Confirmation of Multiple Resistant Chloris radiata Population, Harvested in Colombian Rice Fields

Round 1

Reviewer 1 Report

This is an interesting study exploring herbicide resistance in Chloris radiate. However, in my opinion, the conclusions are overreaching and authors should reevaluate the results and rewrite the discussion accordingly.

The English language needs improvement. In several cases, wording is awkward and confusing, especially in the discussion section.

 

Specific comments

19, 24 and 31: abbreviations should be explained for the first time. Please clarify.

21-22: what do you mean by "the resistance of susceptibility…"? awkward phrasing, please rephrase.

32-34: in my opinion, according to the information presented in this study, there are not enough data to support this assumption. It would be better for the authors to conduct more experiments, validate this resistance and publish another paper.

54: both references should be combined.

62: please erase "photosynthetic".

69-70: this statement is too general and incorrect, although resistance is a world known phenomenon, herbicides are still very effective, please rephrase.

72: please rephrase to "…and may hasten herbicide resistance evolution".

90-92: secondary effects of herbicides may vary between weed species and herbicide within MOA, I suggest removing this part of the sentence.

95-96: finding multiple herbicide resistance populations should not be stated as an objective. Instead, authors should state that they are testing the response of plants originated from a population suspected to be resistance to herbicides from two different MOA. Please rephrase.

97: first time mentioning the R abbreviation. Please elaborate.

103-108: do the farmers commonly use these products or they are just experimental tools?

104: for both, please list the active ingredient and percentage in the product as specified in the table for herbicides. Do you mean Retinol?

105: nonionic surfactant, please correct.

Table 1. I strongly suggest to divide it into two tables one with chemicals used in this study (authors should also add the adjuvants mentioned in the text), and the other, with the results, moved to the proper section. Title should also be rephrased as needed.

114-115: survival should be evaluated by the ability of the plant to produce new leaves or reach to a reproductive stage and set seeds.

119-120: R and S abbreviations were already mentioned above, please erase.

122-123: how many plants were collected at each location? Was there any collection pattern used to represent for the genetic structure of the field population?

126: just a note, if you cover the trays with parafilm the relative humidity in the chamber does not make any difference.

128-129: environmental conditions may have a crucial effect on herbicide activity. What were the temperature and day length during the experiments?

140: references should be combined within the same parentheses.

140-143: I have not seen the injury data, are they a part of a supplementary section?

144-145: please specify the leaf stage to avoid any confusion by the reader.

146: referring to glyphosate experimental rates, it is better to write the recommended rate as part of the dose response in correspondence to Table 1.

149-150: should be moved to the Statistical analysis section.

150: lethal dose of 50% should be referred to as (LD50).

152-153: redundant.

155-170: this whole section is describing very common methodology and should be summarized briefly. I have the same comment regarding both EPSPS enzyme activity and ALS enzyme activity assays.

191-193 and 218-219: should be moved to the Statistical analysis section.

238: the part of greenhouse experiments should come prior to the section of foliar retention tests with and without adjuvants.

241-243: I fail to understand why the authors used the ED50 rate instead of the recommended rate. Would the farmers also use this rate?

260: LD50 should be analysed using a binomial model that describes 1 and 0 as upper and lower limits, see Keshtkar et al. https://onlinelibrary.wiley.com/doi/epdf/10.1002/ps.6268.

280: only one sensitive population.

285-287: this sentence is not clear, it seems akward, please clarify.

296: there is no need to specify the parameters and the equation after they have been presented in the M&M section.

303-308: it is very obvious that the accumulation of shikimic acid has a positive correlation with glyphosate rate for both S vs. R populations. Thus, I suggest to add an additional analyses comparing the shikimic acid amount among glyphosate concentrations for each population separately.

311: authors stated that each experiment was conducted twice using ten replicates, why n=3? Did you average all 20 experimental replicates from both experiments?

324: as results from both figures are presented, authors should be referring to figure 3 and not 3b.

337: as I suggested before, this should come prior to the foliar retention experiment.

348-350: there was only one resistant population. Also, it should be 100% injury.

356: references should be combined within the same parentheses.

357-358: multiple resistance to ALS inhibitors, please rephrase.

358-360: this statement is not supported by the data; authors do not suggest any evident using long-term study to show a vast increase in the frequency of herbicide resistance. Please delete.

363: references should be combined within the same parentheses.

365: we do not kill plants, we control them, please correct.

365-367: I am not certain that the statistical analysis for the LD50 is suitable, thus, upon further analysis this data might change.

378-384: this should come right after the part discussing glyphosate resistance, please rephrase.

384: what should be further examined in order to confirm TS or NTS resistance?

386-387: this is inaccurate, an analysis of the gene sequence is the only direct proof for this, please rephrase.

391: should be "due to an increase in the EPSPS gene copy number", please correct.

393-395: wording is akward, please rephrase. Also, not rice but C. radiate.

400: Lolium was already mentioned, thus, L. rigidum.

401-403: this sentence is very confusing; were there any field tests conducted in this study or is it a part of the cited study?

393-407: this paragraph should be reorganized and reviewed, the explanation on the action of the adjuvant should be moved to the beginning of this section.

406-407: this sentence seems redundant. Suggest to delete.

408-409: awkward phrasing, I suggest; "poor weed control was achieved using both bispyribac-sodium and metsulfuron-methyl, indicating a possible cross-resistance for other ALS inhibitors"

411: "…low control percentages (<20%) were recorded using…"

411-412: in my opinion, at this point, there are not enough data to support this assumption. It would be better for the authors to conduct more experiments, validate this resistance and publish another paper.

414: references should be combined within the same parentheses.

425: using alternative herbicides we intend to reduce the spread of herbicide resistance.

438-440: this statement is inaccurate, authors do not compare between weed management methods and thus should not have claimed that this is one of the best alternatives.

445-448: as I suggested before, validate and publish another paper.

 

Author Response

Thanks for the comments on the manuscript. Next we will answer each of them, please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors here my comments:

1. Introduction: The objectives are clear, however it should be an hypothesis to test to get those objectives. I recommend to elaborate one.

2. Material and Methods:

- Table 1: Please add the meaning of “MOA”, I assume that is “Mode of action”. I would add the meaning of “S” and “R”; In this case it is stated previously in the text, but tables and graphs are independent from the text and some readers may read only tables and graphs.

-Which software do you use to estimate the GR50 and LD50?

-To compare S vs R survival and Dry weight curves I think it is better a non parametric test like Kaplan-Meier.

3. Results:

-In the Survival and Dry weight curves there are not a statistical comparison

-Which is the meaning of the p-value and the *** in the Table 2?

-In figure 3 EPSPS and ALS activity curves should be compared to seek for statistical differences.

4. Discussion: Most of the references used are works from South America, did you check if there are resistance problems in other places of the world?

 

Overall, I think that there is too much information together, I would divide in at least two papers, one for each chemical.

Regards

Author Response

Thanks for the comments on the manuscript. Next we will answer each of them, please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

Overall impression

This study contains important information about an important weed of a staple crop. Besides that, this is a thoroughly designed study with great execution and realistic conclusions. It definitely deserves publication once its minor mistakes or uncertainties are mended and/or clarified. I strongly suggest that there should be a list (or table) of abbreviations.

Language

The English and style of the Manuscript is generally flawless, with some minor mistakes here and there. The word “difficult” in Line 30 for example is used strangely.

Line 170 “to improve the persistence and adherence of plant protection chemical” should be changed either to “to improve the persistence and adherence of plant protection chemicals” OR to “to improve the persistence and adherence of a plant protection chemical”.

Line 351 “there was not decreased in plant growth” – please correct

Line 352 “survival percentage and the dry weight reduction also 352 shown the same” – please correct

Line 406 strange wording “This being an alternative for control”

Title

The Title reflects the content, relevant and is straightforward.

I assume if the Title contained the words “glyphosate-resistant” as well, more readers would be attracted by it. This is just a suggestion.

Authors’ list and affiliations

This section is fine.

Abstract

The Abstract is over the allowed word count limit. It should be reduced to 2/3 of its present length. The Authors have duly provided enough space to represent “Background” and “Methods”, but I think the “Results” are over-, and the “Conclusions” are underrepresented.

I think the appropriate length of the Abstract should be adjusted by shortening the “Results” section.

I would have welcomed some specific suggestions for further research directions and/or some general suggestions as to the management of these resistant weed populations.

I felt the need for an explanation for the abbreviations “EPSP” and “ALS” (Line 19). Perhaps it’s only me, but the “and the reduction of dry weight” (Line 30) is not clear. When I reached “Materials and Methods”, I realized that it referred to the dry weight of weeds, but I think readers must be better informed.

Keywords

The number of keywords is within the limit. I suggest the Authors include “glyphosate”, “rice”, and the Latin name of the weed in question among the keywords. I assume the findings of this paper will attract more readers, so the Authors may want to consider using a few more general keywords, such as “chemical weed control” or “herbicide resistance”.

I think EPSP and ALS-resistance are too specific. But weed control is not exactly my field of research, so I may be wrong here.

Introduction

The Introduction gives a solid background by showing us the importance of rice, and some of the most important weeds of rice worldwide and in the native country of the authors as well.

I like how each paragraph of the Introduction has its clearly defined content. The manuscript suggests that the only approach against weeds is chemical weed control. I miss some explanation here. Why does the manuscript suggest that we only have herbicides as “the” solution? In Line 93, the statement “In recent years, an increase in the distribution and density of C. radiata has been observed…” needs some literature support.

The second half of the sentence (Line 94) explains resistance by attributing it “to possible failures of chemical control in rice crops”. This reasoning has left me wondering as how this is possible. I would like the Authors to elaborate on that. Just a sentence or two. I am thinking of “overuse of the same active ingredient”, or “monoculture”, or “improper crop varieties or sowing time”, but I don’t know if the Authors had something different in their minds.

The main aim of the work is well defined, but the main conclusions are not highlighted as required.

Materials and Methods

Generally speaking, the methods are presented clearly and in sufficient detail, but I have some questions. How do authors define “a single batch” (Line 122-123)? How well does the potting media used for sowing and rearing plants represent the soil of an actual rice field (Line 124)? Can the authors possibly give some literary reference as how the conditions the authors used correspond to the actual conditions usually found in a rice field?

Results

The results are presented in due order, as the experiments were described in the M+M section. Tables and Figures are easy to follow and understand. This section is not really short, but as there were many sub-experiments, this length is justified. Some minor grammatical errors were detected.

Discussion

 

The statement starting at Line 358 “The rapid evolution of resistance” needs some support.

The results of this study are clearly presented in the general context of the research field. The “Discussion” follows the pattern set in the “M+M”, and in the “Results” sections.

The final “paragraph” (starting in Line 437) contains thoughtful future suggestions and conclusions; therefore, it may as well be placed within the “Conclusions”. This paragraph also gives some explanation in a retrospective to the issue of herbicide resistance. I tend to think these suggestions are kind of “common knowledge” by now.

Conclusions, a separate chapter

At its present form, this chapter is rather a summary of the whole manuscript. I suggest the authors add the last “paragraph” of the Discussion, starting at Line 437, with the words “The best strategies for managing” to the beginning of the Discussion.

References

 

Journal articles are not formatted according to the “Instructions to Authors”, which can be found at https://www.mdpi.com/journal/agronomy/instructions

Books, Conference Papers and Websites are correctly presented.

The number of references is satisfactory.

This Manuscript is supported by the most up-to-date literature background. Only 5 of the cited literature were published before 2000.

 

 

Author Response

Thanks for the comments on the manuscript. Next we will answer each of them, please see the attachment

Author Response File: Author Response.pdf

Reviewer 4 Report

My comments are attached as a seperate file

Comments for author File: Comments.pdf

Author Response

Thanks for the comments on the manuscript. Next we will answer each of them, please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Accept

Author Response

Thank you for your comments and acceptation

Reviewer 4 Report

Provided as seperate file

Comments for author File: Comments.pdf

Author Response

Please see the attachment

Author Response File: Author Response.pdf