Detection of Resistance in Echinochloa spp. to Three Post-Emergence Herbicides (Penoxsulam, Metamifop, and Quinclorac) Used in China
and Jun Li 1,2,*
Round 1
Reviewer 1 Report
The manuscript is good enough, I suggest that the authors should include Statistical Analysis as a subsection of Materials and Methods and place Results section as section number 3. Discussion should be numbered as section 4 and the conclusion as section 5.
Author Response
The manuscript is good enough, I suggest that the authors should include Statistical Analysis as a subsection of Materials and Methods and place Results section as section number 3. Discussion should be numbered as section 4 and the conclusion as section 5.
Answer：Thank you for your comments. We had followed your advice and made revision in the manuscript.
Reviewer 2 Report
Dear Editor and authors,
the paper with the title "Detection of resistance in Echinochloa spp. to three post-emergence herbicides used in China" has a good concept and merits acceptance after significant major changes throughout the text. It is an exciting subject for weed scientists and people working with herbicide resistance screening. Authors should answer to all the following comments:
L2: Authors are suggested to name the three herbicides (penoxsulam, metamifop and quinlorac)
L11: "nine populations". It is not clear in the abstract if nine populations (may consider using biotypes?) were assessed in total or per species? Please elaborate.
L11-12: Three species are mentioned here but there is an obvious lack of discussion about the results of the experiments, similarities and differences, and comparisons with literature for these species. This is a general comment that refers to the entire manuscript. Authors should accurately refer to the different species, their susceptibility to the herbicides etc. Changes are expected to be seen in the results and discussion section at least.
L12: What is 1.5 leaf stage? Please erase and state that seedlings were at the 1-2 leaf stage. In the case authors refer to a fully developed leaf and a leaf that has started to formate, then this is not counted as a half. Please justify if you doubt that.
L13: In weed science, rates are preferred over concentrations for the active ingredients. Please justify the use of "concentrations"
L13: Survival rate is more scientific than survivorship. Please replace it throughout the text.
L13-14: Please add the justification that you mention in the materials and methods section for the measurement exactly at 8 days after treatment.
L16: Why is this result important for weed scientists? Please give an extra detail in the text.
L17-18: The addition of two populations confuses the reader. Please be consistent with the total number of assessed biotypes.
L18-21: This output may be well presented in text, but in the abstract is completely subjective and it is not supported by the previous sentences. Please replace "So" with "Therefore".
L26: 1) The references don't follow the format of MDPI Agronomy journal. This is a major point. All references must change throughout the text.
2) Many references are quite old. Please consider to replace them with newer.
L39: "improved human welfare". I doubt that. A lot of environmental effects arise from the use of herbicides that have direct and indirect effects on human health.
L40: "would also bring have". Please rephrase.
L45: "Heap, 1990". Please use the modern version of the Heap, I. The International Herbicide-Resistant Weed Database. Online. Tuesday, January 31, 2023 . Available www.weedscience.org
L49-51: These numbers are out-of-date. Please update with the numbers of Heap.
L56: "were". Are they not yet?
L57: quinclorac, respectively.
L62-63: The most recent publication and listing of rapid herbicide resistance screening tools and methods has been published in 2022. Authors should cite this and read the table describing the tools as well.
Tataridas, A., Jabran, K., Kanatas, P., Oliveira, R. S., Freitas, H., & Travlos, I. (2022). Early detection, herbicide resistance screening, and integrated management of Invasive Plant Species: A review. Pest Management Science, 78(10), 3957-3972. DOI 10.1002/ps.6963
L66-69: Here, authors refer to RISQ tests as tests that are applied to all plant tissues and in the abstract they refer that it as a laboratory assay. Please elaborate and correct.
L81: "two-to three-leaf stage". This is not the rule as some weeds are often sprayed at a later vegetative growth stage or some in the rosette. Please make it clear. For instance, early vegetative growth stages would be an alternative.
L84-86: This method is also conducted after collecting seeds from survived/escaped plants. The herbicide resistant traits are on a case by case basis transferred to the subsequent generations. Therefore, the detection of HR in greenhouses after the termination of weeds' growth stage is accepted as a method to prepare for next year's crop cycle.
L103 / Table 1,4,5,6 / Figures 1,2,3: The coding of the populations is really confusing. The reader can't distinguish between Ech. crusgalli and the remaining two species. Please change that across the text. It is really difficult to follow and certainly, this will lead to a reduced number of citations of your work.
L122: were sprayed
L126-127: Authors mention here and in the statistical analysis that this experiment was repeated twice and the data were pooled. However, there are lacking details about the differences between the runs (statistically, experimental conditions etc.). Please elaborate and improve the text.
Table 2: It is difficult to follow the doses without outlines. Please revise the table.
3. Statistical analysis: The st. analysis part should not be a single part, rather a subsection of materials and methods.
L188: Please use the equation maker of word.
L189-191: Please elaborate about the pooled data. You mention linear relationships. Did the data follow normal distribution? If so, please write the relevant statistics that support the linear regression.
L344: ALS first, ACCase second
Discussion: The discussion section needs to be improved and links to physiology etc. should be also stated. Please consider using more papers. Indicatively:
Wu, L. M., Fang, Y., Yang, H. N., & Bai, L. Y. (2019). Effects of drought-stress on seed germination and growth physiology of quinclorac-resistant Echinochloa crusgalli. PloS one, 14(4), e0214480.
Zhang, C. J., Lim, S. H., Kim, J. W., Song, J. S., Yook, M. J., Nah, G., ... & Kim, D. S. (2015). Quantifying herbicide dose–response and resistance in Echinochloa spp. by measuring root length in growth pouches. Canadian Journal of Plant Science, 95(6), 1181-1192.
Concenço, G., Silva, A. F., Ferreira, E. A., Galon, L., Noldin, J. A., Aspiazú, I., ... & Silva, A. A. (2009). Effect of dose and application site on quinclorac absorption by barnyardgrass biotypes. Planta Daninha, 27, 541-548.
Author Response
L2: Authors are suggested to name the three herbicides (penoxsulam, metamifop and quinlorac)
Answer: Thank you for your comments. We had revised as your request.
L11: "nine populations". It is not clear in the abstract if nine populations (may consider using biotypes?) were assessed in total or per species? Please elaborate.
Answer: Thank you for your comments. As your comments, “biotype” would be more precise than “population”, and we had replaced it through the manuscript. And, all the 9 biotypes are of different susceptibility to penoxsulam, metamifop and quinclorac. To establish the RISQ test, we choose at least 5 biotypes for each herbicide considering to including biotypes of the different species and susceptibility to the herbicide. For example, 5 biotypes (3 from E. crusgalli, one biotype from E. glabrescens and one from E. crusgalli var. zelayenisi) were used in the metamifop test. And this information had been listed in table 1. As we think this background information is too complicated to include in the abstract, so we just mentioned we used 9 biotypes briefly but the detailed background information was not included. But still, we re-written the sentence as “In this study, rapid resistance in-season quick (RISQ) tests were developed for detecting the resistance in Echinochloa spp. to penoxsulam, metamifop and quinclorac, respectively, which are widely used in rice fields to control E. spp. Seedlings in 1-2 leaf stage from nine biotypes of E. crusgalli, E. crusgalli var. zelayensis and E. glabrescens with different susceptibility to the tested herbicides were transplanted to plates containing nutrient agar and different concentrations of herbicides.”
L11-12: Three species are mentioned here but there is an obvious lack of discussion about the results of the experiments, similarities and differences, and comparisons with literature for these species. This is a general comment that refers to the entire manuscript. Authors should accurately refer to the different species, their susceptibility to the herbicides etc. Changes are expected to be seen in the results and discussion section at least.
Answer: Thank you for your comments. The purpose of using three different E. spp is to make the RISQ test applicable to different E. spp., as in rice fields there are always different E. spp occurring at the same time. And RISQ is supposed to be performed at the early vegetative growth of E. spp before the farmers choose what herbicides would be used. At that time, it is hardly impossible to identify exactly what kind of E. spp by visual observation. So our emphasis was put on the rate that could discriminate resistant and susceptible E. spp., but less attention was on the different susceptibility to herbicides of different E. spp. And as you commented, we add some description on the different susceptibility to the three herbicides in these three E. spp. from line 251 to 255.
L12: What is 1.5 leaf stage? Please erase and state that seedlings were at the 1-2 leaf stage. In the case authors refer to a fully developed leaf and a leaf that has started to formate, then this is not counted as a half. Please justify if you doubt that.
Answer: Thank you for your comments. We replaced 1.5 leaf stage as 2 leaf stage
L13: In weed science, rates are preferred over concentrations for the active ingredients. Please justify the use of "concentrations"
Answer: Thank you for your comments. Based on your explaination, we prefer to use “rate” instead of concentration and we replace it throughout the manuscript.
L13: Survival rate is more scientific than survivorship. Please replace it throughout the text.
Answer: Thank you for your comments. We had replaced it as you requested.
L13-14: Please add the justification that you mention in the materials and methods section for the measurement exactly at 8 days after treatment.
Answer: Thank you for your comments. The sentence had been re-written as “Survival rate was recorded at 8 days after treated when no more new roots emerged for all the treatment.”
L16: Why is this result important for weed scientists? Please give an extra detail in the text.
Answer: Thank you for your comments. We added some detail in line 320.
L17-18: The addition of two populations confuses the reader. Please be consistent with the tot Answer: Thank you for your comments.al number of assessed biotypes. The additional two biotypes were used to verify the validity of the developed RISQ test, as the susceptibility of these two biotypes to the herbicides had been determined by whole plant bioassay. And we re-written the sentence as “Two additional biotypes of E. crusgalli collected in fields were used to confirm the validation of the RISQ test and the obtained results by RISQ were consistent with that by the whole-plant bioassay.”
L18-21: This output may be well presented in text, but in the abstract is completely subjective and it is not supported by the previous sentences. Please replace "So" with "Therefore".
Answer: Thank you for your comments. We re-written the sentence as “Therefore, the developed RISQ test would be a possible alternative method to determine the susceptibility of E. spp. to certain herbicides.”
L26: 1) The references don't follow the format of MDPI Agronomy journal. This is a major point. All references must change throughout the text.
2) Many references are quite old. Please consider to replace them with newer.
Answer: Thank you for your comments. The format of the references had been changed. As this manuscript was finished in 2019 and submitted in 2023, most of the references were old, but we had add some new references as alternatives.
L39: "improved human welfare". I doubt that. A lot of environmental effects arise from the use of herbicides that have direct and indirect effects on human health.
Answer: Thank you for your comments. What we try to express is that with the use of pesticides, people would get more agricultural produce, which would supply more food and make the people’s living standard better than before. And we replaced “human welfare” by “people’s living standard”.
L40: "would also bring have". Please rephrase.
Answer: Thank you for your comments. It must be a mistake and we deleted “have”.
L45: "Heap, 1990". Please use the modern version of the Heap, I. The International Herbicide-Resistant Weed Database. Online. Tuesday, January 31, 2023 . Available www.weedscience.org
Answer: Thank you for your comments. We had updated the data and the citation. “To date, at least 515 unique cases of herbicide-resistant weed (including 267 species) had been identified in 72 countries (Heap, 2023).”
L49-51: These numbers are out-of-date. Please update with the numbers of Heap.
Answer: Thank you for your comments. We had update the data and the citation. “To date, at least 515 unique cases of herbicide-resistant weed (including 267 species) had been identified in 72 countries (Heap, 2023).”
L56: "were". Are they not yet?
Answer: Thank you for your comments. We had replaced “were” by “are”
L57: quinclorac, respectively.
Answer: Thank you for your comments. We had add “respectively” after “quinclorac”
L62-63: The most recent publication and listing of rapid herbicide resistance screening tools and methods has been published in 2022. Authors should cite this and read the table describing the tools as well.
Answer: Thank you for your comments. We had added the citation at line 79.
Tataridas, A., Jabran, K., Kanatas, P., Oliveira, R. S., Freitas, H., & Travlos, I. (2022). Early detection, herbicide resistance screening, and integrated management of Invasive Plant Species: A review. Pest Management Science, 78(10), 3957-3972. DOI 10.1002/ps.6963
L66-69: Here, authors refer to RISQ tests as tests that are applied to all plant tissues and in the abstract they refer that it as a laboratory assay. Please elaborate and correct.
Answer: Thank you for your comments. We had re-construct the text.
L81: "two-to three-leaf stage". This is not the rule as some weeds are often sprayed at a later vegetative growth stage or some in the rosette. Please make it clear. For instance, early vegetative growth stages would be an alternative.
Answer: Thank you for your comments. As we had re-construct the text, the sentence containing “two to three-leaf stage” had been deleted. And thank you for clarifying the mistaken phrase we had used.
L84-86: This method is also conducted after collecting seeds from survived/escaped plants. The herbicide resistant traits are on a case by case basis transferred to the subsequent generations. Therefore, the detection of HR in greenhouses after the termination of weeds' growth stage is accepted as a method to prepare for next year's crop cycle.
Answer: Thank you for your comments. What we try to deliver is that the RISQ could be performed before the application of herbicide. Although we use the seeds from the survived plants to establish the RISQ test, but the test is to be used to detect susceptibility of E. spp in the rice field before the application of herbicide. Based on the results of RISQ, we could choose the proper herbicides to be applied. And again, as we re-construct the text, the above description had been deleted.
L103 / Table 1,4,5,6 / Figures 1,2,3: The coding of the populations is really confusing. The reader can't distinguish between Ech. crusgalli and the remaining two species. Please change that across the text. It is really difficult to follow and certainly, this will lead to a reduced number of citations of your work.
Answer: Thank you for your comments. As most of the populations used in the research had been reported in our previous paper, we had continued to use them. And we had re-coded the population in an easy way and changed them across the text as well as in the tables.
L122: were sprayed
Answer: Thank you for your comments. We’ve corrected the mistake.
L126-127: Authors mention here and in the statistical analysis that this experiment was repeated twice and the data were pooled. However, there are lacking details about the differences between the runs (statistically, experimental conditions etc.). Please elaborate and improve the text.
Answer: Thank you for your comments. The whole plant pot assay had been performed twice to see the consistency between different runs. As this work was finished by Mrs. Liu and Miss. Zhang, who had graduated from my lab in 2018 and 2020, respectively, it is difficult to ask them to find the raw data to re-analysis. I’m so sorry for it. And I delete the related description on data pool in the text.
Table 2: It is difficult to follow the doses without outlines. Please revise the table.
Answer: Thank you for your comments.We’ve revised table 2.
Statistical analysis: The st. analysis part should not be a single part, rather a subsection of materials and methods.
Answer: Thank you for your comments. We put this part in materials and methods as section 2.6.
L188: Please use the equation maker of word.
Answer: Thank you for your comments. We’ve re-insert the equation 1 and 2.
L189-191: Please elaborate about the pooled data. You mention linear relationships. Did the data follow normal distribution? If so, please write the relevant statistics that support the linear regression.
Answer: Thank you for your comments. As this work was finished by Mrs.Liu and Miss Zhang, who had graduated from my lab in 2018 and 2020, respectively, it is difficult to ask them to find the raw data to re-analysis. These data were come from Mrs Liu’s dissertation and in her dissertation, only the ED50s and SE were presented. And using a probit regression equation to fit a line regression is also widely used in evaluation the herbicide toxicity to weeds. Still, I’m so sorry for it.
L344: ALS first, ACCase second
Answer: Thank you for your comments. We’ve changed it.
Discussion: The discussion section needs to be improved and links to physiology etc. should be also stated. Please consider using more papers. Indicatively:
Wu, L. M., Fang, Y., Yang, H. N., & Bai, L. Y. (2019). Effects of drought-stress on seed germination and growth physiology of quinclorac-resistant Echinochloa crusgalli. PloS one, 14(4), e0214480.
Zhang, C. J., Lim, S. H., Kim, J. W., Song, J. S., Yook, M. J., Nah, G., ... & Kim, D. S. (2015). Quantifying herbicide dose–response and resistance in Echinochloa spp. by measuring root length in growth pouches. Canadian Journal of Plant Science, 95(6), 1181-1192.
Concenço, G., Silva, A. F., Ferreira, E. A., Galon, L., Noldin, J. A., Aspiazú, I., ... & Silva, A. A. (2009). Effect of dose and application site on quinclorac absorption by barnyardgrass biotypes. Planta Daninha, 27, 541-548.
Answer: Thank you for your comments. We had re-written the discussion part and cited some of the above literatures.
Author Response File: 
Author Response.pdf
Reviewer 3 Report
See attached file
Comments for author File: Comments.pdf
Author Response
The manuscript describes the development of a quick method for the detection of resistance to three herbicides belonging different site of action in Echinochloa spp. The novelty is in the use of a different species and the introduction of a new herbicide site of action (the auxin-like herbicide quinclorac), using a technique which was already developed by other researcher (Kaundun at et. 2011, ref. 25) as discussed also by authors.
Two main limits I saw in the paper. The first one is the English style, I am not a mother tongue, but the manuscript is really difficult to read in many points.
Answer: Thank you for your comments. We’ve had English editing in 2019 and the certification had been uploaded.
The second is linked to the validation of the method (section 2.5 and 4.3) that should be done on blind populations, instead authors set-up the RISQ protocol on 11 populations and then used two of them for the validation.
Answer: Thank you for your comments. We’d use 9 biotypes in the developing of RISQ. And the 2 biotypes used in verification are additional ones.
The literature cited, mainly in the first part of the introduction is really dated. Many more recent papers have been published about Echinochloa spp., use of herbicides to control them and evolution of herbicide resistance in these species. My advice to authors is to improve the quality of introduction by introducing more recent studies to support the need of having quick method to detect herbicide resistance in Echinochloa spp. E.g. the Heap’s website (http://weedscience.org/Home.aspx) has never been cited in the manuscript, but only papers by Heap wrote before 2000 were cited!
Answer: Thank you for your comments. We had replaced these literatures with updated ones.
Check Ref. 33, authors are not correct if this is the cited paper https://doi.org/10.1093/nar/28.12.e63;
furthermore, this is a general paper on LAMP method and not an application of this method to detect herbicide resistance as, for e.g., Pan et al. (2015) (ref. 35).
Answer: Thank you for your comments. We had cited a new literature.
Section 2.1: The names of the populations used for the research are really difficult and to follow the development of the study it is hard, it should possible to simplify them??
Answer: Thank you for your comments. As most of the populations used in the research had been reported in our previous paper, we had continued to use them. And we had re-coded the population in an easy way and changed them across the text as well as in the tables.
Table 1: the column “Resistance profile” derived from your tests or derived from field history of the different populations? This is not clear… in the first case I suggest moving this data to results. Then, I cannot understand the ref indicated in the populations characterized for target-site mechanism (AXXZ-6 and AXXZ-2)… how the authors obtained these data? I believed that the data were included in the ref indicated but they are completely different papers, not by authors of the present manuscript. If you sequenced the ALS gene of those populations, it should be reported in the M&M and Results. Pops JNLS-1 and HYYJ-1 should be indicated as “check S” and “check R” and for the second a ref indicating this resistance profile should be reported.
Answer: Thank you for your comments. Resistance profile means the susceptibility background of these populations, which had been determined in our previous published paper. As to develop the RISQ, we must know the susceptibility of the populations in advance.
Section 2.2 may be omitted, the sentence is already included in the Introduction and herbicides used may be mentioned in the following section 2.3. Please provide the details of herbicide used (commercial product, company, etc.). How many L/ha delivered the sprayer used?
Answer: Thank you for your comments. We add the details of the three herbicides in 2.2. Also, we add the volume we spayed in line 154, which is 600L/ha.
Table 2: put horizontal lines to separate the different herbicides because it is not clear which lines refer to each one. Why not all populations were treated with all herbicides?
Answer: Thank you for your comments. We add the horizontal lines in table 2.
And the populations used in developing a RISQ to one herbicide had two major considerations. One is to include all the species of Echnichloa, and the other is to include known susceptible and resistant populations. Also, we take the workload into account and try to get equilibrium between the scientificalness and efficiency.
Line 149: 1.5-leaf stage???
Answer: Thank you for your comments. We had changed the description of the leaf stage as “early vegetative growth” based on another reviewer’s comments.
Lines 206-210: Indications of different levels of resistance (S, L, M and H) did not correspond with indications in Table 4, 5 and 6 where authors used different indications (S, rr, RR, Rr)… please standardize.
Answer: Thank you for your comments. We had revised it and use S, L, M and H to describe the resistance level.
Heading of section 4.2.2 should be changed in “Selection of RISQ herbicide discriminating concentrations comparing whole plant pot assays data”, or something like that, and section 4.2.3 incorporated because Table 7 included results of both experiments.
Answer: Thank you for your comments. We changed the heading of 4.2.2 as “Selection of discriminating rates of herbicides in the RISQ” and deleted the heading of 4.2.3.
Table 7 needs to be revised for clarity, some suggestions:
“population” have to be moved up, and over there “herbicide dose used” have to be written; 7c) quinclorac moved to left; the survival percentages should have indication of a range (S.E.) based on the different replicates.
Answer: Thank you for your comments. We’d make some revision on table 7 and the S.E. had been added.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Dear Authors, the necessary changes have been made and now the manuscript is much better.
Author Response
Thank you for your comments.
Reviewer 3 Report
Authors wrote: We’ve had English editing in 2019 and the certification had been uploaded."
I don't think that this is enough to write a good paper. It has been improved but not being a native speaker I think a check should be done anyway. The entire work would benefit from it, which is, in any case, from a scientific point of view, a relevant work.
A few editing considerations in the file attached.
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
Thank you for your careful and profensional revision of the manuscript. We'd make a point-by-point response to your comments. And please see the attachment for the detailed response. Thank you very much.
yours,
Jun Li
Author Response File: 
Author Response.docx
