Evaluation of the Potential of Pyrimidine Nucleoside Antibiotics Against Alternaria spp. Resistant to QoIs Fungicides: Insights for the Management of Ginseng Alternaria Leaf and Stem Blight Disease

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is easy to follow except for certain sections, which require re-writing or revision to improve its clarity. The authors must also make sure that everything is in place, and nothing is missing. The specific comments for the respective sections are as follows: 

Please introduce the topic and the research problem to provide a context for the study. The first line in the abstract must be revised for clarity. 

Line 25-28 must be revised to avoid repeating the same information i.e the parameters used for the evaluation 

Line 36 A. spp. - Please spell Alternaria instead of "A." spp.
Line 53-57 - Please separate the points into two sentences for clarity 
Line 84-87 - It would be better to mention the study aim before describing the methodologies used in the study 

Line 198 - Redundant with the previous paragraph. The same goes for the statement on protein extraction. Please revise and avoid repetitive content. 

Table 2 - Please revise the table title and move the statistical description to the bottom of the table. Is the 'note' necessary? The statement is redundant. 

3.5 is difficult to follow. I suggest the authors re-write it. The authors may minimize the numbers in the text (readers can refer to the table) and summarize the results 

Line 540 - Please insert the missing citation, and please check the whole manuscript for any other missing citations 

Comments on the Quality of English Language

The manuscript is written in an easy-to-follow English language 

Author Response

Dear professor,

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Evaluation potential of pyrimidine nucleoside antibiotics used for management of Alternaria spp. resistant to common fungicides” (Manuscript ID: agriculture-3536130, now the revised title as “Evaluation of the potential of pyrimidine nucleoside antibiotics against Alternaria spp. resistant to QoIs fungicides: insights for the management of ginseng Alternaria leaf and stem blight disease”)”. Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.

Best wishes,

Jie Gao

The reviewer 1

Comments and suggestions for authors

The manuscript is easy to follow except for certain sections, which require re-writing or revision to improve its clarity. The authors must also make sure that everything is in place, and nothing is missing. The specific comments for the respective sections are as follows:

Qustion1 Please introduce the topic and the research problem to provide a context for the study. The first line in the abstract must be revised for clarity.

Answer1: Thanks for your valuable suggestion. We have added the sentence “To manage the developing resistance of Alternaria spp. [the causal fungi of ginseng Alternaria leaf and stem blight (GALSB)] to QoIs fungicides” in the first line in the abstract, so that provide a context for the study. Please see Line 13 and 14.

Q2: Line 25-28 must be revised to avoid repeating the same information i.e the parameters used for the evaluation.

A2: Thank you for your suggestions.
We have reorganized and highly condensed this section to avoid redundant information about evaluation parameters. As a result, the overall content is clearer and more logically structured. The revised related section is as follows: “PNA caused morphological changes in A. alternata mycelia and germ tubes, increased cell membrane permeability, and reduced intracellular DNA and protein levels. On detached ginseng leaves, 300 μg/mL PNA achieved mean protective and curative effects of 87.93% and 94.77% against A. alternata 7 days post inoculation, outperforming that of 300 μg/mL kresoxim-methyl. Field trial results showed that PNA (180 g a.i./hm²) achieved mean efficacies of 85.63%, 84.07%, and 72.55% at three sites 7, 15, and 30 days after the last spray, which were 5.28%-37.74% higher than those of control fungicides pyraclostrobin, azoxystrobin, and kresoxim-methyl at corresponding time points. Overall, our findings indicate that PNA are effective agents for the management of Alternaria spp. resistance to QoIs fungicides.”. Please see Line 22-31 in the manuscript.

Q3: Line 36 A. spp. - Please spell Alternaria instead of "A." spp.

A3: Thank you for your valuable suggestion. We have changed “A. spp.” to “Alternaria spp.” in the manuscript.

Q4：Line 53-57 - Please separate the points into two sentences for clarity.

A3: Thank you for your valuable suggestions. We have modified sentences as follows: “Additionally, the efficacy of these fungicides, such as azoxystrobin, pyraclostrobin, and tebuconazole, has decreased over time due to the development resistance in Alternaria spp., and this has led to increases in the dosage and application frequency of fungicides [19, 20]. Consequently, the fungicide residues associated with the excess application of fungicides can result in environmental pollution and residues in plant [21].” Please see Line 60-65 in the manuscript.

Q5: Line 84-87. It would be better to mention the study aim before describing the methodologies used in the study

A5: Thank you for your valuable suggestion. We have mentioned the study aim before describing the methodologies used in the study. “In order to explore whether PNA can be used to manage the resistance of Alternaria spp. to QoIs fungicides, we conducted the following investigations”. And we also supplemented experimental method “mycelial growth rate method” in the comparative analysis of cross-resistance. Please see Line 82-83 and 91 in the manuscript.

Q6: Line 198 - Redundant with the previous paragraph. The same goes for the statement on protein extraction. Please revise and avoid repetitive content.

A6: Thank you for your valuable suggestions. We have removed the redundant content in the first paragraph. Moreover, we have refined the statement and removed some sentence, relevant revised section as follows: “1 mL of precooled 10% TCA-acetone solution (with 0.2% DTT) was added and vortex-mixed. The mixture was left to precipitate overnight at -20℃, then centrifuged at 4℃, 12,000 rpm for 15 min. The precipitate underwent one wash with 100 mmol/L ammonium acetate, 80% methanol, and 80% acetone solutions. Next, 1 mL of SDS/phenol extraction solution (1:1 saturated phenol and SDS buffer) was added, vortex-mixed, and incubated on ice for 5 min. After centrifugation at 4℃, 12,000 rpm for 15 min, the upper phenolic phase was transferred to five volumes of precooled 100 mmol/L ammonium acetate-methanol solution. The solution was precipitated at -20℃ for 4 h and centrifuged again at 4℃ for 15 min. The resulting precipitate was washed with precooled methanol and 80% acetone, freeze-dried (Alpha 1-2 LD plus, Shanghai Bajiu Industrial Co., Ltd., Shanghai, China) to obtain a powder. The powder was dissolved in 1 mL of ddH₂O to determine protein concentration and content. Standard solutions of bovine serum albumin at 0, 100, 200, 300, 400, and 500 μg/mL were prepared in triplicate. OD values at 280 nm were measured to create a standard curve for calculating protein content in different treatments.” Please see Line 215-229.

Q7: Table 2 - Please revise the table title and move the statistical description to the bottom of the table. Is the 'note' necessary? The statement is redundant.

A7: Thank you for your valuable suggestions. We have moved the statistical description to the bottom of the table and refined them. “Note: The data were expressed as mean ± SD (standard deviation) (n = 6). The mean values followed by the same letter in the columns were not significantly different according to Fisher’s LSD test at p = 0.05. By the conidia germination method, DH3-1 isolates showed the highest sensitivity to PNA, followed by XJ2-5 and JY39 isolates.” Please see Line 396-399.

Q8: 3.5 is difficult to follow. I suggest the authors re-write it. The authors may minimize the numbers in the text (readers can refer to the table) and summarize the results

A: Thank you for your constructive feedback. In response to your suggestions, we have rewritten the results section to minimize numerical clutter in the text, directing readers to relevant figures and tables for detailed data. The revised section as follows: “Field trials across Changchun, Baishan, and Yanbian in Jilin Province demonstrated that the control efficacy of PNA 4% AS against GALSB disease increased with higher dosages. Specifically, at 180 g a.i./hm², PNA consistently outperformed other fungicides over 7 days, 15 days, and 30 days post-last spray (PLS) (Figure 8, Table 3, S1, S2, and S3). 7 days PLS: High-dose PNA achieved a mean control efficacy of 85.62%. This was substantially higher than pyraclostrobin (78.65%), azoxystrobin (70.81%), and kresoxim-methyl (74.74%). 15 days PLS: PNA maintained a high mean control efficacy of 84.07%, again surpassing the comparison fungicides (pyraclostrobin: 72.17%; azoxystrobin: 64.58%; kresoxim-methyl: 73.82%). 30 days PLS: PNA still showed a mean control efficacy of 72.55%, while the efficacies of other fungicides dropped below 54% (pyraclostrobin: 52.67%; azoxystrobin: 53.18%; kresoxim-methyl: 53.56%)” Please see Line 447-459.

Q9: Line 540 - Please insert the missing citation, and please check the whole manuscript for any other missing citations

A9: Thank you for your valuable suggestion. We have inserted the reference “McErlean, M.; Liu, X.; Cui, Z.; Gust, B.; Van Lanen, S., Identification and characterization of enzymes involved in the biosynthesis of pyrimidine nucleoside antibiotics. Nat Prod Rep. 2021, 38, 1362-1407. https://doi.org/10.1039/d0np00064g” into the corresponding position in line 540 to supplement the citation for this part. Please see Line 533 in the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Unfortunately for Europeans, your study is no longer of practical importance, as the fungicides you tested have been withdrawn from the market, a large part of them, and for others the approval has not been extended, and is set to expire in a few months. (https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/start/screen/active-substances)

1. The title is in accordance with the study carried out.
2. 2. Abstract. I believe that the abstract is not clear...the purpose, material and method, results are not clearly highlighted...you have introduced many results without us understanding what the experimental variants are, it is not clear under what conditions the study was conducted (laboratory or field)...what was applied in which phenophases? when were the evaluations carried out...we deduce that they were at 7..15..30 days (I think you should redo it so that it is easier to read and quote).

- I suggest introducing the notion of "fungicides" as a keyword

 

3. Introduction. I recommend improving the introduction with information regarding:

- clear description of symptoms,

- damage produced (expressed in percentages),

- presentation of more information related to fungicide resistance of the pathogen Alternaria sp.

- presentation of more information related to the effect (positive or negative) of PNA on other crops and on other pathogens

In these lines:  52, 55; 57, 63,74, 76, 77, 87, 116,  294, 295, 466, 584 correct A. spp. with Alternaria spp.

 

4. Material and research methods described are in accordance with the study. 

Describe - the damaya cultivar (is it sensitive..tolerant to Alternaria)

Specify the distance between the localities where the studies were carried out, are they characterized by the same climatic conditions?

- Enter information related to the climatic conditions in the areas studied

- Enter information regarding the phenophase in which the plants were when you applied the treatments

- What was the pressure/virulence of pathogens in the crop when you carried out the treatments

 

5. Results and discussions

- I believe that the discussions should be improved with information and comparisons from the existing literature that show the importance of the study, its innovative character… the benefits of application in practice.

- Try to be clearer about the results

- Ex. In the text you insist a lot on the efficacy recorded in the 3 regions, but you do not give the same interest to the 3 evaluations (7-15-30 days)… do not discuss in detail which treatment has better efficacy at 7-15-30 days, I believe that it is much more important to know the efficacy and the protection period that a certain treatment has… than to keep highlighting the study locations, whose climatic conditions we do not know and which lead to a decrease in the relevance of your study (Figure 9 and table 9).

 

6. Bibliography. Self-citations were noted, but they are within the field of study

 

 

 

Author Response

Dear professor,

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Evaluation potential of pyrimidine nucleoside antibiotics used for management of Alternaria spp. resistant to common fungicides” (Manuscript ID: agriculture-3536130, now the revised title as “Evaluation of the potential of pyrimidine nucleoside antibiotics against Alternaria spp. resistant to QoIs fungicides: insights for the management of ginseng Alternaria leaf and stem blight disease”)”. Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.

Best wishes,

Jie Gao

The reviewer 2

Comments and suggestions for authors

Question1: Unfortunately for Europeans, your study is no longer of practical importance, as the fungicides you tested have been withdrawn from the market, a large part of them, and for others the approval has not been extended, and is set to expire in a few months. (https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/start/screen/active-substances)

Answer1: We sincerely appreciate the reviewer’s alert regarding the European market situation of the fungicide PNA in our study. Although these fungicides are undergoing regulatory changes in Europe, they maintain significant practical value in the Chinese market. Currently, this multi-component complex-designed fungicide is still registered for use on a wide range of crops in China. It can be legally used across the country (http://www.chinapesticide.org.cn). The multi-component design effectively prevents marginal fungi from developing resistance.
We have supplemented relevant content in the introduction as follows: “In China, the PNA in use mainly contain anisomycin, tetramycin A, tetramycin B, toyocamycin, and nystatin [24, 25]. All these components are approved for crops production. 13 domestic and international manufacturers produce 17 kinds of PNA products, which were registered for 11 crops, these products combat seven major categories of diseases, such as black spot of Chinese cabbage and early blight of tomato caused by Alternaria spp. (http://www.chinapesticide.org.cn)”. Please see Line 72-77.

Q2: Abstract. I believe that the abstract is not clear...the purpose, material and method, results are not clearly highlighted...you have introduced many results without us understanding what the experimental variants are, it is not clear under what conditions the study was conducted (laboratory or field)...what was applied in which phenoplasts? when were the evaluations carried out...we deduce that they were at 7..15..30 days (I think you should redo it so that it is easier to read and quote).

A2: Thank you very much for your meticulous feedback on the Abstract. In response to your concerns, we have made comprehensive adjustments to the Abstract. we have clearly laid out the research purpose, materials, and methods. Information about experimental variants has been added, along with details on whether the experiments took place in the laboratory or field. We try our best to supplement relevant content in the Abstract. However, due to space constraints, we have had to be selective about the information included. Please see Line 13-31 in the Abstract.

Q3: I suggest introducing the notion of "fungicides" as a keyword

A3: Thank you for your suggestion. We have added “fungicides” as a keyword. Please see Line 32 in the keywords.

Q4: Introduction. I recommend improving the introduction with information regarding:
-clear description of symptoms,
-damage produced (expressed in percentages),
-presentation of more information related to fungicide resistance of the pathogen Alternaria sp.
-presentation of more information related to the effect (positive or negative) of PNA on other crops and on other pathogens

A4: Thank you very much for your constructive suggestions. In response, we have added relevant information into the Introduction.
As for description of symptoms and damage caused by the disease, we have revised the section as follows: “Ginseng Alternaria leaf and stem blight (GALSB) caused by Alternaria spp. ranks among the most prevalent and severe diseases in ginseng cultivation. It affects various parts of the ginseng plant, leading to excessive drying and withering of leaves, plant lodging and even death. When infecting fruits, it may cause seed contamination by the pathogen. GALSB typically manifests as circular or irregular necrotic lesions encircled by chlorotic zones on host leaves. It spreads rapidly via copious conidia production, leading to secondary infections [1, 2]. Typically, it has a normal annual incidence rate of 20% to 30%, which can reach up to 70% in severe cases, which lead to serious economic losses to ginseng production [1, 3]. In addition to A. panax and A. tenuissima, A. alternata is one of the main pathogens causing GALSB disease, which has been frequently isolated from ginseng leaves in most regions [2]. A. alternata is an important pathogenic fungus with a broad host range. Besides infecting ginseng/Korea ginseng and American ginseng [2, 4], it can also infect a variety of crops such as tobacco, quinoa, and other plants [5, 6].” Please see Line 39-48 in the introduction.
As for presentation of more information related to fungicide resistance of the pathogen Alternaria spp., we have revised the section as follows: “Research reports indicate that several Alternaria spp., including A. alternata, A. tenuissima, and A. arborescens, which cause Alternaria rot in citrus, Alternaria late blight in pistachios, and early blight in potatoes, have developed varying degrees of resistance to QoIs fungicides including azoxystrobin, kresoxim-methyl, pyraclostrobin, and so on [16-18]”. Please see Line 56-60 in the introduction.
As for presentation of more information related to the effect (positive or negative) of PNA on other crops and on other pathogens, we have supplemented relevant content as follows: “In China, the PNA in use mainly contain anisomycin, tetramycin A, tetramycin B, toyocamycin, and nystatin. All these components are approved for crops production. 13 domestic and international manufacturers produce 17 PNA products, which were registered for 11 crops, these products combat 7 types of diseases, including black spot of Chinese cabbage and early blight of tomato caused by Alternaria spp. (http://www.chinapesticide.org.cn).” Please see Line 72-77 in the introduction.

Q5: In these lines:  52, 55; 57, 63,74, 76, 77, 87, 116, 294, 295, 466, 584 correct A. spp. with Alternaria spp.

A5: Thank you for your valuable suggestions. We have made the necessary corrections as per your instructions, changing “A. spp.” to “Alternaria spp.”. Additionally, I’ve conducted a thorough check of the rest of the manuscript.

Q6: Describe - the damaya cultivar (is it sensitive. tolerant to Alternaria)

A6: Thank you for your valuable suggestions. In this study, the ginseng variety used is Damaya, which is susceptible to Alternaria spp.. Please see Line 235 in the manuscript.

Q7: Specify the distance between the localities where the studies were carried out, are they characterized by the same climatic conditions?
- Enter information related to the climatic conditions in the areas studied
- Enter information regarding the phenoplast in which the plants were when you applied the treatments
- What was the pressure/virulence of pathogens in the crop when you carried out the treatments

A7: Thank you for your valuable suggestions.
Firstly, the shortest distance between any two of the three experimental sites exceeds 180 kilometers, and they have somewhat different climatic conditions.
Secondly, this experiment was conducted during the flowering stage (GALSB disease was in the initial stage) to the fruit ripening stage (the disease was in the decline period of the occurrence) of the ginseng.
Thirdly, the experimental areas we selected are the regions where GALSB disease prevalence often happens, and the GALSB disease occurred severely in the selected experimental plots in the last year.
We have added the relevant geographical coordinates, climatic conditions (Figure S1) in the manuscript. Among them, the data on climatic conditions have been presented in the supplementary file. Please see Line 255-258 and 267-269, and Figure S1 in the supplementary file.

Q10: Results and discussions
- I believe that the discussions should be improved with information and comparisons from the existing literature that show the importance of the study, its innovative character… the benefits of application in practice.
- Try to be clearer about the results
- Ex. In the text you insist a lot on the efficacy recorded in the 3 regions, but you do not give the same interest to the 3 evaluations (7-15-30 days)… do not discuss in detail which treatment has better efficacy at 7-15-30 days, I believe that it is much more important to know the efficacy and the protection period that a certain treatment has… than to keep highlighting the study locations, whose climatic conditions we do not know and which lead to a decrease in the relevance of your study (Figure 9 and table 9).

A10: Thank you for your constructive feedback. In response to your suggestions, we have rewritten the results section to minimize numerical clutter in the text, directing readers to relevant figures and tables for detailed data. We now focus on summarizing key findings, comparing the efficacy and protection periods of different treatments. We have modified the relevant section as follows: “Field trials across Changchun, Baishan, and Yanbian in Jilin Province demonstrated that the control efficacy of PNA 4% AS against GALSB disease increased with higher dosages. Specifically, at 180 g a.i./hm², PNA consistently outperformed other fungicides over 7 days, 15 days, and 30 days post-last spray (PLS) (Figure 8, Table 3, S1, S2, and S3). 7 days PLS: High-dose PNA achieved a mean control efficacy of 85.62%. This was substantially higher than pyraclostrobin (78.65%), azoxystrobin (70.81%), and kresoxim-methyl (74.74%). 15 days PLS: PNA maintained a high mean control efficacy of 84.07%, again sur-passing the comparison fungicides (pyraclostrobin: 72.17%; azoxystrobin: 64.58%; kresoxim-methyl: 73.82%). 30 days PLS: PNA still showed a mean control efficacy of 72.55%, while the efficacies of other fungicides dropped below 54% (pyraclostrobin: 52.67%; azoxystrobin: 53.18%; kresoxim-methyl: 53.56%)” Please see Line 448-460 in the manuscript.
In the discussion, we have added: “QoIs fungicides are currently commonly used to control GALSB. However, due to the resistance development of Alternaria spp., their control efficacy has been continuously declining. The results of three consecutive investigations at three time points in this study all showed that the high-dose PNA was more effective in controlling GALSB than those of the three QoIs fungicides, and it still had a relatively high control efficacy 30 days post-last spray. This indicates that PNA can not only effectively control GALSB disease but also has a relatively long effect period, suggesting that it has the potential to address the development of resistance in Alternaria spp.” Please see Line 550-557.

Q11: Bibliography. Self-citations were noted, but they are within the field of study

A11: Thank you for your review. We understand your concern regarding self-citations. The self-citations have been clearly indicated in this bibliography.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Comments:

The submitted paper ”Evaluation potential of pyrimidine nucleoside antibiotics used for management of Alternaria spp. resistant to common fungicides" by Shuai Shao and colleagues is very interesting and it is well constructed. It is a fair contribution to the area of plant pathology, in particular for the management of the ginseng Alternaria leaf and stem blight disease.

The paper is clear, well written and well organised. But there are things to be improved.

The tittle  should be changed to: “Evaluation of the potential of pyrimidine nucleoside antibiotics against Alternaria spp. resistant to common fungicides: insights for the management of ginseng Alternaria leaf and stem blight disease.” Or a similar one!

The abstract is clear and embodies well the article, pointing out the main outcomes of the paper.

 

Also, keywords were generally well chosen, but I wouldn’t repeat those that are already present in the tittle, as “Alternaria spp.”  or “pyrimidine nucleoside antibiotics”, for example. I would replace those…

 

The Introduction of the paper is in general well written and well organized, but it can be further developed. I would include at least a couple of paragraphs about the Alternaria species complex/sections, namely in the case of Alternaria alternata, as this is the species complex that you tested mostly. I mean, the most part of the isolates come from this species complex. I would include this information/text, for example, just after line #40.

Please note that Alternaria tenuissima species was synonymised under Alternaria alternata. Please see the paper in Studies in Mycology, https://doi.org/10.1016/j.simyco.2015.07.001 by J.H.C. Woudenberg, M.F. Seidl, J.Z. Groenewald, M. de Vries, J.B. Stielow, B.P.H.J. Thomma, P.W. Crous

But for me you can keep the Alternaria tenuissima identification as this is still used/published in very recent papers, in many good journals in the Mycology/Plant Pathology fields… And the authors can also refer to this point…

Line #38. The names of the species are not italicized and they should be.

Please, do not abbreviate “Alternaria sp.” to “A. sp.”

The objectives of the paper are clearly indicated at the end of introduction.

The Materials and Methods section is well organized (the subsections created are useful for the article); the methods are adequately described and with detail. But for the “isolates” subsection, nothing is said about the identification of those isolates. I would like to see a table (even in supplementary materials) where the identification and the references of the isolates are uttered. Were these isolates identified by morphological and molecular tools? Who did this identification? Were these isolates acquired from a reference collection? You mention only the host, the period when they were isolated, and the location… I would complete the information regarding the isolates’ origin and identification.

Please note again that at least some isolates of Alternaria tenuissima species are synonymised under Alternaria alternata. Please see the paper in Studies in Mycology, https://doi.org/10.1016/j.simyco.2015.07.001 as i mentioned already.

If you keep the identifications you have, please make reference to whom identified the isolates at the first time.

All the other sections are written and explained in detail, and I must say that the authors did a tremendous job, and that this is a very complete work.

Moreover, the authors employed adequate tools and also suitable statistics.

Results are also well organised and properly described, with proper figures and tables. Figures and images are of good quality, but some of them are small in size. I would enlarge many of them. Please see my comments below.

In Results, please note that there are some typos (line #304): “(Error! Reference source not found.)”. The same happens in line #344 and also in line #425 and #451 and #475.

Please note that are some “A. alternata” not in italics. For example, line #339 and #363.

Also, I would enlarge graphs of figure 3, for a better visualization.

In addition, I would enlarge a little bit Figure 4 and Figure 5, and even Figures 6 and 7.

If possible, I would also enlarge graphs of Figure 10.

And for sure, you have to enlarge Figure 8 and also Figure 9. They are too small, particularly Figure 8!

Discussion is well constructed and articulated, covering all points of the Results section, and it is well supported by the literature.

“Conclusions” section deserves reformulation. A so important work that you conducted deserves better conclusions. The way they are written seems like a telegram… Authors can develop more the conclusions. Moreover, the last sentence of the conclusions should be more elaborated and concretised.

The list of References is reasonable, but please check over all the references if they are all listed in the text and at the references list at the end.

In summary, this is a fair work in my opinion that can deserve publication in Agriculture journal after some improvements bein made by the authors.

Author Response

Dear professor,

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Evaluation potential of pyrimidine nucleoside antibiotics used for management of Alternaria spp. resistant to common fungicides” (Manuscript ID: agriculture-3536130, now the revised title as “Evaluation of the potential of pyrimidine nucleoside antibiotics against Alternaria spp. resistant to QoIs fungicides: insights for the management of ginseng Alternaria leaf and stem blight disease”)”. Those comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our researches. We have read comments carefully and made corrections in the paper. I hope you are satisfied with the revised version, if there is any question, please tell us it again. We are looking forward to hearing from you again in a not long time! Thank you very much again.

Best wishes,

Jie Gao

The reviewer 3

Comments and Suggestions for Authors

Comments:

The submitted paper “Evaluation potential of pyrimidine nucleoside antibiotics used for management of Alternaria spp. resistant to common fungicides" by Shuai Shao and colleagues is very interesting and it is well constructed. It is a fair contribution to the area of plant pathology, in particular for the management of the ginseng Alternaria leaf and stem blight disease.

Question1: The tittle should be changed to: “Evaluation of the potential of pyrimidine nucleoside antibiotics against Alternaria spp. resistant to common fungicides: insights for the management of ginseng Alternaria leaf and stem blight disease.” Or a similar one!

Answer1: Thank you for your valuable suggestion. We have carefully revised the title, drawing reference from the style you provided. According to your suggestion, we have revised the title as follows: " Evaluation of the potential of pyrimidine nucleoside antibiotics against Alternaria spp. resistant to QoIs fungicides: insights for the management of ginseng Alternaria leaf and stem blight disease". Please see Line 2-5 in the manuscript.

Q2: Also, keywords were generally well chosen, but I wouldn’t repeat those that are already present in the tittle, as “Alternaria spp.” or “pyrimidine nucleoside antibiotics”, for example. I would replace those…

A2: Thank you for your valuable suggestion. We have changed “pyrimidine nucleoside antibiotics” to “fungicides” among the keywords. Please see Line 32 in the manuscript.

Q3: The Introduction of the paper is in general well written and well organized, but it can be further developed. I would include at least a couple of paragraphs about the Alternaria species complex/sections, namely in the case of Alternaria alternata, as this is the species complex that you tested mostly. I mean, the most part of the isolates come from this species complex. I would include this information/text, for example, just after line #40.
Please note that Alternaria tenuissima species was synonymised under Alternaria alternata. Please see the paper in Studies in Mycology, https://doi.org/10.1016/j.simyco.2015.07.001 by J.H.C. Woudenberg, M.F. Seidl, J.Z. Groenewald, M. de Vries, J.B. Stielow, B.P.H.J. Thomma, P.W. Crous
But for me you can keep the Alternaria tenuissima identification as this is still used/published in very recent papers, in many good journals in the Mycology/Plant Pathology fields… And the authors can also refer to this point…

A3: Thank you very much for providing the literature reference. It has indeed enabled me to gain more insights into the pathogen. In response to your suggestion on enriching the Introduction, we incorporated several paragraphs about the Alternaria species complex/sections right after Line 40.
Notably, research conducted in 2021 “Gao, J.; Yang, M.; Xie, Z.; Lu, B.; Tom, H.; Liu, L., Morphological and molecular identification and pathogenicity of Alternaria spp. associated with ginseng in Jilin province, China. Can J Plant Pathol. 2021, 43, 537-550. https://doi.org/10.1080/07060661.2020.1858167”, and we distincted Alternaria alternata and A. tenuissima among the target isolates through morphological and molecular biological identification. For the latter, we used specific primers to achieve accurate distinguishment of the two species. Considering your point, we have also cited reference by Woudenberg et al. We have modified section as follows: “A. alternata and A. tenuissima shared many similarities in terms of morphology and sensitivity to fungicides, and the synonymization of A. tenuissima under A. alternata was once put forward by Woudenberg et al [34].” in discussion.

Q4: Line #38. The names of the species are not italicized and they should be.

A4: Thank you for your valuable suggestion. We have already changed the names of the species in the manuscript to italic. Additionally, we have carefully checked the remaining parts of the manuscript. Please see Line 44.

Q5: Please, do not abbreviate “Alternaria sp.” to “A. sp.”

A5: Thank you for your valuable suggestion. We have conducted comprehensive revisions on the “Alternaria spp.” section in the manuscript.

Q6: The objectives of the paper are clearly indicated at the end of introduction.
The Materials and Methods section is well organized (the subsections created are useful for the article); the methods are adequately described and with detail. But for the “isolates” subsection, nothing is said about the identification of those isolates. I would like to see a table (even in supplementary materials) where the identification and the references of the isolates are uttered. Were these isolates identified by morphological and molecular tools? Who did this identification? Were these isolates acquired from a reference collection? You mention only the host, the period when they were isolated, and the location… I would complete the information regarding the isolates’ origin and identification.
Please note again that at least some isolates of Alternaria tenuissima species are synonymised under Alternaria alternata. Please see the paper in Studies in Mycology, https://doi.org/10.1016/j.simyco.2015.07.001 as i mentioned already.
If you keep the identifications you have, please make reference to whom identified the isolates at the first time.

A6: Thank you very much for your valuable suggestions. First, in our study, we also have found that A. tenuissima and A. alternata exhibit remarkable similarities in growth rates, fungicides sensitivities, and sporulation.
However, in 2021, studies demonstrated that the two can be distinguished using molecular biological methods with specific primers. All isolates were identified through a combination of morphological and molecular techniques by Jie Gao.
We have revised the “Materials” section as follows: “All isolates were identified through morphological approaches, and some isolates were further distinguished via molecular biological methods”. Please see Line 119-121 in the manuscript.

Q7: Results are also well organised and properly described, with proper figures and tables. Figures and images are of good quality, but some of them are small in size. I would enlarge many of them. Please see my comments below.

A7: Thank you very much for your valuable suggestions. You’ve provided highly constructive suggestions. We have addressed your requests regarding the figures and tables. Specifically, we have adjusted the size of the figures as per your recommendations. Additionally, we have made changes to the labels on the horizontal and vertical axes, ensuring enhanced readability and visual appeal.

Q8: In Results, please note that there are some typos (line #304): “(Error! Reference source not found.)”. The same happens in line #344 and also in line #425 and #451 and #475.

A8: Thank you so much for your valuable suggestions. We have carefully revised the cross-references in the table headers and columns in the manuscript. All the issues related to " (Error! Reference source not found.)" in line 304, line 344, line 425, line 451, and line 475 have been fixed. We have double-checked these references to ensure their accuracy and consistency.

Q9 Please note that are some “A. alternata” not in italics. For example, line #339 and #363.

A9 Thank you for your valuable suggestion. We have italicized "A. alternata" as you pointed out in line 339 and 363. Additionally, we have carefully checked the rest of the article to ensure that all occurrences of "A. alternata" are properly italicized. Please see Line 339 and 363 in the manuscript.

Q12: Also, I would enlarge graphs of figure 3, for a better visualization.

A12: Thank you for your valuable suggestion. Thank you for your constructive suggestion regarding Figure 3. We truly appreciate your attention to detail.
I have already enlarged the graphs of Figure 3 as you recommended. Please see Line 359 in the manuscript.

Q10: In addition, I would enlarge a little bit Figure 4 and Figure 5, and even Figures 6 and 7.

A10: Thank you very much for your insightful suggestions. I promptly followed your advice and enlarged Figure 4, Figure 5, Figure 6, and Figure 7. This modification will make the details of the figures more distinguishable, facilitating readers’ understanding. Please see Line 370, 385, 400, and 404 in the manuscript.

Q11: If possible, I would also enlarge graphs of Figure 10.

A11: Thank you for your valuable suggestion. Figure 10 already occupies a relatively large area in the layout. However, to meet your suggestion while maintaining aesthetic appeal and ensuring clarity, we have enlarged the labels on the horizontal and vertical axes. This adjustment makes the figure information more legible. Please see Line 473 in the manuscript.

Q12: And for sure, you have to enlarge Figure 8 and also Figure 9. They are too small, particularly Figure 8!

A12: Thank you for your valuable suggestions. We have now enlarged both Figure 8 as you suggested. Additionally, we have increased the font size of the labels for the horizontal and vertical coordinates in these figures. Moreover, we have adjusted the layout of the figures for better visual coherence. This ensures that the content within them is much clearer and easier to read. We believe these changes will greatly improve the overall presentation and comprehensibility of our figures. Please see Line 424 in the manuscript.

Q13: “Conclusions” section deserves reformulation. A so important work that you conducted deserves better conclusions. The way they are written seems like a telegram… Authors can develop more the conclusions. Moreover, the last sentence of the conclusions should be more elaborated and concretised.

A13: Thank you for your suggestions. You pointed out that the “Conclusions” section required reformulation, the current conclusions were written in a rather telegraphic style, and the last sentence should be more elaborate and concrete. In response to these issues, we have made revisions to the conclusions section. We have modified the section as follows: “In this study, we systematically investigated the potential of pyrimidine nucleoside antibiotics (PNA) against A. alternata. First, we successfully established the baseline sensitivity of A. alternata to PNA. Significantly, its sensitivity to PNA followed a unimodal distribution. This finding offered a crucial method for monitoring the development resistance of A. alternata to PNA in field populations, enabling early intervention in resistance management. Experiments in vitro demonstrated that PNA exhibited remarkable antifungal and biochemical activity against A. alternata PNA effectively inhibited the growth and metabolic activities of the pathogen, highlighting its potential as a potent antifungal agent. Importantly, no cross-resistance between PNA and fungicides used on ginseng were found. This unique characteristic allowed for more flexible and effective fungicide rotation strategies in agricultural settings. Finally, PNA demonstrated high protective and curative efficacy. Field trials further validated its effectiveness, showing longer duration in controlling GALSB diseases. These findings were of great significance for the green management of GALSB disease. They also provided valuable insights into managing the fungicide resistance of Alternaria spp. responsible for GALSB disease, offering new approaches to sustainable disease control”. Please see Line 578-593 in the manuscript.

Q14: The list of References is reasonable, but please check over all the references if they are all listed in the text and at the references list at the end.

A14: Thank you for your suggestion. We have conducted a comprehensive cross-check to ensure all references are correctly cited in the text and included in the reference list at the end.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Comments (second round of review)

Dear Authors,

I do thank you for the efforts and for the fact that you accepted my suggestions. I have no further issues to point out. I think that the article can be accepted now for publication in Agriculture.

Congratulations for this nice piece of work.

Kind regards.