Alternaria alternata and Alternaria koreana, the Causal Agents of Leaf Spot in Celtis sinensis and Their Sensitivity to Fungicides

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Not applicable

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Section 2.6 How many replication for each synthetic fungicide tested against fungal pathogen?

Response 1: Thank you for pointing this out. 3 replicates of each synthetic fungicide tested against fungal pathogens in Section 2.6. On page 7, line 164, a sentence has been added “each treatment was replicated 3 times”.

Comments 2: No statistical analysis for the sensitivity of fungal pathogens to commercial fungicides.

Response 2: Thank you for pointing this out. I agree with this comment. This question is consistent with Comments 5, and the answer has been shown in Response 5.

Comments 3: Section 3.2 last paragraph How to conclude that A. alternata and A. koreana are the pathogens of leaf spot disease of C. sinensis? Only pathogencity test can assume that both fungal isolates can cause leaf spot disease? section 2.1 and 2.2 authors did not identify pathogen yet, right?

Response 3: Thank you for pointing this out. I agree with this comment. Isolated fungal mycelium block as well as suspension (10⁶ conidia/mL) were inoculated onto isolated leaves and C. sinensis seedlings, respectively, both of which caused leaf spot disease (Fig. 2), determining that A. alternata and A. koreana are the pathogens of leaf spot disease of C. sinensis. Currently, all laboratories use pathogenicity assays to identify pathogenic fungi that cause plant diseases. In sections 2.1 and 2.2, it has not been determined pathogen.

Comments 4: Section 3.6 No statistical analysis for this experiment?

Response 4: Thank you for pointing this out. I agree with this comment. This question is consistent with Comments 5, and the answer has been shown in Response 5.

Comments 5: Table 3 No statistical analysis?

Response 5: Thank you for pointing this out. I agree with this comment. I used GraphPad Prism 8 and SPSS to analyze the data of inhibition rates of different isolates, different concentrations and different fungicides. The table were changed as follows:

Table 3. Concentration at 50% of Maximum Effect (EC50 values) of isolates. (Data =mean ± standard error)

Fungicide

EC50 Values (µg/mL)

11

12

13

Prochloraz

2.95 ± 0.31 d

3.96 ± 0.4 c

 4.05 ± 0.42 d

Myclobutanil

84.41 ± 3.33 a

55.14 ± 2.34 a

87.89 ± 3.73 c

Tebuconazole

35.32 ± 5.98 c

13.96 ± 2.52 c

143.17 ± 8.93 b

Pyraclostrobin

50.53 ± 2.05 b

 34.5 ± 7.66 b

 422.26 ± 27.69 a

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Not applicable

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Delete the word “new” on the second line of the first page of the title.

Response 1: Thank you for pointing this out. I agree with this comment. Therefore, I have deleted the word “new” on the second line of the first page of the title.

Comments 2: Change the word “blotch” to “spot” on the tenth line of the first page.

Response 2: Thank you for pointing this out. I agree with this comment. Due to my carelessness, I mistakenly wrote “blotch” instead of “spot”. It has now been corrected to the word “spot”.

Comments 3: Alternaria koreana should be abbreviated.

Response 3: Thank you for pointing this out. I agree with this comment. Due to an error in my writing, the word "Alternaria koreana" on the fourteenth line of the first page should be abbreviated. It has now been corrected to "A. koreana."

Comments 4: Line 40 on the first page, you need a paragraph or sentence that connects leaf spot incidence of Celtis sinensis to Alternaria as pathogen.

Response 4: Thank you for pointing this out. I agree with this comment. Line 40 on the first page, I added a sentence “Currently, there have been no reports of leaf spot in C. sinensis caused by Alternaria.” to keep the context close.

Comments 5: Line 52 on page 2, “At present, the most direct and effective method for the control of diseases caused by Alternaria is chemical control”. With this paragraph and the previous one, it sounds like you already know that the culprit is nothing but Alternaria. But the readers don't know yet. You need to explain why you suspect that it'd be caused by Alternaria.

Response 5: Thank you for pointing this out. In line 44-47, I mentioned that “They can grow and multiply in low-temperature and humid environments, causing a variety of plant diseases, as well as fruit rot, which severely jeopardizes the growth of crops and plants and causes enormous economic losses while destroying natural ecosystems globally”, so it is natural to mention prevention and control methods in the following paragraph. Moreover, the readers can get information that “Alternaria is a pathogen” from title.

Comments 6: Line 65 on page 2, why suddenly use "pathogen" and not “Alternaria”?

Response 6: Thank you for pointing this out. I agree with this comment. Line 65 on page 2, replace the word “pathogen” with “Alternaria” for a more accurate expression, closely related to the context. The word “pathogen” has been changed to “Alternaria”.

Comments 7: Line 76 on page 2, “In June 2022, the disease was observed on the leaves of C. sinensis on the campus of Nanjing Forestry University, Nanjing City, Jiangsu Province (119°46'43"E, 32°02'38"N), and 20 diseased leaves were collected as samples from across 0.75-1.5 meters of C. sinensis.” from one single plant? Why?

Response 7: Thank you for pointing this out. I agree with this comment. Due to my mistake of expression, it caused you a misunderstanding. They were not sampled from a single plant, but from 5 infected plants. Line 76 on page 2, the sentence has been changed to “and 20 diseased leaves were collected as samples from 5 infected C. sinensis plants.”   

Comments 8: Regarding the phylogenetic analysis on line 135 of page 4, why aren't there popular Alternaria species, such as A. arborescens, A. solani, A. brassicola?

Response 8: Thank you for pointing this out. I agree with this comment. I have added A. arborescens, A. solani and A. brassicicola into the phylogenetic analysis.

Comments 9: On line 166 of page 7, “The survey results indicated that nearly 50% of the C. sinensis in Xuanwu District, Nanjing, have shown signs of disease.” Then why you didn't take more samples from many plants?

Response 9: Thank you for pointing this out. I agree with this comment. Due to an error in my expression in line 76 on page 2, which caused you a misunderstanding, it was not sampled from a single plant. They were sampled from many plants.

Comments 10: On line 172 of page 7, “In total, 90 fungal colonies developed from the tissue pieces and were grouped into three types according to their colony characteristics, with frequencies of 84.4% (76 of 90), 10% (9 of 90), and 5.5% (5 of 90).” Morphological characters are not reliable for species identification. You can do 3 isolates for pathogenicity test but you should have sequenced all isolates to make sure that colony morph and gene data are consisitent.

Response 10: Thank you for pointing this out. I agree with this comment. An error in my expression in line 174 on page 8 caused you a misunderstanding. I based my statistical classification on colony characteristics combined with RPB2 gene sequencing results. This sentence was changed to “In total, 90 fungal colonies developed from the tissue pieces and were grouped into three types according to their colony characteristics and RPB2 sequencing results,”.

Comments 11: On line 275 of page 13, according to the EC50 values in Table 3. With these EC50 values, they all look practical resistant to the four fungicides. We don't know yet until tested on plant/leaf sprayed with respective fungicide at the label rate.

Response 11: Thank you for pointing this out. I agree with this comment. At present, I have done the indoor experiment first, and obtained the preliminary data through the fungicide sensitivity experiment, which will guide the completion of the outdoor plant experiment in the later stage.

Comments 12: On line 306 of page 13, “Currently, the classification of Alternaria species primarily relies on the utilization of morphological characteristics and multigene phylogenetic analysis methods.” We no longer rely on morph for identification of Alternaria spp.

Woudenberg et al. (2014) Studies in Mycology 82:1-21

Lawrence et al. (2016) Mycol Progress 15:3

Response 12: Thank you for pointing this out. I agree with this comment. I am sorry to cause you misunderstanding due to my misrepresentation. It is true that I cannot classify Alternaria by morphology only. What I wanted to express is that Alternaria is classified by a combination of morphology and multigene phylogenetic analysis. So, the sentence has changed to “Currently, the classification of Alternaria species primarily relies on the combination of morphological characteristics and multigene phylogenetic analysis methods.”

Comments 13: On line 349 of page 14, “In 2009, researchers conducted in vitro susceptibility tests using six fungicides against the pathogen Alternaria solani. The results showed that prochloraz, with EC50 values ranging from 0.03 to 0.11 µg/ml, was effective in inhibiting the growth of the pathogen [50].” Compared to A. solani, the EC50 values of prochloraz obtained in this study are more than 10 times higher. Discuss.

Response 13: Thank you for pointing this out. I agree with this comment. A. solani、A. alternata and A. koreana are 3 species isolates.

a.     Biological characteristics

Different isolates may have different biological characteristics, such as cell structure, metabolic pathways, and life cycles. These differences may affect how the fungicide interacts with the target cells, leading to varying degrees of inhibition.

b. Genetic variation and evolution

Different isolates may have genetic variations, and some of them may have natural or adaptive resistance to fungicides. Due to the stress of different ecological environments, they may show differences in resistance to fungicides.

c. Mechanism of action of fungicides

The mechanism of action of prochloraz interacts with the target cell structure, and different strains have different sensitivity to these mechanisms.

d. Growth conditions and data errors

Growth temperature, humidity and nutrients and other environmental factors, measurement errors, experimental operations, data analysis, etc., will affect the effectiveness of fungicides.

Comments 14: On line 352 of page 14, “The results showed that prochloraz, with EC50 values ranging from 0.03 to 0.11 µg/ml, was effective in inhibiting the growth of the pathogen [50]. This result is consistent with the results of the present study that prochloraz has the best inhibitory effect on A. alternata and A. koreana, causing leaf spot in C. sinensis.” I disagree. With 10 times high EC50 values, how can you say that "prochloraz" has the "best" inhibitory effect. They use 6 fungicides and in this study 4 fungicides. What other 5 fungicides were used in the study [50]?

Response 14: Thank you for pointing this out. In the study [50], prochloraz was identified as the most effective among six fungicides. In our experiment, prochloraz also demonstrated the best efficacy among the four fungicides tested, which was consistent with the findings of both reports. On page 12, line 271 indicates that prochloraz exhibited the best efficacy among the four fungicides, “The results indicated that prochloraz was the most effective fungicide against Alternaria spp. in this study", On page 14, line 360 further mentions, "Fewer fungicides were selected in this study, and the selection of efficient, environmentally friendly, and economical fungicides needs to be further explored," indicating the need for further exploration in the selection of efficient fungicides.

In the study [50], they used 6 fungicides, which were chlorothalonil, copperoxychloride, mancozeb, difenoconazole, iprodione and prochloraz.

Comments 15: On line 358 of page 14, “In 2023, the EC50 value of fludioxonil was only 0.089 ± 0.020 in Wang et al.'s study [51].” what species?

Response 15: Thank you for pointing this out. I agree with this comment.  It is A. Alternata. The sentence has changed to “In 2023, the EC50 value of fludioxonil for 43 A. alternata was only 0.089 ± 0.020 in Wang et al.'s study [52].”

Comments 16: On line 359 of page 14, “whereas the EC50 value of prochloraz in this study was 2.92, which clearly shows that the inhibitory effect of fludioxonil on A. alternata was stronger. Fewer fungicides were selected in this study, and the selection of efficient, environmentally friendly, and economical fungicides needs to be further explored.” False. We cannot compare the EC50 values among different fungicides/different species, and say fungicide X is more effective than Y because EC50 of X is smaller than Y.

Response 16: Thank you for pointing this out. I agree with this comment. Exactly, “I cannot compare the EC50 values among different fungicides/different species, and say fungicide X is more effective than Y because EC50 of X is smaller than Y.” What I wanted to express is that I just compared the four fungicides but I should explore more because of fewer fungicides were selected in this study. The sentence has changed to “whereas the EC50 value of prochloraz for A. alternata in this study was 2.95 ± 0.31,”.

Comments 17: On line 368 of page 14, “These sensitivity data can serve as a point of reference for assessing potential changes in the sensitivity of Alternaria spp. to various fungicides in the future.” Fungicide sensitivity data from few isolates are not informative enough to state this since fungicide sensitivity varies greatly from one isolate to another within the same species in the same region.

Response 17: Thank you for pointing this out. I agree with this comment. I have deleted this sentence on line 368 of page 14. And it can also explain the questioning of Comments 13 that you expressed “since fungicide sensitivity varies greatly from one isolate to another within the same species in the same region.”

Response to Reviewer X Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Not applicable

Is the research design appropriate?

Can be improved

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: First page, line 40, “Currently, there have been no reports of leaf spot in C. sinensis caused by Alternaria”, Delete: there are much more than Alternaria. Mention leaf spot cuased by Alternaria in plant species/genera similar to Celtis. Then, start to talk about Alternaria.

Response 1: Thank you for pointing this out. I agree with this comment. Therefore, this sentence has been changed to “In August 2023, a report was published on Celtis julianae leaf blotch caused by A. arborescens and A. italica.”

Comments 2: Page 2, line 92, “Three isolates (11, 12 and 13) from the group, with the highest frequency of occurrence were selected for pathogenicity testing and inoculation of leaf blades and seedlings, respectively [19]”, mention species for each isolate name.

Response 2: Thank you for pointing this out. I agree with this comment. This sentence has been changed to “A. alternata (isolates 11, 13) and A. koreana (isolate 12) from the group with the highest frequency of occurrence were selected for pathogenicity testing and inoculation of leaf blades and seedlings, respectively”

Comments 3: Page 2, line 95, “Five isolated leaves were inoculated with each isolate,” should be detached.

Response 3: Thank you for pointing this out. I agree with this comment. This sentence has been changed to “Five detached leaves were inoculated with each isolate”.

Comments 4: Page 2, line 97, “and a 6 mm mycelium block was placed face down on each puncture wound and removed after 24 hours [21]”, which side of leaf?

Response 4: Thank you for pointing this out. Page 2, line 96, the sentence “wounds were created on both sides of the leaf veins with a sterile needle” indicated that on both sides of the leaf.

Comments 5: Line 101 on page 3, “3 leaves were inoculated with spores of one of the 3 isolates by puncturing the leaf with a sterile needle as a wound suspension (106 conidia/mL)”, 1 leaf / 1 plant?? Or 3 leaves / 1 plant??

Response 5: Thank you for pointing this out. Due to my mistake of expression, it caused you a misunderstanding. The sentence has changed to “3 leaves on a plant were inoculated with spores of one of the 3 isolates by puncturing the leaf with a sterile needle as a wound suspension (106 conidia/mL)”.

Comments 6: Line 156 on page 7, title “2.6. Evaluation of fungicides against A. alternata and A. koreana”, how many isolates from each species were tested for this study?

Response 6: Thank you for pointing this out. I agree with this comment. The title has changed to “2.6. Evaluation of fungicides against A. alternata (isolates 11, 13) and A. koreana (isolate 12)”.

Comments 7: Line 196 on page 9, “Figure 2. Pathogenicity of fungal isolates. A. Signs leaves of controls treated with PDA after 4 days. B-D. Signs on leaves inoculated with mycelium blocks of isolates 11, 12 and 13 after 4 days. E. Signs leaves of controls treated with sterile water after 5 days. F-H. Signs leaves of controls handled with sterile water after 5 days. Signs on leaves inoculated with 10 µl of conidial suspension (10⁶ conidia/mL) of isolates 11, 12 and 13 after 5 days.” what species are they? Figures and Tables should stand alone. Explain every time what "isolate 11", 12, 13 mean in the figure leghend or foot note.

Response 7: Thank you for pointing this out. I agree with this comment. The sentence has changed to “Figure 2. Pathogenicity of fungal isolates. A. Signs leaves of controls treated with PDA after 4 days. B-D. Signs on leaves inoculated with mycelium blocks of A. alternata (isolates 11, 13) and A. koreana (isolate 12) after 4 days. E. Signs leaves of controls treated with sterile water after 5 days. F-H. Signs leaves of controls handled with sterile water after 5 days. Signs on leaves inoculated with 10 µl of conidial suspension (106 conidia/mL) of A. alternata (isolates 11, 13) and A. koreana (isolate 12) after 5 days.”

Comments 8: on line 216 of page 10, “Figure 3. Morphological characteristics of fungal isolates.” explain 11=what species etc.

Response 8: Thank you for pointing this out. I agree with this comment. The sentence has changed to “Figure 3. Morphological characteristics of fungal isolates A. alternata (isolates 11, 13) and A. koreana (isolate 12)”.

Comments 9: On line 257 of page 12, “Figure 5. Colony formation of isolates 11, 12, and 13 isolated from Celtis sinensis hatched for 7 days on 6 media at 25°C.” what species are they?

Response 9: Thank you for pointing this out. I agree with this comment. The sentence has changed to “Colony formation of isolates A. alternata (isolates 11, 13) and A. koreana (isolate 12) isolated from Celtis sinensis hatched for 7 days on 6 media at 25°C”.

Comments 10: On line 264 of page 13, “Figure 6. The impact of temperature on the growth of colony diameters in isolates 11, 12, and 13 after 7 days of culture on PDA media” What number is what species?

Response 10: Thank you for pointing this out. I agree with this comment. The sentence has changed to “Figure 6. The impact of temperature on the growth of colony diameters in isolates A. alternata (isolates 11, 13) and A. koreana (isolate 12) after 7 days of culture on PDA media”.

Comments 11: On line 278 of page 14, “Figure 7. The suppression effect on isolates 11, 12 and 13 on plates by various doses of four diverse fungicides (prochloraz, myclobutanil, tebuconazole, and pyraclostrobin) based on fresh PDA for 7 days”, what number is what species?

Response 11: Thank you for pointing this out. I agree with this comment. The sentence has changed to “Figure 7. The suppression effect on isolates A. alternata (isolates 11, 13) and A. koreana (isolate 12) on plates by various doses of four diverse fungicides (prochloraz, myclobutanil, tebu-conazole, and pyraclostrobin) based on fresh PDA for 7 days”.

Comments 12: On line 281 of page 14, “Table 3. Concentration at 50% of Maximum Effect (EC50 values) of isolates” Sensitivity of Alternaria alternata and A. koreana to four fungicides.

In the foot note, indicate which is A. alternata and A. koreana.

Response 12: Thank you for pointing this out. I agree with this comment. I have added the foot note of table 3, “Isolates 11 and 13 were A. alternata; isolate 12 was A. koreana.”.

Comments 13: On line 365 of page 16, “which clearly shows that the inhibitory effect of fludioxonil on A. alternata was stronger”, slightly different nuance, comparing the EC50 values between the 2 fungicides here, we can say that fludioxonil requires less amount to obtain the same inhibitory effect.

Response 13: Thank you for pointing this out. I agree with this comment. The sentence has changed to “which clearly shows that fludioxonil requires a smaller dosage to achieve the same inhibitory effect.”