Systemic Uptake of Rhodamine Tracers Quantified by Fluorescence Imaging: Applications for Enhanced Crop–Weed Detection
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe author demonstrated through experiments that Rhodamine B can be used as a fluorescent tracer for seeds and seedlings, and studied how to alleviate its toxic effects on seeds. This study contributes to the application of Rhodamine B in agricultural production.
（1）The numbers on the bar chart do not need to be marked, but it is best to add the standard deviation on the chart.
（2）The author uses graphs to illustrate the differences between the treatment and control, and it is best to add some data to further explain, such as seedling length and thickness, leaf length, leaf width, leaf area, etc.
Author Response
Response to reviewer #1:
The authors of this manuscript acknowledge general comments and suggestions of our manuscript. Our response (in red) follows:
Comments and Suggestions for Authors
The author demonstrated through experiments that Rhodamine B can be used as a fluorescent tracer for seeds and seedlings, and studied how to alleviate its toxic effects on seeds. This study contributes to the application of Rhodamine B in agricultural production.
Point 1: The numbers on the bar chart do not need to be marked, but it is best to add the standard deviation on the chart.
Response 1: Concerning the reviewer's comment, the authors prefer to retain the values on the charts to present the data clearly. Regarding the addition of standard deviation (SD), we added the standard error of the mean (SE) for each data point (Figures 7 to 10).
Point 2: The author uses graphs to illustrate the differences between the treatment and control, and it is best to add some data to further explain, such as seedling length and thickness, leaf length, leaf width, leaf area, etc.
Response 2: In this investigation, Mean Tracer Digital Count measurements were consistently performed on leaf discs with a diameter of 1.2 cm across all experiments involving bean, hemp, and velvetleaf plants. These measurements were taken at a specific growth stage, eliminating the need for seedling biometric data. The standardized leaf disc size ensured uniformity in the sampling process, allowing for accurate comparisons between different plant species and experimental conditions.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript reports a study on the use of fluorescent “systemic tracer that can be introduced via seed technologies to enhance crop-weed differentiation”, as well as an imaging system developed to detect rhodamine fluorescence in seedlings, whole leaves, and leaf discs.
The study was well designed, the methodology is suitable for the study and the subject is relevant. Therefore, I suggest that the manuscript be accepted with minor suggestions to improve the presentation of the M&M and Results sections, listed below. Please review the text for typos, misspelling and use of the plural form.
The summary needs to be improved to clarify the purpose of the study and the main conclusions. Similarly, the purpose of the study is not clear in the introduction. The research purpose was presented in a very fine way in the Discussion and Conclusions sections, so I suggest rewriting the Introduction to show in a clear way the objectives of the study.
Does WT stand for water tracer? What is the difference between Rhodamine B and Rhodamine WT? Please explain why WT is “a potential alternative tracer to RB”. What would be the advantage? Please clarify this in the introduction.
Line 17: Did you mean “applied as a seed treatment”?
Line 44: please describe what ST and IF stand for
Line 146: Did you mean Acros Organics?
Please shorten the titles of the subsections in the M&M section. This section is confusing, with many treatments and experiments. I suggest describing the treatments in a table, for better visualization. The same applies to the Results subsections. Please present the results using the same subsection titles as in the M&M.
Line 306-307: this sentence has no verb. Please rewrite this part of the text.
Line 319: please delete the word “well”.
Lines 320-321: Please rewrite this sentence: “The RB treatment solution was applied that the active ingredient was deposited in the outer region of the pellet”
Line 329: Figure 3. Please review this title for the plural form.
Figure 7. Please provide a main title, describing what the graph is about. Then describe the samples and treatments for A (snap bean) and B (hemp) (no need to repeat the description for both plant species). The title should explain the figure without the need to go back to the main text, therefore, you should explain the treatments (RB, SB and WT).
Line 411: what is FLIR?
Line 417: seeds, in the plural
Figures 9a and 10a were not cited in the text.
Line 470: hemp pelleted* seeds
Lines 475-478: it is not common to describe the objectives of the research in the Discussion section. Is this a requirement of this journal? If not, please move this paragraph to the end of the introduction.
Also, it is not common to describe the results again in the Discussion section, referring to figures and tables. Please rewrite the Discussion section to not repeat what was presented in the Results section. Or the authors could consider bringing together the discussion with the results section. However, my suggestion would be to keep the Discussion section separated from the Results section. The Discussion section is well written, bringing clear and relevant information.
Comments on the Quality of English LanguagePlease review the text for typos, misspelling and use of the plural form.
Author Response
Response to reviewer #2:
We would like to thank Reviewer #2 for the careful and detailed reading of our manuscript and for the thoughtful comments and constructive suggestions, which help to improve the quality of our manuscript. Our response (in red) follows:
Comments and Suggestions for Authors
The manuscript reports a study on the use of fluorescent “systemic tracer that can be introduced via seed technologies to enhance crop-weed differentiation”, as well as an imaging system developed to detect rhodamine fluorescence in seedlings, whole leaves, and leaf discs.
The study was well designed, the methodology is suitable for the study and the subject is relevant. Therefore, I suggest that the manuscript be accepted with minor suggestions to improve the presentation of the M&M and Results sections, listed below. Please review the text for typos, misspelling and use of the plural form. Spelling and grammar check completed.
The summary needs to be improved to clarify the purpose of the study and the main conclusions.
The Abstract was revised on lines 10-38 with additional Keywords added (Lines 39-41).
Similarly, the purpose of the study is not clear in the introduction. The research purpose was presented in a very fine way in the Discussion and Conclusions sections, so I suggest rewriting the Introduction to show in a clear way the objectives of the study.
The Introduction was revised and condensed to better align with the objectives of the paper. One paragraph on weed management moved from the Introduction to the Discussion
Point 1: Does WT stand for water tracer? That is correct WT stand for water tracer
What is the difference between Rhodamine B and Rhodamine WT? They are chemically different, as stated in line 136. Also note that the CAS # are different for the three rhodamine compounds (RB line 108, WT line 134, SB line 144). Please explain why WT is “a potential alternative tracer to RB”. What would be the advantage? Please clarify this in the introduction. Explanation on WT is now in the Introduction and Methods. Lines 64-74 were revised for clarification.
Point 2: Line 17: Did you mean “applied as a seed treatment”?
Line 13: edited as “seed treatment”
Point 3: Line 44: please describe what ST and IF stand for?
Line 54: Edited as “applied as a seed treatment (ST) or in-furrow (IF) soil treatment”
Point 4: Line 146: Did you mean Acros Organics?
Line 135: Correct Acros Organics is now Thermo Scientific Chemicals and edited as “Acros Organics”
Point 5: Please shorten the titles of the subsections in the M&M section. This section is confusing, with many treatments and experiments. I suggest describing the treatments in a table, for better visualization. The same applies to the Results subsections. Please present the results using the same subsection titles as in the M&M.
All subheadings were shortened throughout Methods and Results. With the shortened subtitles, we disagree that need tables are needed to describe each study.
Revised subtitles line numbers: 98, 106, 132, 156, 180, 194, 208, 248, 269, 288, 317, 323, 351, 364, 426, 433, 449, 495.
Point 6: Line 306-307: this sentence has no verb. Please rewrite this part of the text.
Line 291: edited as “The study utilized ‘Vega’ hemp seeds, a dual-purpose variety sourced from UNISeeds Inc., Cobden, Ontario, Canada.”
Point 7: Line 319: please delete the word “well”.
Line 303:The word “well” deleted.
Point 8: Lines 320-321: Please rewrite this sentence: “The RB treatment solution was applied that the active ingredient was deposited in the outer region of the pellet”
Line 304-305: edited as “The RB treatment solution was applied to the pellet, the pellet quickly absorbed the solution and after drying the RB distributed in the pellet.”
Point 9: Line 329: Figure 3. Please review this title for the plural form.
Line 312-314: Figure 3. Six pelleted hemp seeds surrounding three velvetleaf seeds marked with arrows. (a) Nontreated pellets on the left and (b) pelleted seeds treated with RB on the right. Germination media was a mixture of sand and perlite (2:1 v:v).
Point 10: Figure 7. Please provide a main title, describing what the graph is about. Then describe the samples and treatments for A (snap bean) and B (hemp) (no need to repeat the description for both plant species). The title should explain the figure without the need to go back to the main text, therefore, you should explain the treatments (RB, SB and WT).
Line 389-393: Figure 7-caption edited as “Figure 7. Fluorescence intensity (mean tracer digital count) of the three tracers (Rhodamine B, WT, and SB) from (a) bean leaf disc of 1st true leaf, 1st, and 2nd trifoliate and (b) hemp leaf disc of 1st, 2nd and 3rd true leaf obtained 14, 21 and 28 days after planting using Image J software. Seedlings grown in a mixture of sand and perlite (2:1 v:v). Statistical analysis of mean tracer digital count (MTDC) within each leaf type (significance level: α=0.05).”
Point 11: Line 411: what is FLIR?
Line 401: edited as “The Fluorescence intensity ratio (FLIR)”
Point 12: Line 417: seeds, in the plural
Line 422: edited as “bean seeds”
Point 13: Figures 9a and 10a were not cited in the text.
“Figure 9a cited in M&M line 282” The Fluorescence intensity ratio (FLIR)
“Figure 10a cited in line 498”
Point 14: Line 470: hemp pelleted* seeds
Line 512: edited as “hemp pelleted seeds”
Point 15: Lines 475-478: it is not common to describe the objectives of the research in the Discussion section. Is this a requirement of this journal? If not, please move this paragraph to the end of the introduction.
Text in the Discussion section revised not to repeat the term objectives in the first paragraph
Also, it is not common to describe the results again in the Discussion section, referring to figures and tables. Please rewrite the Discussion section to not repeat what was presented in the Results section.
A brief description of results in the Discussion section was used to make comparisons and link our results with literature and to improve readability.
Or the authors could consider bringing together the discussion with the results section. However, my suggestion would be to keep the Discussion section separated from the Results section.
Results and Discussion are kept as separate sections.
The Discussion section is well written, bringing clear and relevant information.
Point 15: Comments on the Quality of English Language
Please review the text for typos, misspelling and use of the plural form.
The authors edited the typos and misspelled words throughout the manuscript.
Three papers were deleted as references from Taylor’s Seed Science and Technology at Cornell.
