Confirmation of the Mechanisms of Resistance to ACCase-Inhibiting Herbicides in Chinese Sprangletop (Leptochloa chinensis (L.) Nees) from South Sulawesi, Indonesia
Round 1
Reviewer 1 Report (Previous Reviewer 3)
Thanks to the authors for their previous revision. However, I still think the authors should carefully determine whether the species of Sidrap population belongs to Leptochloa chinensis, because such a big difference was not reasonable between ACCase genes amplied from two populations of same species, even between the two copies of ACCase gene. Also, if the Cys2088Met mutation was the first report in ACCase, its chromatogram should be added to demonstrate it. Or I suggest the authors delete relevant results about the Sidrap population.
Other minor comments:
Line 14: Change “Acetyl CoA Carboxylase” into “acetyl-CoA carboxylase”.
Line 21: change "butyl-cyhalofop“ to "cyhalofop-butyl”, check it throughout the paper.
Line 30: Change “Acetyl-coenzyme Carboxylase” into “acetyl-CoA carboxylase”.
L43: change "Chinese sprangletop (Leptochloa chinensis)" to "Chinese sprangletop (Leptochloa chinensis (L.) Nees)"
Line 71: change “cyhalofop” into “cyhalofop-butyl”. Check it throughout the paper.
Line 177: 2.44
Table 2.: Change “b2” into “b”.
Line 221: delete the redundant “and”.
Author Response
Dear reviewers,
I have revised all the comments from the reviewer and I have also proofread the manuscript by qualified proofreaders from Padjadjaran University (proofread certificate is attached).
Author Response File: 
Author Response.pdf
Reviewer 2 Report (Previous Reviewer 4)
The manuscript has been reviewed and I agree to accept its current form.
Author Response
Dear Reviewer,
I have proofread the manuscript
Author Response File: Author Response.pdf
Reviewer 3 Report (Previous Reviewer 1)
Please find my comments in the attached file.
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
I have proofread the manuscript (the proofread certificate is attached)
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report (Previous Reviewer 1)
Dear authors,
Good job. I am satisfied with the changes, however, I still feel you must rewrite the conclusion section in a better way. Also, the authors must provide a justification and highlight the novelty very briefly (preferably in the abstract as well as the conclusion section), the mutation is not novel. Please find my other comments in the following attachment.
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
I have revised the manuscript according to your comments
Author Response File: Author Response.pdf
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
Please find attached my comments in the attached PDF. Substantial revision is required.
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
I have already done all comments from reviewer and I have also attached my response.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript ‘Confirmation the Resistant and Mechanisms to ACCase Inhibitor Herbicides in Chinese sprangletop (Leptochloa chinensis) From South Sulawesi, Indonesia’ by Kurniadie et al. describes the understanding of the mechanisms of ACCase inhibitor herbicides resistance in L. chinensis. The manuscript is well-written. I have a few observations on the manuscript which may be useful for further improvement;
Line-1: You may keep ‘Article’ only
Abstract
Line-10: Please introduce the weed with the correct nomenclature Leptochloa chinensis (L.) Nees.
Introduction:
The introduction is well-written with rationale, justification, and aim. Please mention one line of future implications of the study.
Materials and Methods
Line 82: What is paddy soil?
Line 83: Were the pots earthen pots? Please mention…as you have exposed to 120oC.
Line 84: seedswere….please check the spacing between the words throughout the text.
Line 102: ‘square root transformation (square root of data + 0.5)’??? Actually, square root transformation [square root of (data + 0.5)]. Please check your calculation and delete (square root of data + 0.5), it is understood.
Line 104: p-value < 0.05 or p-value ≤0.05..please check.
Line 115: Quick DNA Plant/Seed Miniprep Kit” (Zymo Research, D6020)…please mention, (make, city, country) of the consumables and equipment used in the study.
Please elaborate on the DNA sequencing part in detail including bioinformatics analysis such as translation of DNA sequences to amino acid sequences and the software/methods used.
Line 117: Please write the gene name in italics ALS genes throughout the text.
Results
The authors presented the results nicely.
Table 1: Please mention the unit of doses. Tukeys’ values may be presented as superscript. If the data has been square root transformed, please mention the transformed data in the parenthesis.
e.g., 01,C (0.71)
Line 158: Please check the scientific name L. chinensis is italics through out the text.
Line 193: Please make different sections within the result.
Figure 3 depicts the amino acid (protein) sequences. Please clarify the translation of DNA to amino acid sequences in the materials section and subsequently in the result section.
Whether the sequences were submitted to NCBI, Please mention the ID.
I could not find the mechanisms of resistance in the result section, which has been discussed in the discussion section. Please emphasize the mechanism of ACCase inhibitor herbicide resistance.
Discussion
-The results were well-discussed.
Overall, it is a well-written manuscript that contains valuable information and may be considered for publication with minor revision.
Good luck with the revision.
Author Response
Dear Reviewer,
I have already done all the comments from the reviewers and I have attached my response.
Author Response File: Author Response.docx
Reviewer 3 Report
Leptochloa chinensis is a noxious weed worldwide, and the herbicide resistance evolution in this weed seriously threats cereal production. This current study by Kurniadie et al. reported the resistance of L. chinensis populations from South Sulawesi, Indonesia. Potential resistance mutation was also determined in the ACCase of resistant biotypes. However, I have some major concerns regarding the current manuscript.
1. More details should be added to the backgrounds
L40 and 61: The authors emphasized that resistant biotypes of L. chinensis have been reported in Asia or China, but without citing important literatures (doi.org/10.1016/j.pestbp.2022.105165, doi.org/10.1002/ps.6734). I do not understand why citing Wang et al. (2018) for proving the sentence.
L67: The first letter should be lowercase for acetolactate synthase, deoxy-xylu-67 lose phosphate synthase. Such errors should be revised throughout the manuscript.
2. Confusion in the ACCase gene mutation
Generally, the ACCase genes of gramineous weeds were aligned to the full-length black-grass (Alopecurus myosuroides Huds.) plastidic ACCase. Up to now, there are seven mutation sites in ACCase that contributed resistance to ACCase inhibitors. Obviously, the ACCase genes amplified in this study were not spanning all the known mutation sites reportedly.
By comparing the Trp-177-Cys mutation of ACCase identified by the Authors with that of A. myosuroides (GenBank accession no. AJ310767), the mutation should be Trp-2027-Cys mutation of ACCase gene in the resistant Maros population. I recommend the authors using Trp-2027-Cys for describing the mutation sites. Also, I suggest the authors amplifying the ACCase genes spanning all the known mutation sites again by using primers reported (doi.org/10.1002/ps.6734) to confirm if there were other resistance mutations contributing to the resistance.
3. It might be beneficial to the discussion
In the discussion, the authors emphasized that there were 33 amino acid substitutions in the ACCase of Sidrap population when compared to that of the susceptible population. However, the results were not convincing, because the ACCase amplified were not spanning all the known mutation sites reportedly in your paper.
L242-251: The authors discussed the NTSR, but without citing importing reports relevant to L. chinensis. As far as I know, NTSR has been also reported in L. chinensis species (doi.org/10.1002/ps.6734, doi.org/10.1002/ps.6297).
In addition, the Sidrap population was also resistant to the ALS inhibitor bispyribac-sodium. Please clarify that there may be TSR or NTSR potentially involved in the resistance.
L258: Trp-2027-Leu mutation in ACCase was firstly reported in L. chinensis from China.
3. There are many common language or expression errors. Therefore, the language of the paper needs to be polished by an English native speaker or a professional language polishing company. Also, to correct the common errors in terms, please have relevant experts revise the manuscript thoroughly.Some examples:
L2: Change the “resistant” to “resistance”.
L54 and L67: It is a repeat for the full name of ALS
L88-92: Herbicide at doses
L110: The b in the equation should be superscript.
L117: Change “ALS” into “ACCase”. Also, it should be italic for genes.
L147: In Figure 1, changing the “0, 0.25, 0.5, 1, 2, 4, 8” to “0×, 0.25×, 0.5×, 1×, 2×, 4×, 8×”.
L110, 166, 187: GR50. The error should be checked throughout the manuscript.
L198: Confusion in the Trp177Cys (GGA; susceptible, GTA; Maros) …, GGA codons come into Gly, GTA codons come into Val. Please have a check thoroughly.
Author Response
Dear reviewer,
I have already done all the comments from the reviewers, and I have also attached my response.
Author Response File: Author Response.docx
Reviewer 4 Report
The manuscript entitled "Confirmation of Resistance and Mechanisms for ACCase Inhibitor Herbicides in Chinese sprangletop (Leptochloa chinensis) from South Sulawesi, Indonesia" is well written and suitable for publication in Agronomy. However, the corrections below are necessary.
Abstract:
The brief introduction is on weed distribution, but should be on resistance and/or chemical control.
Line 18: ALS resistance too? You didn't write that in the title and objective of the study. Change "bispiribak" to "bispyribac-sodium".
The brief conclusion does not reflect your study. You should rewrite it.
Introduction:
Line 40. You must write the common name of Leptochloa chinensis.
Lines 47-51. What about Indonesia, are there resistance cases? You should write to improve the reader's understanding. I read that you wrote in the next paraphrase.
line 54. You have already described ACCase.
line 55. Does the species you study have reported cases of resistance in the world?
line 67. Have you already described ALS.
You should write about the mechanisms of resistance NTSR and TSR.
Objective: Why was resistance assessed for clomazone (DOXP, carotenoid inhibitor) and bispyribac-sodium (ALS)? You have not commented on these 2 herbicide modes of action.
Material and Methods:
Line 93. Why did you use such a high spray volume?
Line 100. What does © mean?
Lines 101-108. The formulas are the same. Did you use one to find C50 and one for GR50?
Line 117. Genetic sequencing was performed only for the ALS enzyme. What about ACCase and DOXP?
Results:
Line 130. You must write ANOVA.
Table 1. You should use only regression and not a test of means, because they are doses. Even Table 2 is the regression of these data. You should plot the regression figures and make tables for the C50 and GR50 parameters.
Line 158. Scientific name always in italic.
Line 172. Fig. 2. The herbicide name is wrong.
Fig. 2. You must enter the error bar or standard deviation of all averages.
In the legend you must use the city, but biotypes Suceptible, Resistant (Maros) and Resistant (Sidrap). Idem Fig. 3.
Discussion:
Does Clomazone inhibit DOXP or PDS? I think this is wrong and it only acts on DOXP.
Lines 242-251. It should be discussed in the introduction.
Conclusion:
It says that the mechanism of resistance is TSR related.
Have you studied the NTSR mechanism in relation to absorption, translocation and metabolism?
How will your study help the grower in controlling this weed?
References:
All are suitable for the manuscript.
Author Response
Dear reviewer,
I have already done all comments from the reviewers and I have also attached my response.
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Please find my comments in the attached file.
Comments for author File: Comments.pdf
Author Response
Reviewer 1
|
No. |
Comment |
Answer |
Line |
|
1 |
ACCase-inhibiting herbicides |
We extended the abbreviations of ACCase based on suggestions from other reviewers become Acetyl CoA Carboxylase (ACCase) inhibiting herbicides |
14 |
|
2 |
ACCase gene |
We have rewritten based on this recommendation |
119 |
|
3 |
10 µg, This the concentration after dilution? Because in mixture you are using almost 22 µL of water, which is too much. So, to me the concentration of the template DNA is not clear, and also please mention which DNA was used: is it genomic DNA or complementary DNA? |
We have revised and checked based on this recommendation |
123 |
|
4 |
SnapGene Version |
We have rewritten based on this recommendation |
125 |
|
5 |
Times recommended dose means in table 1 |
The information has the meaning of the number written in the lower column, namely 0; 0.25; 0.50; 1; 2; 4 and 8 times the recommended dose of each herbicide (Metamifop, butyl-cyhalofop, byspiribac-sodium, and clomazone) |
- |
|
6 |
Provide standard error values |
We have rewritten based on this recommendation |
- |
|
7 |
100 nucleotide changes |
The statement explains that there are 100 nucleotide changes that occur in the DNA accase-gene sequence, but the 100 nucleotide substitutions only cause 41 amino acid substitutions. |
196 |
|
8 |
Change 1 to single |
We have rewritten based on this recommendation |
197 |
|
9 |
Already described in many articel |
We agree with this, but we cited TSR and NTSR because they are important for further explanation of the resistance mechanism in L. chinensis and other grasses weeds that have been reported in the world, this paragraph is also one of the inputs proposed by other reviewers. |
- |
Reviewer 3 Report
Although the authors have made some revisions, I believe that the manuscript must be further improved before it can be accepted.
Some questions:
Abstract: All the abbreviations firstly mentioned in this section should provide their full names.
L66: change “ALS” to “acetolactate synthase (ALS)”
Line 71: change “deoxy-xylulose phosphate synthase” to “1-deoxy-d-xylulose-5-phosphate synthase (DOXP)”.
Line 223: Aligned to the full-length black-grass (Alopecurus myosuroides Huds.) plastidic ACCase, the sequence of ACCase gene…
Line257: adding “P450s” after the “…cytochrome”, also citing reference [36].
Line260: Change “ALS” into “ACCase and ALS”.
Line261: Change “ALS” into “ACCase”, also should be italic.
Line303: If the Cys2088Met mutation was the first report in ACCase, its chromatogram must be added. As reported previously, there are two copies of ACCase presented in L. chinensis species. Thus, a total of 41 substitutions presented in the ACCase fragment of Sidrap biotype may indicate that the ACCase amplified could be a different copy from that of the other two biotypes. Have the authors tested that if the Sidrap biotype was L. chinensis? Please refer to the “Section 2.1” in doi.org/10.1002/ps.6733 for detailed methods of species identification.
The ACCase sequences of L. chinensis spanning all the known mutation sites can be obtained using the primers reported by Zhao et al. (2022, https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/ps.6734?download=true). The experiment should be conducted again to obtain more convincing sequencing results.
The manuscript must be polished by a native English speaker or a professional language editing company since there are many errors in English writing and grammar.
Comments for author File: Comments.pdf
Author Response
Reviewer 3
|
No. |
Comment |
Answer |
Line |
|
1 |
Abstract: All the abbreviations firstly mentioned in this section should provide their full names. |
We have rewritten based on this recommendation |
- |
|
2 |
L66: change “ALS” to “acetolactate synthase (ALS)” |
We have rewritten based on this recommendation |
71 |
|
3 |
Line 71: change “deoxy-xylulose phosphate synthase” to “1-deoxy-d-xylulose-5-phosphate synthase (DOXP)”. |
We have rewritten based on this recommendation |
72 |
|
4 |
Line 223: Aligned to the full-length black-grass (Alopecurus myosuroides Huds.) plastidic ACCase, the sequence of ACCase gene… |
We have rewritten based on this recommendation |
266-270 |
|
5 |
Line257: adding “P450s” after the “…cytochrome”, also citing reference [36]. |
We have rewritten based on this recommendation |
264 |
|
6 |
Line260: Change “ALS” into “ACCase and ALS”. Line261: Change “ALS” into “ACCase”, also should be italic |
We have rewritten based on this recommendation |
- |
|
7 |
Line303: If the Cys2088Met mutation was the first report in ACCase, its chromatogram must be added. As reported previously, there are two copies of ACCase presented in L. chinensis species. Thus, a total of 41 substitutions presented in the ACCase fragment of Sidrap biotype may indicate that the ACCase amplified could be a different copy from that of the other two biotypes. Have the authors tested that if the Sidrap biotype was L. chinensis? Please refer to the “Section 2.1” in doi.org/10.1002/ps.6733 for detailed methods of species identification. |
Based on the morphology of L. chinensis which we used as samples, there was no difference between susceptible, Maros and Sidrap biotypes. Because this finding is the first time found, the sequence differences found in the Sidrap biotype need to be studied further, and this input can be a good suggestion for us in future research to identify and confirm the sequence changes that occur in the Sidrap biotype. The suggestion to provide a chromatogram in this paper is a good suggestion, but we did not include the chromatogram because the chromatogram is not written in the confirmation and weed resistance mechanisms in most of the articles that we refer. |
- |
|
8 |
The ACCase sequences of L. chinensis spanning all the known mutation sites can be obtained using the primers reported by Zhao et al. (2022) The experiment should be conducted again to obtain more convincing sequencing results. |
We have answered this in a previous review. We have also referenced the journal (Zhao et. al., 2022) (DOI: 10.1002/ps.6734). We have reviewed the amplified sequence results in our article, and based on the references, at least the sequences in this article cover 6 of the 7 mutations that have been confirmed to cause resistance to ACCase inhibitors. |
- |
|
9 |
suggestions for deleting multiple sentences in pdf files |
We have rewritten based on this recommendation |
- |
Reviewer 4 Report
I recommend the acceptance of the manuscript, as the authors make all the requested corrections increasing the quality of the paper for the reader.
Author Response
The were not revised comments from Reviewer 4
