorganisms CFU has been defined.

  Reviewer - Line 173 – What is IAFE?

Answer: The correct term is LAI, which means leaf area index.

• Reviewer- I do not understand the distinction between sections 3.5.3 and 3.5.6. Both seem to be measuring root biomass but section 3.5.6 has a specific storage at 20 deg C for two days prior to drying. What is the purpose of this hold time? And, there is reference to the 20 deg C being in a “cold chamber” whereas it is typically assumed that room temperature is approximately 20 deg C. Did the authors consider biological degradation of a wet sample “stored” at 20 deg C?

Answer: Section 3.5.6 has been removed. It was not carried out.

• Reviewer-Lines 205-206: There is some text here that makes no sense: “initial temperature of 100 °C, left for 20 min and gradually increased from 50 °C to 50 °C until reaching 350 °C.” I am guessing that the second mention of “50 °C” was meant to be a time unit and this is a typo but it is unclear.

Answer: This was a mistake. The samples were shaken and then placed in a digester block at an initial temperature of 100 °C, left for 20 minutes and gradually increased until reaching 350 °C. The samples were left in the block until digestion (light green colouration); after cooling, distillation began.

• Reviewer-Table 1 has many issues:

  Why is colony length reported in units of area (cm²)? Is not length typically reported in cm, m, or comparable unit?

Answer: The correct is cm.

What does “No Conidia mL^-1” refer to? As stated, it says “no conidia” while I am guessing the authors intend for this to be number of conidia.

Answer: The number of conidia has been changed.

What is “UFC” as reported in the table? Is this supposed to be “CFU”?

Answer: The correct term is CFU. It has been changed.

In both this table and subsequent tables it is unclear what “F” and “CV (%)” are referring to at the bottom of the table. My apologies if I missed this in the text but it is very unclear to me.

Answer: CV has been changed to VC, which means variation in the coefficient. F has been removed.

• Tables 2 and 3

These tables could likely be combined to make comparisons between the before and after soils more apparent.

Answer: Both tables were combined.

• Why is the units associated with pH listed as “CaCl₂”?

Answer: There are two methods to measure the pH. The first used water, and the second used CaCl₂. We used the second method.

• As listed, all of the concentrations appears to be provided in units of volume (e.g., mg dm^-3). Is this intentional? How was the volume of soil used for each analysis measured as this is not described in the methods.

Answer: Yes, it is. These units are the correct way to show the values. The volume of soil was 10 g per sample.

• What is “CTC”?

Answer: The correct term is CEC – cation exchange capacity.

• Why is there not mention at all of variability in the measurements? Were replicates run for these analyses and how much variability was observed?

Answer: The soil for trial was collected, and a representative amount was analysed regarding its fertility. After the incubation period, another representative amount was analysed. There was no repetition of each incubation period.

• Reviewer- The text associated with these tables suggests that both OM and phosphorus increases with addition of rock powder but it seems challenging to believe that a small addition of powdered rock is going to actually increase soil OM by approximately 15%. If the rock powder was basalt I can’t imagine there being a large amount of OM inherent to the powder itself. Furthermore, why would adding a small amount of rock powder decrease Cu, Mn, and Zn concentrations? It seems more likely that variability in the analytical results could be the cause.

Answer: The reviewer is correct. It was a surprise. However, the soil samples were representative according to soil fertility analysis. There is scarce information about the use of rock powder and its benefits. Some authors have proposed that rock powder remineralizes the soil, activates many microorganisms and increases organic matter and fertility. Certainly, the increase in organic matter and phosphorus content did not come from rock powder. However, it may have increased the soil microbes’ solubilization of phosphorus. The present study has shown interesting results that need to be confirmed by other studies.

    Reviewer   Lines 296-298: This section highlights why the data from these experiments need to be reported. For instance, this section suggests that there is no significant difference between values of 0.60 and 1.13. This is likely only explainable by having very large variability in the replicates of each treatment type and showing that variability would be very helpful to the reader.

      Answer: The reviewer is right. There was no difference between the values 0.60 and 1.13 due to high variability. It can be shown by the variation of coefficient VC that is shown in the table.

•  

Reviewer -   Tables 5, 7, and 9 all list results/data for “No.” and “Q”. What are these? Is this a repeated typo in which the authors are actually referring to nitrogen (N) and phosphorus (P)?

      Answer: This was a mistake. No change was observed for N (nitrogen) and Q for P (phosphorus).

• Reviewer -Lines 384-385: This is a very confusing sentence as it appears to say that treatments 3, 4, and 5 achieved better results than treatments 3, 4, and 5 which does not make sense.

      Answer: The correct statement is “ it was observed that the treatments that were composed of microorganisms, fertilization (50%) and rock powder (treatments 3, 4 and 5) achieved better results than treatments 3, 4 and 5. 1 with 100% of the recommended fertilization, and 2, which in addition to the fertilization also had rock powder in its composition.

• Reviewer -Tables 4, 6, 8 – all of these tables list the stem diameter for maize grown to 40 days post germination as being on the order of approximately 0.6 to 1.2 mm. I want to question the authors on this as 0.6 mm stem diameter for a 40-day-old maize plant is extremely small and I would argue that many maize plants exceed this diameter at emergence. Furthermore, I do not believe that a maize plant over 1 m tall would have a stem diameter less than a mm. Care needs to be taken and the tables thoroughly reviewed for accuracy.

      Answer: The correct unit is cm, not mm.

• Reviewer -Tables 4, 5, 6, 7, 8 – There is a lot of repeated data in these table as the same controls are used in each case. This results in the tables taking up a lot more room than needed and may be confusing to the reader. I would recommend condensing Tables 4, 6, and 8 into one, combined table and likewise condensing Tables 5, 7, and 9 into one, condensed table to improve overall readability.

Answer: I would agree with the reviewer to condense Tables 5, 7 and 9. However, I cannot. The experiments were carried out separately. For each experiment, there was a randomized spatial distribution among the pots. I cannot analyse the statistic as if it were just an experiment. Certainly, joining the tables will improve the reading, but statistically it is incorrect.

 Reviewer  - I presented here some of the more glaring issues I had with the manuscript. Again, I believe the scientific motivation behind this experiment is sound. However, a large number of potential errors in the data presentation, lack of data on measurement variability, sparce information on the motivation and nature of the added rock powder, and related issues make the current version of the manuscript challenging to follow and raises questions regarding the stated outcomes. It is also challenging to evaluate some of the conclusions presented without having a better understanding of the data owing to what appear to be errors in the data (as reported) and not being able to evaluate the variability in the presumed results from experimental replicates.

Answer - I thank the reviewer for showing concerns about the manuscript and for the opportunity given to us to improve it. Except for table 2, all the tables show the significance index p and the variation coefficient VC%. With VC, it is possible to verify the percentage of variation and calculate the number varied within the repetitions for each analysed parameter. When the VC is high, there is usually no difference among the treatments. However, the differences shown by the statistical analysis show that they are truly different. Regarding soil fertility, there was no repetition. However, the soil sample was collected according to Malavolta et al., 1980 when twenty soil samples were collected. These samples were mixed and formed one composite soil sample that was analysed for fertility. The analysis of fertility was carried out twice, before and after the application of rock powder. We hope that all concerns have been solved.

 

Author Response File: Author Response.docx

Reviewer 2 Report

agronomy-2055881: "Trichoderma harzianum and Bacillus subtilis in association with rock powder for the initial development of maize plants".

Research carried out by the authors seems to be important to the development and enhancement of existing information on this subject. Title is consistent with the content of work. Abstract, keywords are prepared in a clear manner and contain the necessary information. The methods used are appropriated. The work was constructed logically and the study contains a large amount of data. This paper is very interesting. But, some issues must be improved.

1. The importance and innovative aspects of the paper should be more evident.

2. Please justify the purpose of the research more.

3. The methodology lacks soil characterization.

4. The type of soil used in the experiment and where it came from was not specified.

5. Soil classification according to the World Reference Base of Soil Resources and methods used for the determination of sand, silt and clay fractions in soil should be given in the "Materials and Methods" section.

6. In the section "Material and methods" not specified how many replications performed analyzes of soil.

7. Please, specify where the rock powder is coming from.

8. It is unclear what previously there was a type of land use where the soils were collected? Please mention about it. Plus, general practices of land should be explained.

9. Describe and expand your conclusions in more detail.

10. Not all latin names are written in italics.

11. There are double literature numbers in the literature list.

12. Please, be sure that all the references cited in the manuscript are also included in the reference list and vice versa.

 

Author Response

Reviewer2

1. The importance and innovative aspects of the paper should be more evident.

Answer: Global agriculture is facing many challenges. One of them is the lack of available fertilizers due to military conflicts. This study has shown that the use of rock powder with B. subtilis and T. harzianum improved soil fertility and plant growth. In addition, the use of rock powder with these microorganisms could reduce the production cost and environmental impact without reducing the yield. These are innovative aspects.

 

2. Please justify the purpose of the research more.

Answer: There are few studies using rock powder with Bacillus subtilis and Trichoderma harzianum. Therefore, this study aimed to verify the benefits generated with their use.

3. The methodology lacks soil characterization.

Answer: This has been added. The soil from the experimental area was classified as clayed eutrophic red latosol.

 

4. The type of soil used in the experiment and where it came from was not specified.

Answer: The soil came from the Farm of University.

 

5. Soil classification according to the World Reference Base of Soil Resources and methods used for the determination of sand, silt and clay fractions in soil should be given in the "Materials and Methods" section.

Answer: This has been added. The soil was classified as a clayed eutrophic red latosol.

 

6. In the section "Material and methods" not specified how many replications performed analyses of soil.

Answer: We collected 20 soil samples. These samples were mixed and formed one compost soil sample that was analysed for fertility.

 

7. Please specify where the rock powder is coming from.

Answer: This has been added. The rock powder was acquired directly from its manufacturer. The brand is Coplan, produced in the Embaúba city Sao Paulo State Brazil. Rock powder is a residue of basalt rock extraction and has the ability to remineralize soil. Their physical and chemical attributes are shown in Table 2.

 

8. It is unclear what previously there was a type of land use where the soils were collected? Please mention about it. Plus, general practices of land should be explained.

Answer: There was no crop planted previously in this soil. It was collected from the ground rest area.

 

9. Describe and expand your conclusions in more detail.

Answer:

 

10. Not all Latin names are written in italics.

It has been corrected.

 

11. There are double the number of studies in the literature list.

Answer: It happens when the same citation is citated.

 

12. Please, be sure that all the references cited in the manuscript are also included in the reference list and vice versa.

Answer: It has been verified.

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript presented here (Trichoderma harzianum and Bacillus subtilis in association with rock powder for the initial development of maize plants) is a revision to a previous submission. At its core, the manuscript describes an interesting experiment designed to test the degree to which a crushed rock material can improve soil fertility for maize growth. The authors then also attempt to demonstrate whether adding in two different microbes (one a bacterium and one a fungi) further improve crop yields. While the scientific concept being tested is interesting, there remain some serious concerns with the manuscript which I note below. Please note that all line numbers listed in these comments relate to the line number in the ‘agronomy-2055881-coverletter.docx’ file as I realize these line numbers differ from those in the “agronomy-2055881-peer-review-v2.pdf” file but had to choose one file for commenting.

·       It is generally not considered the review’s responsibility to proof read and spend time helping to identify typos in a submitted manuscript. In the previous version of the manuscript I identified some of the more egregious typos observed and some of these appear corrected while some are not. I am unsure whether the incorrect version of the manuscript was submitted or whether I am missing something, but I make the following comments on the version I was provided as a reviewer and note that these issues really need to be resolved:

o   Tables 3 and 5 both list the stem diameter of grown maize plants as being less than or right around 1 mm. This is simply wrong and to publish this would be embarrassing to both the authors and the journal. It is completely inconceivable that a maize plant would be one and a half meters tall and have a stem diameter of less than a mm (as is reported, for instance, in Table 3 for treatment 3). This error was previously provided to the authors and they seemed to correct the issue in Table 7 but did not take the time to fix the issue in other locations. Note that this error also persists in the text of the manuscript with line 190 stating that stem diameter data is ”expressed in millimeters” which seems to contradict the author’s statement in their response to the reviews (which states the correct unit is cm).  

o   Note: units seem to be a challenge in general as line 204 suggests the authors report plant dry biomass in units of milligrams while Table 3 provides no units for “root dry matter” and Tables 5 and 7 use units of grams (not milligrams).

o   Table 5, 6, 7, and 8 appear to have incorrect microbe names under the “Treatment” column. For instance, the caption to Table 5 lists the bacteria being tested while the actual table lists on the fungi being test (similar issues with Tables 6, 7, and 8). Again, to publish this would be embarrassing to both the authors and the journal and it should not be the reviewer’s job to identify these blatant errors.

o   Line 312: there is a statement that there is “5.5 mg dm^-1 more potassium” in the soil after addition of the rock powder but this is completely inconsistent with what is listed in Table 2 which shows a difference of only 0.6 mmol dm^-1. Where did the authors get this 5.5 number? Were they erroneously reporting the phosphorus data and listing it as potassium? Again, issues like this would be embarrassing to the authors and journal if published and these should be captured by the authors in a careful review of their own work.

o   Table 2 lists values for Na as “-“ and values for Al as “0”. What is the difference for these two values? Was sodium not measured and aluminum measured to be zero? If so, what is the limit of detection and why list sodium if it wasn’t even measured?

o   Multiple tables: There appears to be a huge inconsistency in the number of significant figures used in the tables overall. For instance, in Table 3 the aerial dry matter is reported using one or three significant figures. Is this another example of a typo/error in the manuscript or is there a reason for this? The same issue is present within the text where, for instance, line 330 lists values of “151.66, 136, and 122.33 centimeters”. In this case, why do the authors use five significant figures for two measurements and only three for one of the measurements?

·       Table 2 remains a bit concerning as there is no mention of replication of the soil data. In the response from the authors they describe that they combined twenty soil samples into one for analysis. This is completely fine. BUT, the authors provide no indication of the analytical precision or variation of the subsequent analyses which is critical to evaluating if there is any chemical changes in the soil following the addition of rock powder. For instance, the authors claim the soil increases in OM from 1.74 to 2 g/dm (again there is an issue with significant figures and if the authors choose to report only one significant figure then both of these values are 2 so not different). Yet, we have no idea what the expected variability on these numbers may be. For example, if the OM measurements typically varies (in analytical replicates) by a value of ±0.3 then these values are technically the same. Without having an assessment of the analytical variability associated with the data it makes the text in lines 306 through 321 questionable at best.

·       Table 2 and throughout – please explain why these units were chosen and define what is meant by the subscript “c” for some of the analytes (the mmol_c).

·       Line 318: What is meant by “relevant value”. What is an irrelevant value?

·       Many of the statistical analyses appear to claim a p value of 0. I am not familiar with this, can the authors please explain how they arrive at this extremely high level of confidence in the differentiation of the reported averages. In looking at the p values provided I am assuming a p value listed as zero is actually a rounding issue and that the actual p value is extremely small – could this be the case? If so, could the authors describe how they choose the number of significant figures to report the p values.

·       I am completely surprised that the authors do not provide their data anywhere in the manuscript nor do they report any measure of variability in their individual measurement of replicates. I am aware that the authors list both “VC” and “CV” in some of their tables which appears to be an attempt to provide some measure of variability but the authors 1) seem to have a typo as some tables list VC and other CV, 2) never define how they are using this term or how they calculate the values, and 3) only provide this aggregated term without providing an indication of the analytical variability within one sample measurement.
The lack of data reporting and measures of analytical variability make it extremely difficult for a reader to evaluate the composite statistical analyses provided. It is often considered good practice to make data available to readers in supplemental information and/or to provide the reader some indication of the spread of analytical replicates (to at least provide the standard deviation or standard error of the replicate measurements and the number of replicates).

·       Lines 436-437 state that three of the treatments had values greater than 5 g. Was this statistically verified? The only statistical comparison I can find is between the different treatments.

·       Lines 450-451 claim that the calcium index did not exceed 20 g/kg. What is the calcium index? The actual calcium values appear to stay below 1.55g/kg so it is interesting that the authors claim they never got greater than 20 (which is true, but still an odd choice of number for comparison).

·       Line 511 lists “this effect” in the first sentence of a paragraph without defining what “this effect” is referring to.

·       Line 511: what is meant by “fail in this proposal”? This text is unclear, what is the proposal?

·       In a previous version the authors were asked to provide more information on the soil they used in the experiment. They did list that the soil “came from the farm of university” which I assume to mean “came from the farm on the university campus”. My question is whether there are any previously published papers that could be referenced here to provide more context on this soil, its location, previous land use, etc.

·       Line 124: what is meant by the term “has properties to remineralize soil”? I am used to thinking about remineralizing a particular component of soil but what is meant when referring to this in the larger, integrated context? Or, is this just a typo and meant to more specifically refer to soil OM or some other constituent?

·       Lines 129-130: It is appreciated that the authors provide a bit more context on the nutrient content of the added rock powder but it looks like they forgot to add the units to the zinc and manganese results.

·       Lines 142-143 mention irrigation “up to 70% of field capacity”. Does this mean that moisture level was maintained at 70% field capacity? How often was water added and how dry did the soil get before water addition?

 

I reiterate that I believe the topic of this work is interesting and would be of interest to a broader scientific community. However, the lack of care with which this manuscript was constructed, the persistent typos and inconsistencies even in the second submitted version, and the lack of detail with which the data and its analysis is presented leave serious questions about the readiness of this work for publication.

Author Response

Answer- The correct names are: Pedro Henrique Vinha Silva; Ane Gabriele Vaz Souza; Lilian Dutra Araújo; Gabriel Vinicius Lima de Souza; Cesar Vinicius Martoreli da Silva.

Reviewer: Please add the rest authors’ email after their affiliation

Answer: The e-mails of the authors have been added after their affiliation.

Reviewer: Please add zip code for all the affiliations.

Answer: The zip codes have been added.

Reviewer: Please check ALL the titles and subtitles. I am not sure if the logic is right, especially those in Section 4.

Answer: many titles and subtitles were removed. Some of them were replaced.

Reviewer: Please fil these parts with your own info.

Answer: The information have been added.

Reviewer: There are more than 20 refs with just one author name. Please check if the author name part is complete

Answer: There are two references with one author name.

First of all, I am not sure if this study will be accepted or not. But I want to thank the reviewer for his excellent job. I want to thank the reviewer for his patience twice and for the opportunity given to us to improve this study.

All changes were written in red color. 

Reviewer 1: Tables 3 and 5 both list the stem diameter of grown maize plants as being less than or right around 1 mm. This is simply wrong and to publish this would be embarrassing to both the authors and the journal. It is completely inconceivable that a maize plant would be one and a half meters tall and have a stem diameter of less than a mm (as is reported, for instance, in Table 3 for treatment 3). This error was previously provided to the authors and they seemed to correct the issue in Table 7 but did not take the time to fix the issue in other locations. Note that this error also persists in the text of the manuscript with line 190 stating that stem diameter data is ”expressed in millimeters” which seems to contradict the author’s statement in their response to the reviews (which states the correct unit is cm).  

Answer: I apologize. Now it has been correct again.

o   Note: units seem to be a challenge in general as line 204 suggests the authors report plant dry biomass in units of milligrams while Table 3 provides no units for “root dry matter” and Tables 5 and 7 use units of grams (not milligrams).

      Answer: The correct unit is gram. It has been changed.

• Table 5, 6, 7, and 8 appear to have incorrect microbe names under the “Treatment” column. For instance, the caption to Table 5 lists the bacteria being tested while the actual table lists on the fungi being test (similar issues with Tables 6, 7, and 8). Again, to publish this would be embarrassing to both the authors and the journal and it should not be the reviewer’s job to identify these blatant errors.

Answer: The names of the microbes were correct accordingly.

• Line 312: there is a statement that there is “5.5 mg dm^-1more potassium” in the soil after addition of the rock powder but this is completely inconsistent with what is listed in Table 2 which shows a difference of only 0.6 mmol dm^-1. Where did the authors get this 5.5 number? Were they erroneously reporting the phosphorus data and listing it as potassium? Again, issues like this would be embarrassing to the authors and journal if published and these should be captured by the authors in a careful review of their own work.

Answer: The text has been corrected for phosphorus, potassium, calcium and sulfur were observed, for example, the 5.5 mg dm-3 more phosphorus, 0.60 for potassium and the 2.4 mmolc dm-3 for calcium that were noted in the second analysis (Table 02).

o   Table 2 lists values for Na as “-“ and values for Al as “0”. What is the difference for these two values? Was sodium not measured and aluminum measured to be zero? If so, what is the limit of detection and why list sodium if it wasn’t even measured?

      Answer:  We made a mistake. The correct is zero.

• Multiple tables: There appears to be a huge inconsistency in the number of significant figures used in the tables overall. For instance, in Table 3 the aerial dry matter is reported using one or three significant figures. Is this another example of a typo/error in the manuscript or is there a reason for this? The same issue is present within the text where, for instance, line 330 lists values of “151.66, 136, and 122.33 centimeters”. In this case, why do the authors use five significant figures for two measurements and only three for one of the measurements?

Answer: The number of figures were standardized.

• Table 2 remains a bit concerning as there is no mention of replication of the soil data. In the response from the authors they describe that they combined twenty soil samples into one for analysis. This is completely fine. BUT, the authors provide no indication of the analytical precision or variation of the subsequent analyses which is critical to evaluating if there is any chemical changes in the soil following the addition of rock powder. For instance, the authors claim the soil increases in OM from 1.74 to 2 g/dm (again there is an issue with significant figures and if the authors choose to report only one significant figure then both of these values are 2 so not different). Yet, we have no idea what the expected variability on these numbers may be. For example, if the OM measurements typically varies (in analytical replicates) by a value of ±0.3 then these values are technically the same. Without having an assessment of the analytical variability associated with the data it makes the text in lines 306 through 321 questionable at best.

      Answer: Previously composite soil samples were analyzed. But according to suggestion of reviewer we analyzed the soil samples which we had at the laboratory. Now we analyzed four repetitions for each moment before and after application of powder rock. The results were analyzed according to Scott knott test 5%.

• Table 2 and throughout – please explain why these units were chosen and define what is meant by the subscript “c” for some of the analytes (the mmol_c).

      Answer: mmolc means the value of mol divided by 1000.

• Line 318: What is meant by “relevant value”. What is an irrelevant value?

      Answer: It has been removed.  

• Many of the statistical analyses appear to claim a p value of 0. I am not familiar with this, can the authors please explain how they arrive at this extremely high level of confidence in the differentiation of the reported averages. In looking at the p values provided I am assuming a p value listed as zero is actually a rounding issue and that the actual p value is extremely small – could this be the case? If so, could the authors describe how they choose the number of significant figures to report the p values.

      Answer: To standardize we adopt the significance value of 5%. The statistical test was Scott Knott 5%.

• I am completely surprised that the authors do not provide their data anywhere in the manuscript nor do they report any measure of variability in their individual measurement of replicates. I am aware that the authors list both “VC” and “CV” in some of their tables which appears to be an attempt to provide some measure of variability but the authors 1) seem to have a typo as some tables list VC and other CV, 2) never define how they are using this term or how they calculate the values, and 3) only provide this aggregated term without providing an indication of the analytical variability within one sample measurement.
  The lack of data reporting and measures of analytical variability make it extremely difficult for a reader to evaluate the composite statistical analyses provided. It is often considered good practice to make data available to readers in supplemental information and/or to provide the reader some indication of the spread of analytical replicates (to at least provide the standard deviation or standard error of the replicate measurements and the number of replicates).

      Answer: For the soil fertility were collected four replicates. Please see 3.3 Soil description.

      Each treatment was carried out four repetitions too.

      Answer: In all tables were added the CV% and Standard Error values.

• Lines 436-437 state that three of the treatments had values greater than 5 g. Was this statistically verified? The only statistical comparison I can find is between the different treatments.

      Answer: Here, we are comparing the aerial dry matter of the treatments inside experiment 3 only. We are not comparing the experiments.  

• Lines 450-451 claim that the calcium index did not exceed 20 g/kg. What is the calcium index? The actual calcium values appear to stay below 1.55g/kg so it is interesting that the authors claim they never got greater than 20 (which is true, but still an odd choice of number for comparison).

      Answer: This phrase has been removed.

• Line 511 lists “this effect” in the first sentence of a paragraph without defining what “this effect” is referring to.

      Answer: It has been changed to “The effect of mineralization…

• Line 511: what is meant by “fail in this proposal”? This text is unclear, what is the proposal?

      Answer: The phrase was changed to “ Plant growth-promoting microorganisms have several abilities that contribute to increasing plant growth, and these microorganisms have been used for this purpose. However, in some situations, these microorganisms may fail in this proposal

• In a previous version the authors were asked to provide more information on the soil they used in the experiment. They did list that the soil “came from the farm of university” which I assume to mean “came from the farm on the university campus”. My question is whether there are any previously published papers that could be referenced here to provide more context on this soil, its location, previous land use, etc.

      Answer: Regarding the soil, please see the citation -  DE ANDRADE BARBOSA, Marcelo et al. Multivariate analysis and modeling of soil quality indicators in long-term management systems. Science of the Total Environment, v. 657, p. 457-465, 2019.

• Line 124: what is meant by the term “has properties to remineralize soil”? I am used to thinking about remineralizing a particular component of soil but what is meant when referring to this in the larger, integrated context? Or, is this just a typo and meant to refer to soil OM or some other constituent?

      Answer: The correct is remineralizing. But it has been removed.

• Lines 129-130: It is appreciated that the authors provide a bit more context on the nutrient content of the added rock powder but it looks like they forgot to add the units to the zinc and manganese results.

      Answer: The percentages have been added.

• Lines 142-143 mention irrigation “up to 70% of field capacity”. Does this mean that moisture level was maintained at 70% field capacity? How often was water added and how dry did the soil get before water addition?

      Answer: The soil was collected with 12% of humidity and when the irrigation started the humidity was maintained at 70% with aid of automated sprinkler.

I reiterate that I believe the topic of this work is interesting and would be of interest to a broader scientific community. However, the lack of care with which this manuscript was constructed, the persistent typos and inconsistencies even in the second submitted version, and the lack of detail with which the data and its analysis is presented leave serious questions about the readiness of this work for publication.

Answer: The reviewer pointed out many concerns; we changed the manuscript accordingly and hope that all concerns have been solved and this manuscript may be accepted for publication.  

 

 

 

Author Response File: Author Response.pdf