Baseline Sensitivity to and Succinate Dehydrogenase Activity and Molecular Docking of Fluxapyroxad and SYP-32497 in Rice Sheath Blight (Rhizoctonia solani AG1-IA) in China

Round 1

Reviewer 1 Report

The research work attempted to evaluate the baseline sensitivity of sheath blight-causing pathogen R. solani to fluxapyroxad and SYP-32497. The study is incomplete and limited information has been provided in this manuscript. The morphological and molecular identification of sheath blight fungus is not studied. The data analysis is superficial. There are no photographs of mycelial inhibition and no record of disease mitigation on the host plants. It will be better to conduct additional experiments based on the suggested lines. Some additional suggestions for further improvement

1.      The abstract has revealed very limited information. It must be elaborated for a better understanding of different sets of experiments.

2.      The information provided in the abstract is not able to critically differentiate the aim of the work. It is better to rephrase and emphasize on comparative analysis of two fungicides.

3.      There is no information on mycelial pure culture establishment, etiology and anastomosis group analysis. Were the representative samples submitted in culture collection?

4.      Determination of enzyme efficiency: On which isolate or isolates?

5.      Molecular docking: How it was correlated with the rest of the experiments.

6.      Lines 64-67 “establish the baseline sensitivity of R. solani isolates towards fluxapyroxad and SYP- 32497 in China, (b) determine how the fungicides affect succinate dehydrogenase activity in R. solani, and (c) to analyze the relationship between fluxapyroxad, SYP-32497, and protein binding properties by molecular docking” represent the central theme so the title and abstract must be prepared likewise.

7.      The comparative analysis of both fungicides is required.

8.      Several latest references on similar aspects are missing.

 

 

Author Response

Revision list according to the comments from Reviewer 1.

Each comment will be directly addressed regrading the modified manuscript with changes highlighted in red.

1. The abstract has revealed very limited information. It must be elaborated for a better understanding of different sets of experiments.

Response 1: Thank you for your rigorous consideration. We have changed it.

Line 15-29

2. The information provided in the abstract is not able to critically differentiate the aim of the work. It is better to rephrase and emphasize on comparative analysis of two fungicides.

Response 2: We have rewritten this part according to the Reviewer’s suggestion.

Line 15-29

3. There is no information on mycelial pure culture establishment, etiology and anastomosis group analysis. Were the representative samples submitted in culture collection?

Response 3: Thank you for your rigorous consideration. In this part, we have confirmed that all the strains tested belong to the AG1-IA. But in order to better explain the theme of the article: to establish the sensitive baseline of rice bacterial wilt to SYP-32497 and fluxapyroxad, we did not show this part of the content. And we also added photos of SYP-32497 and fluxapyroxad inhibiting mycelial growth on a petri dish.

 

4. Determination of enzyme efficiency: On which isolate or isolates?

Response 4: We totally understand the reviewer's concern. From an economic point of view, we only tested the succinate dehydrogenase activity of SYP-32497 and fluxapyroxad against YZ1. YZ1 is isolated from Yizhou, Hunan Province.

5. Molecular docking: How it was correlated with the rest of the experiments.

Response 5: We totally understand the reviewer's concern. Molecular docking allows us to better understand the interactions between compounds and target proteins. The results also explain the differences in activity between different molecules and targets. The binding force of SYP-32497 to the target enzyme was stronger than that of fluxapyroxad. This result is consistent with the results of mycelial growth inhibition activity and enzyme activity inhibition test.

6. Lines 64-67 “establish the baseline sensitivity of R. solani isolates towards fluxapyroxad and SYP- 32497 in China, (b) determine how the fungicides affect succinate dehydrogenase activity in R. solani, and (c) to analyze the relationship between fluxapyroxad, SYP-32497, and protein binding properties by molecular docking” represent the central theme so the title and abstract must be prepared likewise.

Response 6: We totally understand the reviewer's concern.Our title has been changed to “Baseline sensitivity, succinate dehydrogenase activity and molecular docking to Rhizoctonia solani on rice in China”.

7. The comparative analysis of both fungicides is required.

Response 7: Thank you for pointing out this problem in manuscript. In the discussion part, we made a comparison.

8. Several latest references on similar aspects are missing.

Response 8: We have added some new literature related to the experiment that we can consult in the part References.

 We gratefully thanks for the precious time the reviewer spent making constructive remarks. We have made some changes. If we can't achieve the effect you want, please be sure to contact me again.

Reviewer 2 Report

I my review of the manuscript titled “A baseline sensitivity study of Rhizoctonia solani to fluxapy- 2 roxad and SYP-32497 in China”, following points of concerns were spotted.

·        The title of the manuscript will continue to be viewed as general until name of the plant disease is included

·        The abstract is all over the place:  The first two sentences of the abstract could be merged

·        R. solani is fungal pathogen affecting many plant species. Authors may specify the disease and isolation plants in the abstract

·        Line 23….is considered instead of is consider

·        Line 24…source of substance….Which substance?

·        Line 61…. Anhui Province, China….space needed

·        In the introduction, the structural elucidation of Fluxapyroxad is missing especially when authors already put the structure of SYP-32497

·        The use of English language needs substantial improvement and a check by native English speaker is suggested

·        Why plant samples collected from areas where no SDHIs have been applied before?

·        What was the number of plan samples? Which provinces were targeted?

·        For isolation, did the authors use any antibiotic in PDA media?

·        How did the authors isolate single hyphae?

·        0.1010 g SYP-32497 and 0.1020 g quantities were dissolved in how much DNSO to reach the 104 ppm concentration?

·        Section 2.3: Authors did not mention touch upon any reference control in fungicidal sensitivity assays?

·        Why only YZ1 isolates of R. solani is used for succinate dehydrogenase assay?

·        Why authors have used high concentration like 0, 0.08, 0.4, 2, 10, 50 ug/mL for SDH assay especially when they already have set quite low concentrations as baseline sensitivity?

·        Mycelia were washed and dried after 12 h to 102 determine SDH enzyme activity…the authors should further describe how they processed samples for enzyme inhibition activity measurement. The protocol should be fully elucidated.

·        Section 2.6: Statistical analysis should touch upon the methods used in SPSS v. 18?

·        The legend of Figure 2 does not sound good…Please revise to make the massage clear

·        In the figure 2, titles on X and Y exis needs spaces before parentheses. Same issue exists in Figure 3

·        The authors isolated a total of 360 R. solani isolates from differences provinces of China, how did they identify the isolates to confirm their taxonomy

·        From Figure 2 and Figure 3, It seems that some of the isolated got very high EC50 values, how did the authors conclude that no resistance to fungicides is found?

·        Section 3.2..Figure 4 space needed

·        Figure 4…spaces needed on X and Y exis before parenthesis

·        How enzyme inhibition efficiency was calculated? The authors need to put formula in the M&M section

·        The authors need to check the whole MS for spaces. At many points, spaces are not used especially before parentheses

 

·        Discussion part looks weak to me; the authors mostly illuminated their results and missed the proper implications retrieved from results and appropriate comparisons of these results with related studies.

Author Response

　　Revision list according to the comments from Reviewer 2

Each comment will be directly addressed regrading the modified manuscript with changes highlighted in red.

1. The title of the manuscript will continue to be viewed as general until name of the plant disease is included.

Response 1: We gratefully appreciate for your valuable suggestion. Our title has been changed to “Baseline sensitivity, succinate dehydrogenase activity and molecular docking to Rhizoctonia solani on rice in China”.

2. The abstract is all over the place:  The first two sentences of the abstract could be merged

Response 2: Thank you. We have changed it.

Rice sheath blight caused by Rhizoctonia solani occurs worldwide and mainly controlled by fungicides. SYP-32497 is a novel succinate dehydrogenase inhibitor, which interferes with the succinate ubiquinone reductase in the mitochondrial respiratory chain of fungi. This study aimed to evaluate the baseline sensitivity of R. solani from 13 major rice producing areas in China to SYP-32497 and fluxapyroxad. The study also explored the cause for the activity discrepancy between SYP-32497 and fluxapyroxad via enzyme activity inhibition test and molecular docking. A total of 360 R. solani isolates were sensitive to SYP-32497 and fluxapyroxad. Baseline sensitivities were unimodally distributed with mean values of 0.00667 ± 0.00475 and 0.0657 ± 0.0250 μg ml⁻¹, respectively, for SYP-32497 and fluxapyroxad. Enzyme activity assays and molecular docking results revealed that SYP-32497 exhibited a much higher SDH inhibition (IC₅₀ = 0.300 μg ml⁻¹) than to fluxapyroxad (IC₅₀ = 1.266 μg ml⁻¹) due to its excellent SDH binding ability via hydrogen bonding, π-cation, and hydrophobic interactions. These results suggest that SYP-32497 is a good suitable control agent for alternative rice sheath blight.

3. solani is fungal pathogen affecting many plant species. Authors may specify the disease and isolation plants in the abstract.

Response 3: Thank you.We have changed it.

Rice sheath blight caused by Rhizoctonia solani occurs worldwide and mainly controlled by fungicides. SYP-32497 is a novel succinate dehydrogenase inhibitor, which interferes with the succinate ubiquinone reductase in the mitochondrial respiratory chain of fungi. This study aimed to evaluate the baseline sensitivity of R. solani from 13 major rice producing areas in China to SYP-32497 and fluxapyroxad. The study also explored the cause for the activity discrepancy between SYP-32497 and fluxapyroxad via enzyme activity inhibition test and molecular docking. A total of 360 R. solani isolates were sensitive to SYP-32497 and fluxapyroxad. Baseline sensitivities were unimodally distributed with mean values of 0.00667 ± 0.00475 and 0.0657 ± 0.0250 μg ml⁻¹, respectively, for SYP-32497 and fluxapyroxad. Enzyme activity assays and molecular docking results revealed that SYP-32497 exhibited a much higher SDH inhibition (IC₅₀ = 0.300 μg ml⁻¹) than to fluxapyroxad (IC₅₀ = 1.266 μg ml⁻¹) due to its excellent SDH binding ability via hydrogen bonding, π-cation, and hydrophobic interactions. These results suggest that SYP-32497 is a good suitable control agent for alternative rice sheath blight.

4. Line 23….is considered instead of is consider

Response 4: We are very sorry for our incorrect writing. We have changed it. Rice (Oryza sativa L.) is considered to be a globally important and highly nutritious crop

5. Line 24…source of substance….Which substance?

Response 5: We are very sorry for our incorrect writing. We have changed it. Rice (Oryza sativa L.) is considered to be a globally important and highly nutritious crop

6. Line 61…. Anhui Province, China….space needed

Response 6: We are very sorry for our negligence. We've increased it.

7. In the introduction, the structural elucidation of Fluxapyroxad is missing especially when authors already put the structure of SYP-32497

Response 7: We are very sorry for our negligence.We've increased it.

8. The use of English language needs substantial improvement and a check by native English speaker is suggested

Response 8：Thank you. We've had a professional team touch it up

 

9. Why plant samples collected from areas where no SDHIs have been applied before?

Response 9:Thank you for your rigorous consideration. In general, cross-resistance may occur between fungicides with the same mode of action, so to establish a baseline sensitivity of pathogens to a new fungicide, pathogens that have not been applied should be selected. In addition, we need to establish a sensitive baseline to monitor changes in the sensitivity of pathogens to fungicides in the future, and pathogens that have not been applied are very important for the establishment of sensitive baselines.

10. What was the number of plan samples? Which provinces were targeted?

Response 10: Thank you for your rigorous consideration. A total of 360 plants sample were obtained from numerous Chinese provinces, including Jiangsu, Henan, Jilin, Hunan, Jiangxi, Anhui, Sichuan, Inner Mongolia, Zhejiang, Heilongjiang, Liaoning, Hubei, and Tianjin (Table 1)

In M&M we added it as well.

11. For isolation, did the authors use any antibiotic in PDA media?

Response 11: We are very sorry for our negligence. Yes, we did ,PDA modified with streptomycin, In M&M we added it as well.

12. How did the authors isolate single hyphae?

Response 12: Thank you for your rigorous consideration. After isolation, the faster growing hyphae will appear on the PDA. We operate in the microscope and cut off the small pieces with single hyphae.

13. 1010 g SYP-32497 and 0.1020 g quantities were dissolved in how much DNSO to reach the 10⁴ ppm concentration?

Response 13: We are very sorry for our negligence. 10mL DNSO to reach the 10⁴ ppm concentration. In M&M we added this as well.

14. Section 2.3: Authors did not mention touch upon any reference control in fungicidal sensitivity assays?

Response 14: We are very sorry for our negligence. The concentration of DMSO in the medium was adjusted to 0.1% in volume. In MM we added this as well.

15. Why only YZ1 isolates of solani is used for succinate dehydrogenase assay?

Response 15: Thank you for your rigorous consideration. From an economic point of view, we only tested the succinate dehydrogenase activity of SYP-32497 and fluxapyroxad against YZ1. YZ1 is isolated from Yizhou, Hunan Province.

16. Why authors have used high concentration like 0, 0.08, 0.4, 2, 10, 50 ug/mL for SDH assay especially when they already have set quite low concentrations as baseline sensitivity?

Response 16: Considering that the enzyme activity may be different from the mycelium growth inhibition activity, in order to see the difference in enzyme inhibition activity more significantly, we chose a higher concentration of fungicide treatment.

17. Mycelia were washed and dried after 12 h to 102 determine SDH enzyme activity…the authors should further describe how they processed samples for enzyme inhibition activity measurement. The protocol should be fully elucidated.

Response 17: We have rewritten this part in M&M according to the Reviewer’s suggestion.

18. Section 2.6: Statistical analysis should touch upon the methods used in SPSS v. 18?

Response 18: Yes, we use SPSS v.18.0 to analyze whether our sensitivity baseline data conforms to the normal distribution.

19. The legend of Figure 2 does not sound good…Please revise to make the massage clear

Response 19: We are sorry, we don't quite understand what you mean, but our title and label have been changed appropriately.

20. In the figure 2, titles on X and Y exis needs spaces before parentheses. Same issue exists in Figure 3

Response 20: Thank you for your thoughtfulness in raising such detailed questions for us. We have listened to your suggestion and made changes.

21. The authors isolated a total of 360 solani isolates from differences provinces of China, how did they identify the isolates to confirm their taxonomy

Response 21: Thank you so much for your careful check. We selected 1-2 strains from each province for sequencing, and morphologically confirmed that our strain was R.solani.

22. From Figure 2 and Figure 3, It seems that some of the isolated got very high EC50 values, how did the authors conclude that no resistance to fungicides is found?

Response 22: Thank you for pointing out this problem in manuscript. In our opinion, on the one hand, the strains we isolated came from places where succinate dehydrogenase inhibitor fungicides had not been used, on the other hand, we analyzed the data, and the sensitive baseline data conformed to the normal distribution. Therefore, we do not think that there are any strains resistant to these two fungicides.

23. Section 3.2..Figure 4 space needed

Response 23: Thank you for your thoughtfulness in raising such detailed questions for us. We have listened to your suggestion and made changes.

24. Figure 4…spaces needed on X and Y exis before parenthesis

Response 24: We are very sorry for our negligence. We have listened to your suggestion and made changes.

25. How enzyme inhibition efficiency was calculated? The authors need to put formula in the M&M section

Response 25: We totally understand the reviewer's concern. We added the formula in M&M.

SDH activity (U/G mass) = [(Δ A determination-Δ A blank) / (ε × d) × V anti-total × 109] / (V sample / V total × W) / T = 1603.175 × (Δ A determination-Δ A blank) / W. (V anti-total: the total volume of the reaction system; ε: 2,6-dichlorophenol-indophenol extinction coefficient, 2.1×104 L/mol/cm; d: cupola light diameter, 1 cm ; V sample: the added sample volume (0.01 mL); V total: the added reagent 1 and reagent 2 volume (1.01 mL); T: Reaction time (1 min); W: Sample quality).

SDH activity inhibition (%) = (C-T)/C X100, where C and T represent SDH activity in the control and treatment groups, respectively.

26. The authors need to check the whole MS for spaces. At many points, spaces are not used especially before parentheses

Response 26: Thank you for your thoughtfulness in raising such detailed questions for us. We have listened to your suggestion and made changes.

• 27. Discussion part looks weak to me; the authors mostly illuminated their results and missed the proper implications retrieved from results and appropriate comparisons of these results with related studies.

Response 27: We gratefully thanks for the precious time the reviewer spent making constructive remarks. We have made some changes. If we can't achieve the effect you want, please be sure to contact me again.

 

Round 2

Reviewer 1 Report

The manuscript has been substantially improved on the suggested lines. but there are some minor issues which should be addressed

The modified title still seems inappropriate

The details of isolates GenBank accession numbers are not provided which could have firmly established the taxonomy of the pathogen. 

 

 

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

The authors have put in a good effort to substantially improve the manuscript; however, I would still go with the following concerns to reach a conclusive statement:

1) Although the authors have added the chemical structure of Fluxapyroxad in the introduction, I would still suggest to a proper source citation.

2) Figure 5 legend: Replace the colony morphology with mycelial inhibition

3) If authors have sequenced the ITS of some of the sampled R. solani, wouldn't it be better to reveal some sequence information such as accession numbers phylogenetic analysis?

Probably authors may wanna add a phylogenetic tree based on ITS sequences they got