Co-Ensiling Whole-Plant Cassava with Corn Stalk for Excellent Silage Production: Fermentation Characteristics, Bacterial Community, Function Profile, and Microbial Ecological Network Features

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript introduces the silage bacterial community and network interaction, which is benefit for the future research on the silage and agricultural development. However, I have to say that the writing of the manuscript is far from meeting the requirements of scientific papers. Firstly, the authors totally misunderstand the meaning of discussion section. Section discussion aims to reveal the study novelty through comparing with other literatures rather than just conclude the findings. Conversely, the findings are included in result section, and the references are barely showed in this section. The current writing is not the style of standard scientific report. Secondly, the figures in the manuscript are also flawed! What the numbers mean in Figs 1A and 1B? I assume they are p values, but the elaboration is required. In the Y-axis of Figs 2A and 2B, “%” is redundant because the unit has shown in the title “relative abundance (%)”. The letters in Fig 2C are too small, and I barely see the details. Where are the Y-axis titles in Fig 3, and similar issues are found in Fig 5. Besides, the network figures in Fig 5 are totally meaningless because most nodes have completely overlapped and connected into one piece, and nothing can be seen clearly in the figures. Also in Fig 6, I can't see anything clearly, including X- and Y-axis, legends, and other elements. The overlapping problem are also found in Fig 8B. I cannot tell the accuracy of the result statements with the blurred images. Thirdly, the format of references is not unified. I hope that the authors improve the quality of the writing, figure, and other details of this manuscript for meeting the requirements of scientific papers.

Author Response

Reviewer 1

Thank you for your kindly and professional comment.Your comments are very helpful to improve the quality of our manuscript.We will do our best to revise the paper to meet the publishing requirements.

1. Firstly, the authors totally misunderstand the meaning of discussion section. Section discussion aims to reveal the study novelty through comparing with other literatures rather than just conclude the findings. Conversely, the findings are included in result section, and the references are barely showed in this section. The current writing is not the style of standard scientific report.

Response: We have reorganized the paper structure according to the template.

2. Secondly, the figures in the manuscript are also flawed! What the numbers mean in Figs 1A and 1B? I assume they are p values, but the elaboration is required. In the Y-axis of Figs 2A and 2B, “%” is redundant because the unit has shown in the title “relative abundance (%)”. The letters in Fig 2C are too small, and I barely see the details. Where are the Y-axis titles in Fig 3, and similar issues are found in Fig 5. Besides, the network figures in Fig 5 are totally meaningless because most nodes have completely overlapped and connected into one piece, and nothing can be seen clearly in the figures. Also in Fig 6, I can't see anything clearly, including X- and Y-axis, legends, and other elements. The overlapping problem are also found in Fig 8B. I cannot tell the accuracy of the result statements with the blurred images.

Response: We have added information in Figure 1 and revised Figure 2, 3 and 5. We updated the original drawing of Figure 6, that clean enough. We have made every effort to modify Figure 8, and key information can be seen.

3. Thirdly, the format of references is not unified.

Response: We have revised the references in the whole paper.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The article meets an important problem, which is efficient forage production. Composing plant mix with efficient microbial support makes that research reasonable. In my opinion Authors did important work, which valuably supports farmers of warm regions. The article has an applicable potential. However, I found some points, which need to be corrected/improved for better understanding for everyone interested in this subject.

1. In many points, mostly in the abstract, discussion, etc., I found, that the Latin names of organisms were not written in italics. Please write all Latin names in italics.

2. There are some not explained shortcuts like in Table 2, in brackets are shortcuts FM, DM; I imagine that it may be explained by readers that they mean: Fresh weight/mass and dry weight/mass, however, there is no place for any guesses in scientific articles.  

3. Reading the article text in which authors use many shortcuts e.g. CP, WSC, etc. makes the article hard to read. All of the shortcuts have to be remembered by readers and believe me it is not nice during reading. Instead, it would be better to use full names/terms. Thus, please change shortcuts e.g. CP, WSC, etc. in the text into full terms.  

4. line 414-416: "The co-occurrence networks of the bacterial communities varied with the silage. Fig. 5 shows that the networks of Group CF had the greatest edges, network density, and average degree." - please explain the terms: "edges", network density, degree, etc. - what they really mean for the soundness of the results. Remember that not every reader is very familiar with such analyses. This should be explained in the part describing analytical methods.

5. I found that figures 5 to 8 are impossible to read them. The pictures are not enough sharp. Please consider bigger figures, or adding them as supplementary materials, or another way to make them easier to see.  

6. Part Results in fact is Results and Discussion; the Discussion part seems to be a Conclusion. Please name the article sections correctly and add the part Conclusions.

7. I tried to find an accession PRJNA1011842 in SRA but it was impossible. Please make sure that it was successfully submitted.

 

References used in the article seem to be appropriate.

 

 

Author Response

Reviewer 2 

Thank you for your kindly and professional comment.Your comments are very helpful to improve the quality of our manuscript.We will do our best to revise the paper to meet the publishing requirements.

 

1. In many points, mostly in the abstract, discussion, etc., I found, that the Latin names of organisms were not written in italics. Please write all Latin names in italics.

Response: We have revised the written issue.

2. There are some not explained shortcuts like in Table 2, in brackets are shortcuts FM, DM; I imagine that it may be explained by readers that they mean: Fresh weight/mass and dry weight/mass, however, there is no place for any guesses in scientific articles.  

Response: We added the note under Table 2.

3. Reading the article text in which authors use many shortcuts e.g. CP, WSC, etc. makes the article hard to read. All of the shortcuts have to be remembered by readers and believe me it is not nice during reading. Instead, it would be better to use full names/terms. Thus, please change shortcuts e.g. CP, WSC, etc. in the text into full terms.  

Response: Thank you for your reminder. Due to the limitation of the length of the paper, we have to use abbreviations. As usual, we have explained them in the first place they appear.

4. line 414-416: "The co-occurrence networks of the bacterial communities varied with the silage. Fig. 5 shows that the networks of Group CF had the greatest edges, network density, and average degree." - please explain the terms: "edges", network density, degree, etc. - what they really mean for the soundness of the results. Remember that not every reader is very familiar with such analyses. This should be explained in the part describing analytical methods.

Response: We have added the information at 2.3.

5. I found that figures 5 to 8 are impossible to read them. The pictures are not enough sharp. Please consider bigger figures, or adding them as supplementary materials, or another way to make them easier to see.  

Response: We updated the original drawing of Figure 5 and 6, that clean enough. We have made every effort to modify Figure 8, and key information can be seen.

6. Part Results in fact is Results and Discussion; the Discussion part seems to be a Conclusion. Please name the article sections correctly and add the part Conclusions.

Response: We have reorganized the paper structure according to the template.

7. I tried to find an accession PRJNA1011842 in SRA but it was impossible. Please make sure that it was successfully submitted.

Response: With the consent of all authors, the uploaded data will be released after the official publication of the paper. Please understand.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors explored the effect of whole-plant cassava to corn stalk ratio on the fermentation profile, chemical composition and bacterial community of silage, which was interesting and fell in the scope of the journal agronomy. However, the authors should deal with the comments or problems below before being considered for acceptance.

First, the title (30 words in total) of this paper is a little long and is to some extent redundant. I suggest using “Co-ensiling whole-plant cassava with corn stalk for excellent silage production: Fermentation characteristics, bacterial community, function profile and microbial ecological network features”

Second, the authors carried out discussion (L217-225, L237-279, L338-370, L394-412 and L483-509) in “Results” section, which is generally not allowed. Inversely, the authors should move these paragraphs to the section “Discussion”, accordingly.

Third, partial Tables and Figures are not normative, such as Tables 3 and 4. As to Figures 1A and 1B, p value (<0.05) can be shown when only significance is stated; Figures 5A, 5B, 5C, 5D, 5E, 5F and 5G, they appeared to be vague, namely too many parameters and nor clear, the authors should improve this; Figure 6 also appeared to be vague and the authors should enhance the dpi of the document and update them; Many parameters are overlapped in Figure 8 and the authors should separate them clearly in the figure.

Last, the language quality of the whole manuscript should be improved.

 

Comments in detail are as follows.

L19-20 and 137-138, on a fresh weight basis ? please define it in the text

L22, considered

L24, ammonia nitrogen

L25, compared with

L26-27 and 33, “Acetobacter fabarum”, “Pseudomonas aeruginosa”, “Levilactobacillus brevis”, “Lactiplantibacillus plantarum”, “Lactobacillus harbinensis” and “Pseudomonas aeruginosa” should be italic

L57, "Manihot esculenta" should be italic

L76, "Zea mays" should be italic

L142, please keep in accordance between "water-soluble carbohydrates" and "water soluble carbohydrates" (L216-217)

L142-143, did the authors use amylase during NDF analysis ?

L154, please show the full words of the abbreviation for the first appearance in the text

L215, please use the abbreviations in Table 2 and put the full words of these abbreviations below Table 2 in the form of table annotation

L233 and 280 (also in the remaining text), “p” should be lowercase and italic

L281-283, put the full words of these abbreviations below Table 4 in the form of table annotation

L349, please keep in accordance between "lactic acid bacteria" and "LAB" (L97)

L514, ammonia nitrogen; the whole paragraph of this section should be brief and improve it

L543, please move this section to L537-540

Comments on the Quality of English Language

L19, ratios

L22, considered

L25， compared with, ensiled alone

L49-50, However, the shortage of high-quality forage became accentuated due to the limited production of existing grasslands (Fang et al., 2018)

 

 

 

 

Author Response

Reviewer 3 

Thank you for your kindly and professional comment.Your comments are very helpful to improve the quality of our manuscript.We will do our best to revise the paper to meet the publishing requirements.

 

1. First, the title (30 words in total) of this paper is a little long and is to some extent redundant. I suggest using “Co-ensiling whole-plant cassava with corn stalk for excellent silage production: Fermentation characteristics, bacterial community, function profile and microbial ecological network features”

Response: Based on your suggestion,we changed the title.

2. Second, the authors carried out discussion (L217-225, L237-279, L338-370, L394-412 and L483-509) in “Results” section, which is generally not allowed. Inversely, the authors should move these paragraphs to the section “Discussion”, accordingly.

Response: We have reorganized the paper structure according to the template.

3. Third, partial Tables and Figures are not normative, such as Tables 3 and 4. As to Figures 1A and 1B, p value (<0.05) can be shown when only significance is stated; Figures 5A, 5B, 5C, 5D, 5E, 5F and 5G, they appeared to be vague, namely too many parameters and nor clear, the authors should improve this; Figure 6 also appeared to be vague and the authors should enhance the dpi of the document and update them; Many parameters are overlapped in Figure 8 and the authors should separate them clearly in the figure.

Response: We have replaced new tables. We revised Figure 1 and 5(A-G) . We updated the original drawing of Figure 6, that clean enough. We have made every effort to modify Figure 8, and key information can be seen.

4. Last, the language quality of the whole manuscript should be improved.

Response: Thank you for your comments, after the other content of the paper is revised, we will invite native English speakers to improve the quality of our paper.

Comments in detail are as follows.

L19-20 and 137-138, on a fresh weight basis ? please define it in the text

Response: The mixed ratio was fresh weight basis, and we have added it at the two places.

L22, considered

Response: We have revised it.

L24, ammonia nitrogen

Response: We have revised it.

L25, compared with

Response: We have revised it.

L26-27 and 33, “Acetobacter fabarum”, “Pseudomonas aeruginosa”, “Levilactobacillus brevis”, “Lactiplantibacillus plantarum”, “Lactobacillus harbinensis” and “Pseudomonas aeruginosa” should be italic

L57, "Manihot esculenta" should be italic

L76, "Zea mays" should be italic

Response: We have rewrite these Latin names in italics

L142, please keep in accordance between "water-soluble carbohydrates" and "water soluble carbohydrates" (L216-217)

Response: We have revised it.

L142-143, did the authors use amylase during NDF analysis ?

Response: Yes we used the amylase, and we added the information at 2.2.

L154, please show the full words of the abbreviation for the first appearance in the text

Response: We have check and revise the whole paper.

L215, please use the abbreviations in Table 2 and put the full words of these abbreviations below Table 2 in the form of table annotation

Response: We have revised the Table 2.

L233 and 280 (also in the remaining text), “p” should be lowercase and italic

Response: We have check and revise the whole paper.

L281-283, put the full words of these abbreviations below Table 4 in the form of table annotation

Response: We have revised the Table 4.

L349, please keep in accordance between "lactic acid bacteria" and "LAB" (L97)

Response: We have check and revise the whole paper.

L514, ammonia nitrogen; the whole paragraph of this section should be brief and improve it

Response: We have revised it and improved the conclusion.

L543, please move this section to L537-540

Response: We have revised it.

Comments on the Quality of English Language

L19, ratios

L22, considered

L25， compared with, ensiled alone

L49-50, However, the shortage of high-quality forage became accentuated due to the limited production of existing grasslands (Fang et al., 2018)

Response: We have revised them.

Reviewer 4 Report

Comments and Suggestions for Authors

Comments on the article submitted for review:

1. Title too long

2 Latin names in italics

3. Wrong citations used- names should be replaced by figures

"References: References must be numbered in order of appearance in the text (including table captions and figure legends) and listed individually at the end of the manuscript. We recommend preparing the references with a bibliography software package, such as EndNote, ReferenceManager or Zotero to avoid typing mistakes and duplicated references."

 

4. 1 year of research? Is that not too little after all? In terms of field observations?-key question

5) Figs 1 A,B,D are not very legible,  they should be corrected and maybe placed vertically in pairs?

6. fig 2 C also not very legible

7. fig 5 A-J - not readable at all

8. fig 8 B - illegible

9. References - in a different font from the written article

10. Punctuation marks to be checked

11. In the Results section, there is a ''Discussion of results'' with other reports, and in the Discussion section, there is not a single reference to other reports

12. 2.2 Chemical composition and fermentation index - how many replicates per site/object?

13. Only in the results does a table of abbreviations appear, should this not appear in the methods? In my opinion to be improved

14.Lack of summary - conclusion

Author Response

Reviewer 4 

Thank you for your kindly and professional comment.Your comments are very helpful to improve the quality of our manuscript.We will do our best to revise the paper to meet the publishing requirements.

 

Comments on the article submitted for review:

1. Title too long

Response: We have revised it.

2 Latin names in italics

Response: We have revised them.

3. Wrong citations used- names should be replaced by figures

"References: References must be numbered in order of appearance in the text (including table captions and figure legends) and listed individually at the end of the manuscript. We recommend preparing the references with a bibliography software package, such as EndNote, ReferenceManager or Zotero to avoid typing mistakes and duplicated references."

 Response: We have revised the references in the whole paper.

4. 1 year of research? Is that not too little after all? In terms of field observations?-key question.

Response: I understand the issue you are concerned about, this study was conducted in a laboratory, not a field experiment. Many similar studies are one-year trials, such as follow studies:

• Bai, J., Ding, Z., Su, R., Wang, M., Cheng, M., Xie, D., et al. (2022a). Storage Temperature Is More Effective Than Lactic Acid Bacteria Inoculations in Manipulating Fermentation and Bacterial Community Diversity, Co-Occurrence and Functionality of the Whole-Plant Corn Silage. Microbiol Spectr 10(2), e0010122. doi: 10.1128/spectrum.00101-22.
• Du, Z., Sun, L., Lin, Y., Chen, C., Yang, F., and Cai, Y. (2022). Use of Napier grass and rice straw hay as exogenous additive improves microbial community and fermentation quality of paper mulberry silage. Animal Feed Science and Technology 285, 115219. doi: https://doi.org/10.1016/j.anifeedsci.2022.115219
• Zeng, T., Li, X., Guan, H., Yang, W., Liu, W., Liu, J., et al. (2020). Dynamic microbial diversity and fermentation quality of the mixed silage of corn and soybean grown in strip intercropping system. Bioresource Technology 313, 123655. doi: https://doi.org/10.1016/j.biortech.2020.123655.

5. Figs 1 A,B,D are not very legible,  they should be corrected and maybe placed vertically in pairs?

Response: We revised Figure 1.

6. fig 2 C also not very legible

Response: We updated the original drawing of Figure 2, that clean enough.

7. fig 5 A-J - not readable at all

Response: We revised Figure 5.

8. fig 8 B - illegible

Response: We have made every effort to modify Figure 8, and key information can be seen.

9. References - in a different font from the written article

Response: We have revised the references(字体).

10. Punctuation marks to be checked

Response: We have checked and revised the punctuation.

11. In the Results section, there is a ''Discussion of results'' with other reports, and in the Discussion section, there is not a single reference to other reports

Response: We have reorganized the paper structure according to the template.

12. 2.2 Chemical composition and fermentation index - how many replicates per site/object?

Response: All the indexed had three replicates.We have added at 2.2.

13. Only in the results does a table of abbreviations appear, should this not appear in the methods? In my opinion to be improved

Response: We have revised the Table 2,3 and 4, and added abbreviations at table note.

14.Lack of summary - conclusion

Response: We have rewrote the conclusion.

Reviewer 5 Report

Comments and Suggestions for Authors

The investigation of this article is devoted to search of a better silage mixture. It is a well-known fact that very often singe plant silage is not sufficient with nutrients and for enhancing quality of silage, co-ensiling of different types of plants is used. In this study, authors investigated the advantages of mixing corn stalk and whole-plant cassava for producing good silage. Also authors analyzed several ratios of mixing cassava with corn, and it was revealed that co-ensiling of 30% of whole-plant cassava and 70% of corn stalk is the most efficient in production of silage. Besides the structure of microbial community, function profile and co-occurrence of microbial networks of all variants of corn and cassava silage were analyzed, that give valuable information microorganism relationship during ensiling of single and mixed plant species.

The results are useful and perspective, however I have some comments and questions:

What is SC7 and Huamei? Is it a cultivar or a variety?

All species names of plants and microorganism have to be in Italic.

Check the style of the references, the style of doi and the name of journals (some of the names are short and some are full).

Why some of the references in the text are blue?

There is only Li et al, 2021 in the list of references, there are no Li et al, 2021a and Li et al., 2021b.

Lines 103-104 – “Many studies have shown…” – What are these studies?

Figures 5 and 6 are unreadable. The text on pictures 5 A-G is too small. Zooming on the screen did not help. Figure 6 is nice but I can’t evaluate it because of the small text.

“Part 4 Discussion” is not a discussion. This is a conclusion. The discussion is presented through the text of “part 3 Results”, because part 3 contains references to other researches and comparisons with other studies. Either name part 3 “Results and Discussion” or make a separate part of “Discussion”.

In my opinion in Abstract it should be noted that co-ensiling restricted Acetobacter of corn and Pseudomonas of cassava. These two plants have different undesirable bacteria.

I would like authors to explain why chemical composition of mixed variants were not analyzed before ensiling. Table 4 show the influence of co-ensiling on chemical properties of silage but it would be good to know chemical characteristics of mixtures before ensiling.

Lines 512-513 – this is too simple and does not show the reason why this conclusion was made. In my opinion, it is better to say “Analysis of several kinds of ratio mixtures of corn stalk and whole-plant cassava revealed that the best silage was produced from 30% of cassava and 70% of corn”.

 

Author Response

Reviewer 5 

Thank you for your kindly and professional comment.Your comments are very helpful to improve the quality of our manuscript.We will do our best to revise the paper to meet the publishing requirements.

 

1. What is SC7 and Huamei? Is it a cultivar or a variety?

Response: Both of them are cultivars. We added the information at 2.1.

All species names of plants and microorganism have to be in Italic.

Response: We have checked and revised them.

Check the style of the references, the style of doi and the name of journals (some of the names are short and some are full).

Response: We have revised the references

Why some of the references in the text are blue?

Response: We have revised them.

There is only Li et al, 2021 in the list of references, there are no Li et al, 2021a and Li et al., 2021b.

Response: We have revised it.

Lines 103-104 – “Many studies have shown…” – What are these studies?

Figures 5 and 6 are unreadable. The text on pictures 5 A-G is too small. Zooming on the screen did not help. Figure 6 is nice but I can’t evaluate it because of the small text.

Response: We revised Figure 5 and we updated the original drawing of Figure 6, that clean enough.

“Part 4 Discussion” is not a discussion. This is a conclusion. The discussion is presented through the text of “part 3 Results”, because part 3 contains references to other researches and comparisons with other studies. Either name part 3 “Results and Discussion” or make a separate part of “Discussion”.

Response: We have reorganized the paper structure according to the template.

 

In my opinion in Abstract it should be noted that co-ensiling restricted Acetobacter of corn and Pseudomonas of cassava. These two plants have different undesirable bacteria.

Response: We have revised it at Abstract.

I would like authors to explain why chemical composition of mixed variants were not analyzed before ensiling. Table 4 show the influence of co-ensiling on chemical properties of silage but it would be good to know chemical characteristics of mixtures before ensiling.

Response: Unfortunately, we only analyzed the chemical composition of two raw materials before ensiling, and did not analyze the mixed materials before ensiling. Analyzing the changes in chemical composition of mixed raw materials before and after ensiling is more accurate, and we will use this method in future research

Lines 512-513 – this is too simple and does not show the reason why this conclusion was made. In my opinion, it is better to say “Analysis of several kinds of ratio mixtures of corn stalk and whole-plant cassava revealed that the best silage was produced from 30% of cassava and 70% of corn”.

Response: We have revised this sentence.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed my concenrs and comments. I now humbly endorse the publication of this manuscript.

Author Response

All authors deepest gratitude goes to the anonymous reviewers for their careful work and thoughtful suggestions that have helped improve this paper substantially.

Reviewer 5 Report

Comments and Suggestions for Authors

The text of the article is improved and almost suitable for publishing. I have only one wish. Replace figure 2 C to supplementary materials and make bigger. The text in this figure is unreadable now. Figure 6 is still unreadable. This figure is so nice but it impossible to read the text. Put it to supplementary materials and make large. Let it occupy the whole space of a sheet.

Author Response

We deepest gratitude goes to the anonymous reviewers for their careful work and thoughtful suggestions that have helped improve this paper substantially. We fully understand your concerns, and in order to present our results more comprehensively to the author, we prefer to keep these two figures. In order for the author to accurately understand our results, we have uploaded their detailed information as supplementary materials based on your kindly reminder. Thank you for your understanding.

Author Response File: Author Response.pdf