The Inhibitory Mechanism of Eugenol on Lasiodiplodia theobromae and Its Induced Disease Resistance of Passion Fruit

Round 1

Reviewer 1 Report

Dear authors, although the topic is important and topical, the manuscript need to be improved.

Considering the title, I expected a comparison of eugenol efficacy on different passion fruit pathogens. For the same reason when in Materials and Methods you described the isolation of pathogenic fungi on naturally infected passion fruits, I expected the isolation of different pathogens. Therefore, I suggest changing the title specifying that studies are carried out only on Lasiodiplodia theobromae.

In my opinion, the description of isolation and identification of pathogenic fungi should be reduced, as it is excessive compared to the topic of the paper, unbalancing the sections. Instead, it would be necessary to implement the description of the methodology used to study the effectiveness of eugenol in reducing pathogen growth, which is only mentioned in some sections.

Line 76 Symptoms of fruit naturally infections observed, should be described in details.

Lines 81-83 How many plates were incubated? How many pathogens/strains were isolated? Which criteria were used to choose a specific strain?

Lines 85-91 The description of the method used is not clear. How many inoculations per fruit? Why was control not wounded?

Lines 96-100 Where is it described that different strains were isolated?

Lines 102-103 Please add a brief description of the method used.

Line 110 Write the full meaning of an acronym the first time you mention it.

Lines 123-125 Please briefly explain the meaning of cross-method.

Lines 146-151 Please rewrite the period because it is not clear, and it lack details. For example: the supernatant was centrifugated at which speed, for how many minutes? Why did you obtain again the fungal mycelium?

Line 157 Please write the full meaning of MDA the first time you mention it.

Line 197 Please write the full meaning of PI staining.

Line 200 Bacterial filaments?

Line 223 Can you explain the meaning of B-spore?

Line 248 Please transform the sentence in the passive form.

Line 297-308 Please add references.

Lines 338-343 In my opinion this section should be transferred in Materials and Methods since describe symptoms of naturally occurring fruit, taking for the isolations of fungi.

Section 3.1 Too many details about Passion fruit rot pathogen isolated. It is not the objective of the study.

Line 345-346 What does bacteria cake mean?

Figure 3B Bacterial diameters?

Section 3.3.1. The results should be rewritten using a more scientific language. For example, in line 402-403 “the spore germination rate…… was higher”, was it significantly higher? Did you measure spore germination? In what manner?

Lines 473-475 The sentence is not clear "The control fruit group was infected" (with pathogen?), "four days after inoculation with pathogens". Please rewrite in a clearer way.

I suggest major revision before the publication of the manuscript.

Author Response

Dear Editor and Reviewers,

Thank you very much for your overall comprehensive and positive comments on this manuscript, and thank you for giving us the chance to revise. We have fully considered all the opinions and requirements in the letter, and have made detailed replies and modifications on an item-by-item basis.Notably, all my responses are marked in blue, the original contents are marked in green, and revised portions are marked in red. Please find my itemized responses below and my corrections in the “Manuscript File”.

Thanks again!

Responds to the Editor’s comments:

1.Comments: Considering the title, I expected a comparison of eugenol efficacy on different passion fruit pathogens. For the same reason when in Materials and Methods you described the isolation of pathogenic fungi on naturally infected passion fruits, I expected the isolation of different pathogens. Therefore, I suggest changing the title specifying that studies are carried out only on Lasiodiplodia theobromae.

Response: First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript.We have made modifications to the title, changing it from "Antifungal Activity of Eugenol Against Passion Fruit Rot Pathogens and Its Induction of Resistance to Fruit Rot in Passion Fruit" to "The Inhibitory Mechanism of Eugenol on Lasiodiplodia theobromae and Its Induced Disease Resistance". Please find my itemized responses and my detailed modifications in the “Manuscript File”.

2.Comments: In my opinion, the description of isolation and identification of pathogenic fungi should be reduced, as it is excessive compared to the topic of the paper, unbalancing the sections. Instead, it would be necessary to implement the description of the methodology used to study the effectiveness of eugenol in reducing pathogen growth, which is only mentioned in some sections.

Response: :We are very grateful and agree with the important issues and suggestions you have pointed out. After carefully sorting and analyzing the logic of the isolation and identification of pathogenic bacteria, we have condensed and refined the content of the isolation and identification of pathogenic bacteria. The specific modifications are in the reply below and the revised manuscript:

Lines 74-85 and Lines 86-97: The narratives of the two paragraphs were combined, summarized and condensed into one paragraph. The original two paragraphs of “ Naturally infected passion fruits were disinfected with a 75% ethanol solution for 30 s and removed from the solution to dry. These fruits were then placed in clean water for 30 s to wash away excess ethanol solution and dried on a laminar flow bench. Tissue (approximately 5 mm2) was collected from the margin between naturally infected and healthy passion fruit; this tissue was used for pathogenic isolation, and was transferred to a PDA plate. These plates were then placed in a 25 °C incubator and monitored regularly for colony growth. Colonies were separated on PDA plates every three days until a pure strain was obtained; this was labelled strain A.Pathogen was tested using Koch's postulates. The pathogenic culture with a diameter of 5 mm was inoculated on the PDA plate and cultured until the mycelium grew effectively. Nine fresh and healthy passion fruits were inoculated with the pathogenic culture, and the same number of fruits were inoculated without wounding to act as controls. Subsequently, the inoculated fruits were maintained at a constant temperature of 28 °C and a relative humidity of 95% for 7 days, followed by continuous observation and recording of symptom development. Once symptoms manifested, the infected area of the fruit was collected and re-isolated to complete Koch's postulate test and confirm whether the re-isolated fungus was the pathogen.” were replaced by “Naturally infected passion fruits were disinfected with a 75% ethanol solution for 30 s and removed from the solution to dry. These fruits were then placed in clean water for 30 s to wash away excess ethanol solution and dried on a laminar flow bench. Tissue (approximately 5 mm²) was collected from the margin between naturally infected and healthy passion fruit; The tissue was used for pathogen isolation and transferred to potato dextrose agar (PDA) plates. We used a total of 50 PDA plates for the isolation and purification of different pathogens These plates were then placed in a 25 °C incubator and monitored regularly for colony growth. Colonies were separated on PDA plates every three days until different purified strains..The pathogenicity of the isolated pathogen was tested using Koch's postulates. The pathogenic culture with a diameter of 5 mm was inoculated on the PDA plate and cultured until the mycelium grew effectively. Take ninety fresh healthy passion fruit, scrub the surface with 75% alcohol to disinfect, rinse the alcohol with distilled water and dry, traumatize the center of the passion fruit surface with a hole punch, receive the pathogenic bacteriophage cake to the skin trauma site and make the side with mycelium touch the fruit wound part; And inoculate the same amount of non pathogenic bacterial cake as a control. Subsequently, the inoculated fruits were maintained at a constant temperature of 28 °C and a relative humidity of 95% for 7 days, followed by continuous observation and recording of symptom development. Once symptoms manifested, the infected area of the fruit was collected and re-isolated to complete Koch's postulate test and confirm whether the re-isolated fungus was the pathogen.”(Lines 74-97, Page 3-4)

3.Comments: Line 76 Symptoms of fruit naturally infections observed, should be described in details.

Response: First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. We have added a detailed description of the symptoms of natural infection of passion fruit in the newly submitted 'manuscript file'.The specific modifications are in the reply below and the revised manuscript:

Line 76 : This paragraph provides a more detailed description, with the original paragraph “Naturally infected passion fruits were disinfected with a 75% ethanol solution for 30 s and removed from the solution to dry. These fruits were then placed in clean water for 30 s to wash away excess ethanol solution and dried on a laminar flow bench. Tissue (approximately 5 mm2) was collected from the margin between naturally infected and healthy passion fruit; this tissue was used for pathogenic isolation, and was transferred to a PDA plate. These plates were then placed in a 25 °C incubator and monitored regularly for colony growth. Colonies were separated on PDA plates every three days until a pure strain was obtained; this was labelled strain A.” replaced by “Naturally infected passion fruits were disinfected with a 75% ethanol solution for 30 s and removed from the solution to dry. These fruits were then placed in clean water for 30 s to wash away excess ethanol solution and dried on a laminar flow bench. Tissue (approximately 5 mm²) was collected from the margin between naturally infected and healthy passion fruit; The tissue was used for pathogen isolation and transferred to potato dextrose agar (PDA) plates. We used a total of 50 PDA plates for the isolation and purification of different pathogens These plates were then placed in a 25 °C incubator and monitored regularly for colony growth. Colonies were separated on PDA plates every three days until different purified strains..We used criteria such as pathogenicity assay, morphological observation and ITS sequence analysis of pathogenic fungi to select specific strains and named the selected strains as strain A.” (Lines 74-85, Page 2)

4.Comments: Lines 81-83 How many plates were incubated? How many pathogens/strains were isolated? Which criteria were used to choose a specific strain?

Response: First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. We used a total of 50 petri dishes for the incubation of pathogenic bacteria, and a total of 9 strains of pathogenic bacteria were isolated. We used criteria such as pathogenicity determination, morphological observation, and ITS sequence analysis of pathogenic fungi to select a specific strain. The specific modifications are in the reply below and the revised manuscript:

 Lines 81-83: This paragraph provides a more detailed description, with the original paragraph “Naturally infected passion fruits were disinfected with a 75% ethanol solution for 30 s and removed from the solution to dry. These fruits were then placed in clean water for 30 s to wash away excess ethanol solution and dried on a laminar flow bench. Tissue (approximately 5 mm²) was collected from the margin between naturally infected and healthy passion fruit; this tissue was used for pathogenic isolation, and was transferred to a PDA plate. These plates were then placed in a 25 °C incubator and monitored regularly for colony growth. Colonies were separated on PDA plates every three days until a pure strain was obtained; this was labelled strain A.” replaced by “Naturally infected passion fruits were disinfected with a 75% ethanol solution for 30 s and removed from the solution to dry. These fruits were then placed in clean water for 30 s to wash away excess ethanol solution and dried on a laminar flow bench. Tissue (approximately 5 mm²) was collected from the margin between naturally infected and healthy passion fruit; The tissue was used for pathogen isolation and transferred to potato dextrose agar (PDA) plates. We used a total of 50 PDA plates for the isolation and purification of different pathogens. These plates were then placed in a 25 °C incubator and monitored regularly for colony growth. Colonies were separated on PDA plates every three days until different purified strains. We used criteria such as pathogenicity assay, morphological observation and ITS sequence analysis of pathogenic fungi to select specific strains and named the selected strains as strain A.” (Lines 75-85, Page 2)

5.Comments: Lines 85-91 The description of the method used is not clear. How many inoculations per fruit? Why was control not wounded?

Response: Thank you very much for your suggestion. We have made modifications to the method description and fruit inoculation description. The specific modifications are as follows:

Lines 85-91 : This paragraph provides a more detailed description, with the original paragraph “The pathogenicity of the isolated pathogen was tested using Koch's postulates. The pathogenic culture with a diameter of 5 mm was inoculated on the PDA plate and cultured until the mycelium grew effectively. Nine fresh and healthy passion fruits were inoculated with the pathogenic culture, and the same number of fruits were inoculated without wounding to act as controls. Subsequently, the inoculated fruits were maintained at a constant temperature of 28 °C and a relative humidity of 95% for 7 days, followed by continuous observation and recording of symptom development. Once symptoms manifested, the infected area of the fruit was collected and re-isolated to complete Koch's postulate test and confirm whether the re-isolated fungus was the pathogen.” replaced by “The pathogenicity of the isolated pathogen was tested using Koch's postulates. The pathogenic culture with a diameter of 5 mm was inoculated on the PDA plate and cultured until the mycelium grew effectively. Take ninety fresh healthy passion fruit, scrub the surface with 75% alcohol to disinfect, rinse the alcohol with distilled water and dry, traumatize the center of the passion fruit surface with a hole punch, receive the pathogenic bacteriophage cake to the skin trauma site and make the side with mycelium touch the fruit wound part; And inoculate the same amount of non pathogenic bacterial cake as a control. Subsequently, the inoculated fruits were maintained at a constant temperature of 28 °C and a relative humidity of 95% for 7 days, followed by continuous observation and recording of symptom development. Once symptoms manifested, the infected area of the fruit was collected and re-isolated to complete Koch's postulate test and confirm whether the re-isolated fungus was the pathogen. ” (Lines 86-97, Page 2-3)

6.Comments: Lines 96-100 Where is it described that different strains were isolated?

  Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have described the isolation of different strains in section 2.1.1, but these descriptions may not be clear enough, so we have made modifications. The specific modifications are as follows:

Lines 75-85: This paragraph provides a more detailed description, with the original paragraph “Naturally infected passion fruits were disinfected with a 75% ethanol solution for 30 s and removed from the solution to dry. These fruits were then placed in clean water for 30 s to wash away excess ethanol solution and dried on a laminar flow bench. Tissue (approximately 5 mm²) was collected from the margin between naturally infected and healthy passion fruit; this tissue was used for pathogenic isolation, and was transferred to a PDA plate. These plates were then placed in a 25 °C incubator and monitored regularly for colony growth. Colonies were separated on PDA plates every three days until a pure strain was obtained; this was labelled strain A.” replaced by “Naturally infected passion fruits were disinfected with a 75% ethanol solution for 30 s and removed from the solution to dry. These fruits were then placed in clean water for 30 s to wash away excess ethanol solution and dried on a laminar flow bench. Tissue (approximately 5 mm²) was collected from the margin between naturally infected and healthy passion fruit; this tissue was used for pathogenic isolation, and was transferred to a PDA plate. We used a total of 50 PDA plates for the isolation and purification of different pathogens These plates were then placed in a 25 °C incubator and monitored regularly for colony growth. Colonies were separated on PDA plates every three days until different purified strains. We used criteria such as pathogenicity assay, morphological observation and ITS sequence analysis of pathogenic fungi to select specific strains and named the selected strains as strain A” (Lines 75-85, Page 2)

7.Comments: Lines 102-103 Please add a brief description of the method used.

  Response: thank you very much for your overall approval and comprehensive comments on this manuscript. We have added a brief description of the CTAB method for extracting DNA. The specific modifications are as follows:

Lines 103-109: This paragraph provides a more detailed description, with the original paragraph “Edwards’ cetyltrimethylammonium bromide (CTAB) method was used to extract DNA from this strain [17]. Following the PCR product analysis, agarose gel electrophoresis was performed by Sangon Biotech Co., Ltd (Guangzhou, China). The sequencing results were subjected to Basic Local Alignment Search Tool (BLAST, U.S. National Library of Medicine) comparative analysis using the NCBI database; phylogenetic tree construction was performed with species that exhibited similar homology, using Molecular Evolutionary Genetics Analysis (MEGA).” replaced by “Edwards’ cetyltrimethylammonium bromide (CTAB) method was used to extract DNA from this strain [17]. The universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3’) of the fungal rDNA-ITS were used as PCR amplification primers. The amplification system was 25 µL 2×Taq PCR Master Mix, 1 µL DNA, 2 µL 10 mmoL/L upstream primer and 2 µL 10 mmoL/L downstream primer, 20 µL ultrapure water. PCR cycling conditions were 95°C pre-denaturation for 5 min, 95°C for 30s, 58°C for 30s, 72°C for 30s for a total of 35 cycles, 72°C repair extension for 5 min, and stored at 4°C. Following the PCR product analysis, agarose gel electrophoresis was performed by Sangon Biotech Co., Ltd (Guangzhou, China). The sequencing results were subjected to Basic Local Alignment Search Tool (BLAST, U.S. National Library of Medicine) comparative analysis using the NCBI database; phylogenetic tree construction was performed with species that exhibited similar homology, using Molecular Evolutionary Genetics Analysis (MEGA).” (Lines 105-119, Page 3)

8.Comments: Line 110 Write the full meaning of an acronym the first time you mention it.

    Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have made changes to the noun that first appeared, written its full name, and highlighted it in red in the submitted 'manuscript file'.

The specific modifications are as follows:

Lines 111 : This sentence provides a comprehensive explanation, replacing "Mahdi's method was used to determine the MIC " with "Mahdi's method was used to determine the minimal inhibitory concentration (MIC)" (Lines 121-122, Page 3)

9.Comments: Lines 123-125 Please briefly explain the meaning of cross-method.

    Response: thank you very much for your overall approval and comprehensive comments on this manuscript. We have provided a brief explanation of the cross-method. The specific modifications are as follows:

Lines 125-127: This sentence provides a comprehensive explanation, replacing “The diameter of the fungal colony was measured using the cross-method, and the inhibition rate was calculated according to Equation (1).” with “The diameter of the fungal colony was measured using the cross-method(The cross method is to use a vernier caliper to cross measure the colony diameter and take the average value for counting.), and the inhibition rate was calculated according to Equation (1).” (Lines 137-140, Page 3)

10.Comments: Lines 146-151 Please rewrite the period because it is not clear, and it lack details. For example: the supernatant was centrifugated at which speed, for how many minutes? Why did you obtain again the fungal mycelium?

Response: thank you very much for your overall approval and comprehensive comments on this manuscript. We have made more detailed revisions to the methods used. The specific modifications are as follows:

(1). Lines 146-151 : This paragraph provides a more detailed description, replacing the original “The filtrate was filtered through four layers of cheesecloth to remove the fungal filament, the supernatant was centrifuged to remove the precipitate, the fruit rot pathogen spores were suspended in PBS solution, the spore concentration was adjusted to 10⁶ CFU/mL using a haemocytometer, and a spore suspension was prepared. ” with “Filter the obtained solution through four layers of gauze to remove hyphae. Centrifuge the filtrate at 10000 r/min for 10 minutes to remove precipitates. Resuspend the spores of fruit rot pathogens in a phosphate buffer salt (PBS) solution. Use a blood cell counting plate to adjust the spore concentration to 10⁶ CFU/mL, and prepare a spore suspension for later use.” (Lines 159-163, Page 4)

11.Comments: Line 157 Please write the full meaning of MDA the first time you mention it.

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have made changes to the noun that first appeared, written its full name, and highlighted it in red in the submitted 'manuscript file'.

The specific modifications are as follows:

Lines 157-159 : This sentence provides a comprehensive explanation, replacing "To measure cell membrane permeability, relative conductivity was measured according to Liu’s method [20], intracellular nucleic acid leakage was determined using Tao’s method [21], intracellular protein release and soluble sugar release were determined using Chen’s method [22], and MDA content was determined using Wei’s method [23]. " with "To measure cell membrane permeability, relative conductivity was measured according to Liu’s method [20], intracellular nucleic acid leakage was determined using Tao’s method [21], intracellular protein release and soluble sugar release were determined using Chen’s method [22], and malondialdehyde (MDA) content was determined using Wei’s method [23]." (Lines 171-173, Page 4)

12.Comments: Line 197 Please write the full meaning of PI staining.

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have made changes to the noun that first appeared, written its full name, and highlighted it in red in the submitted 'manuscript file'.

The specific modifications are as follows:

Lines 197-198 : This sentence provides a comprehensive explanation, replacing "PI staining to Observe the Effect of Eugenol on the DNA of Passion Fruit Rot–Causing Fungi " with " Propidium iodide (PI) staining to Observe the Effect of Eugenol on the DNA of Passion Fruit Rot–Causing Fungi." (Line 212, Page 5)

13.Comments: Line 200 Bacterial filaments?

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have made changes to inappropriate terms, and the changed terms will be marked red in the submitted "manuscript file".The specific modifications are as follows:

Lines 200 : This sentence provides a comprehensive explanation, replacing "Cultured bacterial filaments were collected, dried, and stored for later use." with "Collect the cultured mycelium, dry it, and store it for future use " (Line 215, Page 5)

14.Comments:Line 223 Can you explain the meaning of B-spore?

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have made modifications to the inappropriate wording, and the revised sentences will be highlighted in red in the submitted 'manuscript file'. The specific modifications are as follows:

Lines 248-250, : This sentence provides a comprehensive explanation, replacing "After natural air-drying, 10 μl (10⁶ CFU/mL) of the B-spore suspension was inoculated into each wound." with "After natural air-drying, 10 μl (10⁶ CFU/mL) of the Lasiodiplodia theobromae spore suspension was inoculated into each wound." (Lines 240, Page 6)

15.Comments: Line 248 Please transform the sentence in the passive form.

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have made modifications to the inappropriate wording, and the revised sentences will be highlighted in red in the submitted 'manuscript file'. The specific modifications are as follows:

Lines 248-250, : This sentence provides a comprehensive explanation, replacing "A crude enzyme extract was prepared to determine the levels of the defence enzymes catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) [25-26]." with "The prepared crude enzyme extract is used to determine the levels of defense enzymes catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) [25-26]." (Lines 266-267, Page 6)

16.Comments: Line 297-308 Please add references.

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have added references in the corresponding positions and highlighted them in red in the submitted 'manuscript file'. The specific modifications are as follows: Lines 297-308, : This sentence provides a comprehensive explanation, replacing "For 4-coumarate Coenzyme a Ligase (4CL), the reaction system was 0.1 mL of enzyme solution, 0.3 mL of 50 μmol/L adenosine triphosphate (ATP), 0.3 mL of 5 μmol/L coenzyme A (CoA), and 0.15 mL of 0.3 μmol/L p-coumaric acid. This reaction solution was heated in a water bath at 40 °C for 10 min, and the absorbance of the reaction solution was measured at 333 nm. The increase in absorbance of the reaction system at 333 nm by 0.01 per minute was considered as one enzyme activity unit (U).The β-1,3-glucanase (GLU) activity was determined by a colorimetric method using 3,5-dinitrosalicylic acid. The reaction system included 100 μL of 0.1% kombucha polysaccharide (prepared with 0.05 mol/L pH 5.5 sodium acetate-acetic acid buffer), 100 μL of enzyme solution; this was mixed well and maintained at 37 °C for 30 min. This experiment was repeated three times. One unit of β-1,3-glucanase activity was defined as 1 μmol of reducing sugar per g of tissue per h." with " For 4-coumarate Coenzyme a Ligase (4CL), the reaction system was 0.1 mL of enzyme solution, 0.3 mL of 50 μmol/L adenosine triphosphate (ATP), 0.3 mL of 5 μmol/L coenzyme A (CoA), and 0.15 mL of 0.3 μmol/L p-coumaric acid. This reaction solution was heated in a water bath at 40 °C for 10 min, and the absorbance of the reaction solution was measured at 333 nm. The increase in absorbance of the reaction system at 333 nm by 0.01 per minute was considered as one enzyme activity unit (U).[31]

The β-1,3-glucanase (GLU) activity was determined by a colorimetric method using 3,5-dinitrosalicylic acid. The reaction system included 100 μL of 0.1% kombucha polysaccharide (prepared with 0.05 mol/L pH 5.5 sodium acetate-acetic acid buffer), 100 μL of enzyme solution; this was mixed well and maintained at 37 °C for 30 min. This experiment was repeated three times. One unit of β-1,3-glucanase activity was defined as 1 μmol of reducing sugar per g of tissue per h.[32]." (Lines 313-324, Page 7)

17.Comments: Lines 338-343 In my opinion this section should be transferred in Materials and Methods since describe symptoms of naturally occurring fruit, taking for the isolations of fungi.

Response: Thank you for your targeted suggestions and questions,

which are important guides for us to improve the quality of this revised manuscript and for future scientific work.This section describes the symptoms of naturally occurring passion fruit rots fruit symptoms are used to verify that the isolated and purified strains are pathogenic and to complete the verification of Koch's Postulates. Therefore, we believe that it is correct to include the natural onset symptoms in the conclusion.

18.Comments: Section 3.1 Too many details about Passion fruit rot pathogen isolated. It is not the objective of the study.

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. After carefully sorting and analyzing the logic of the isolation and identification of pathogenic bacteria, we have condensed and refined the content of the isolation and identification of pathogenic bacteria. The specific modifications are in the reply below and the revised manuscript:

Lines 365-380 : The narratives of the two paragraphs were combined, summarized and condensed into one paragraph. The original two paragraphs of “The colonies of strain A are shown in Figure 2A. This fungus grows rapidly on potato dextrose agar (PDA) plates, and can completely occupy a culture dish within 4 days of culturing at 25 °C. Initially, the colonies were white, but eventually turned light black. The colony texture was dry and easy to lift, the aerial mycelia were fluffy and dense, and a light black colour was observed at the base that deepened over time. Using an optical microscope, the small conidia were determined to be ellipsoidal (Figure 2B). PCR amplification of the ribosomal DNA internal transcribed sequence (rDNA-ITS) of strain A of the isolated pathogen was performed using universal primers ITS1 and ITS4. The corresponding electrophoresis profile indicated that the rDNA-ITS of the strain was approximately 500–700 bp in length (Figure 2C). The neighbour-joining phylogenetic tree of strain A was constructed using MEGA-X; this indicated a 99% homology between strain A and Lasiodiplodia theobromae (Figure 2D). The combined morphological characteristics and molecular identification results suggest that strain A is the same pathogen that causes guava fruit rot, L. theobromae.” were replaced by “ Identification of The Pathogenic Bacteria of Fruit rot of Passion Fruit. The colonies of strain A are shown in Figure 2A. This fungus grows rapidly on potato dextrose agar (PDA) plates, and can completely occupy a culture dish within 4 days of culturing at 25 °C. Initially, the colonies were white, but eventually turned light black. The colony texture was dry and easy to lift, the aerial mycelia were fluffy and dense, and a light black colour was observed at the base that deepened over time. Using an optical microscope, the small conidia were determined to be ellipsoidal (Figure 2B).The corresponding electrophoresis pattern of the pathogen strain indicates that the rDNA ITS length of the strain is approximately 500-700 bp (Figure 2C).The neighbour-joining phylogenetic tree of strain A was constructed using MEGA-X; this indicated a 99% homology between strain A and Lasiodiplodia theobromae (Figure 2D). The combined morphological characteristics and molecular identification results suggest that strain A is the same pathogen that causes guava fruit rot, L. theobromae." (Lines 383-395, Page 9)

19.Comments: Line 345-346 What does bacteria cake mean?

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. The name expression here is not very accurate. We have changed "bacterial cake" to "pathogenic fungal cake". The term 'pathogenic fungal cake' refers to a circular cake shaped substance taken from a purified pathogenic bacterial culture medium using a cake extractor. The specific modifications are in the reply below and the revised manuscript:

Lines 345-346 : This sentence provides a comprehensive explanation, replacing "The isolated and purified pathogen was grafted onto healthy fruit using a sterile bacterial cake as a control." with "The control group was inoculated with blank PDA fungal cake, and the treatment group was inoculated with purified pathogenic fungal cake and transplanted onto healthy fruits." (Lines 362-363, Page 8)

20.Comments: Figure 3B Bacterial diameters?

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. The name expression here is not very accurate. We have changed "Bacterial diameters" to "colony diameter". We have also changed figure 3-B, please check it in the newly submitted "manuscript file".The changed figure is shown below

Figure 3. Effect of eugenol on (A) mycelial growth, (B) colony diameter, and (C) mycelial inhibition rate of pathogenic fungi. Different lowercase letters in the same sampling are significantly different to each other (p < 0.05).

21.Comments: Section 3.3.1. The results should be rewritten using a more scientific language. For example, in line 402-403 “the spore germination rate…… was higher”, was it significantly higher? Did you measure spore germination? In what manner?

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. We have rewritten this paragraph in more detail and added pictures of spore germination rates, please see the newly submitted "manuscript file". The specific modifications are in the reply below and the revised manuscript:

(1). Lines 402-413 : This paragraph provides a more detailed explanation, replacing "As shown in Figure 4A, compared with the control group, the spore germination rate of fruit rot pathogens in the control group after eugenol treatment was higher, and the bud tube elongation was evident.. Spore germination and bud tube elongation were inhibited in the eugenol-treated group. In addition, with an increase in eugenol concentration, the spore germination rate of fruit rot pathogenic fungi decreased. As shown in Figure 4B, the cell membrane structure of the fruit rot pathogen in the control group was complete, ordered, and relatively full; the cell membrane in this control group also contained high water content and high morphological integrity, displaying little propidium iodide (PI) staining (red) and a low degree of cell membrane damage. With an increase in eugenol concentration, the cell membrane structure began to deform, fold, and curl, the cell gap gradually increased, and the cell wall was clearly perforated, indicating that the integrity of the cell membrane of the fruit rot pathogen was damaged." with "As shown in Figure 4A, compared with the control group, the spore germination rate of fruit rot pathogens in the control group after eugenol treatment was higher, and the bud tube elongation was evident.. Spore germination and bud tube elongation were inhibited in the eugenol-treated group. In addition, with an increase in eugenol concentration, the spore germination rate of fruit rot pathogenic fungi decreased. After 4 hours of treatment with 1/2 MIC and MIC concentrations of eugenol, the spore germination rates of pathogenic fungi were 13.97% and 9.87%, respectively, compared to 20.05% in the control group; After 8 hours of treatment, the spore germination rate of the control group was 36%, which was 1.4 times and 2.4 times higher than the spore germination rate of pathogenic fungi treated with 1/2 MIC and MIC mass concentrations of eugenol (Figure 4C).As shown in Figure 4B, the cell membrane structure of the fruit rot pathogen in the control group was complete, ordered, and relatively full; the cell membrane in this control group also contained high water content and high morphological integrity, displaying little propidium iodide (PI) staining (red) and a low degree of cell membrane damage. With an increase in eugenol concentration, the cell membrane structure began to deform, fold, and curl, the cell gap gradually increased, and the cell wall was clearly perforated, indicating that the integrity of the cell membrane of the fruit rot pathogen was damaged(Figure 4B). (Lines 431-450, Page 11)

(2). The changed figure is shown below:

Figure 4. (A) The effect of eugenol on spore germination and the hyphal cell membrane of the passion fruit rot pathogen. (B) The effect of spore germination and PI dye on the hyphal cell membrane of the passion fruit rot pathogen. (C)Effect of different mass concentrations of eugenol treatment on the germination rate of spores.

22.Comments: Lines 473-475 The sentence is not clear "The control fruit group was infected" (with pathogen?), "four days after inoculation with pathogens". Please rewrite in a clearer way.

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. Both the control fruit group and the treatment group were inoculated with pathogenic fungi, but the treatment group was soaked in distilled water without eugenol. The treatment group was soaked in MIC concentrations of eugenol solution. This treatment is to better determine the inhibitory effect of different concentrations of eugenol on the pathogenic fungi of passion fruit rot, so the control group was also inoculated with pathogenic fungi.This method has been described in Section 2.5.1, Treatment II. It may be that our description is not accurate enough and has been modified. Please check the newly submitted "manuscript file" for the newly modified content. The specific modifications are in the reply below and the revised manuscript:

Lines 219-228: The method description in this paragraph has been more carefully rewritten, changing from "Treatment II: Passion fruit were rinsed in sterile water and MIC eugenol solution for 15 min before air-drying. The same method as Treatment I was conducted, using 75% ethanol to disinfect the surface and naturally air-drying the fruit.  Using sterile needles, wounds were created on the equatorial region of the passion fruit. After natural air-drying, 10 μl (10⁶ CFU/mL) of the B-spore suspension was inoculated into each wound. After natural drying, the fruit was stored at 25±1 °C and 90±5% relative humidity for 12 days in an incubator; the diameter of the lesion was measured every 2 days based on the cross-shape formula. A lesion closer to 1 cm was used to determine defence enzyme activity and the concentration of antifungal substances. The induction rate was calculated as follows" to "Select fresh and healthy passion fruits and divide them into two groups. The control group and the treatment group are immersed in distilled water without eugenol and MIC concentration of eugenol for 15 minutes, then air dry and set aside. After air drying, the two sets of fruits are treated using the same method as treatment I. The surface is disinfected with 75% ethanol and the fruits are naturally air dried. Using sterile needles, wounds were created on the equatorial region of the passion fruit. After natural air-drying, 10 μl (10⁶ CFU/mL) of the Lasiodiplodia theobromae spore suspension was inoculated into each wound. After natural drying, the fruit was stored at 25±1 °C and 90±5% relative humidity for 12 days in an incubator; the diameter of the lesion was measured every 2 days based on the cross-shape formula. A lesion closer to 1 cm was used to determine defence enzyme activity and the concentration of antifungal substances. The induction rate was calculated as follows" (Lines 234-245, Page 5-6)

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript entitled “Antifungal activity of eugenol against passion fruit rot pathogens and its induction of resistance to fruit rot in passion fruit” was reviewed. The work is interesting and was developed in sufficient depth to ensure that it makes an important contribution. Without detracting from the rigor of the study and its importance, some comments are made below that may help improve the manuscript.

    Introduction

I think that the problem statement is well stablished.

Materials and Methods

·       How was eugenol obtained?

·       Eugenol is not soluble in water, so its incorporation into the PDA medium had to be addressed with an emulsifier. How was the preparation of the emulsions carried out?

·       What were the characteristics of the eugenol stock solution?

·       What equipment was used to measure conductivity?

·       The treatment 1 of the evaluation of the effect of eugenol on the incidence of fruit rot and spot diameter in passion fruit, used packaging in polyethylene bags (see lines 215-216). How was the effect of the modified atmosphere created by packaging in polythene bags evaluated or ruled out?

·       Why treatment II did not use the same methodology?

·       The study of the effect of eugenol treatment on the shriveling of passion fruit used packaging in polyethylene bags. How was the effect of the modified atmosphere created by packaging in polythene bags evaluated or ruled out? In this case, packaging surely caused high relative humidity, thus, shriveling was masked.

·       Data analysis. It is stated that comparisons were made with the support of Duncan's test, but the corresponding statistical design was not explained. For the cases in which statistical tools were used, the comparison of results was made at certain times, but the analysis within each treatment over time seems to be lacking. In this sense, when declarations of increase or decrease of the value of some parameter are made, there is no corresponding endorsement. Without neglecting the fact that time is a random effects factor from the statistical point of view and that the analysis should have been conducted as a mixed model, at least time should have been considered as a variation factor.

Results and Discussion

·       In the study of the inhibitory effect of eugenol on the passion fruit rot pathogen, a statistical analysis over time within each treatment seems to be lacking (see Figure 3).

·       The same situation occurs with other variables (see Figures 5 and 6).

·       In Figure 7, the meaning of one or two asterisks is not clear.

 General

·       It is necessary to explain the meaning of the abbreviations the first time they are mentioned: MIC, PDA, MDA, PI, etc.

Author Response

Dear Editor and Reviewers,

First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. Once again, we appreciate for your warm work earnestly, and hope that this revised manuscript will be approved by the editors and reviewers. Notably, all my responses are marked in blue, the original contents are marked in green, and revised portions are marked in red. Please find my itemized responses below and my corrections in the “Manuscript File”.

Thanks again!

Responds to the Editor’s comments:

1.Comments: How was eugenol obtained?

Response: First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. Eugenol was purchased from Shanghai Yuanye Biological Co. We have revised the manuscript file to include the source of eugenol in the first citation, please check in the file. The specific modifications are in the reply below and the revised manuscript:

Lines 112: This sentence has been changed replacing "A stock solution of 10.00 mg/mL eugenol  was prepared and diluted in PDA to obtain solutions with concentrations of 0 (control), 0.05, 0.15, 0.30, 0.45, and 0.60 mg/mL." with "A solution of 10.00 mg/mL of eugenol was prepared by weighing 0.1 g of eugenol dissolved in 10 ml of anhydrous ethanol; A certain amount of 10.00 mg/mL of eugenol solution was aspirated in PDA medium to obtain PDA medium with concentrations of 0 (control), 0.05, 0.15, 0.30, 0.45 and 0.60 mg/mL, respectively." (Lines 122-126, Page 3)

2.Comments: Eugenol is not soluble in water, so its incorporation into the PDA medium had to be addressed with an emulsifier. How was the preparation of the emulsions carried out?

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. Our description of the problem with the preparation of the eugenol solution may not be accurate, so we have revised it in the newly submitted "manuscript document". Eugenol solution is dissolved and configured with anhydrous ethanol solution as follows: weigh 0.10 g of eugenol stock solution, dissolve in 10 ml of anhydrous ethanol solution and configure into eugenol solution with a concentration of 10 mg/mL. Then the corresponding eugenol solution was dissolved in PDA medium to make the medium containing 0 (control), 0.05, 0.15, 0.30, 0.45, and 0.60 mg/mL of eugenol concentration.

The specific modifications are in the reply below and the revised manuscript:

Lines 122-126: This sentence has been changed replacing "A stock solution of 10.00 mg/mL eugenol was prepared and diluted in PDA to obtain solutions with concentrations of 0 (control), 0.05, 0.15, 0.30, 0.45, and 0.60 mg/mL." with "A solution of 10.00 mg/mL of eugenol was prepared by weighing 0.1 g of eugenol dissolved in 10 ml of anhydrous ethanol; A certain amount of 10.00 mg/mL of eugenol solution was aspirated in PDA medium to obtain PDA medium with concentrations of 0 (control), 0.05, 0.15, 0.30, 0.45 and 0.60 mg/mL, respectively." (Lines 122-126, Page 3)

3.Comments: What were the characteristics of the eugenol stock solution?

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We will provide a brief explanation for the eugenol stock solution here: Eugenol stock solution is a highly concentrated solution of eugenol configured by directly weighing eugenol dissolved in anhydrous ethanol solution. The PDA medium containing eugenol and the distilled water containing eugenol for soaking passion fruit used later in the paper were both configured from eugenol stock solution.

4.Comments: What equipment was used to measure conductivity?

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We used the DDS-11A conductivity (Shanghai Yidian Scientific Instruments Co., Ltd.) meter to measure conductivity. We have made changes to the use of the instrument in the newly submitted document and marked it in red, please check it. The specific modifications are in the reply below and the revised manuscript:

Lines 179-181: This sentence has been changed replacing "To determine relative conductivity, the extracellular conductivity L/(μS/cm) of the shaking culture treated for 2, 4, 6, and 8 hours were first measured" with "To determine the relative conductivity, the extracellular conductivity L/(μS/cm) of shaken cultures treated for 2, 4, 6 and 8 h was first measured using a DDS-11A conductivity meter (Shanghai Raycom Xinjing Instruments Co., Ltd.)" (Lines 179-181, Page 4)

5.Comments: The treatment 1 of the evaluation of the effect of eugenol on the incidence of fruit rot and spot diameter in passion fruit, used packaging in polyethylene bags (see lines 215-216). How was the effect of the modified atmosphere created by packaging in polythene bags evaluated or ruled out?

Response: Thank you very much for your overall comprehensive and positive

comments on this manuscript, and thank you for giving us the chance to revise.

For this question, we have the following answer:

After pre-treatment of passion fruit in the laboratory directly in the fruit frame for storage, we found that the passion fruit would lose water and crumple rapidly, which affected the observation of fruit changes and quality. Therefore, we used polyethylene bags to reduce fruit wrinkling after treating passion fruit for subsequent observations and experiments.

For the effect of modified atmosphere produced by packing passion fruit with polyethylene bags, both the treatment and control groups were in the same nature of the bags and their gas environment was consistent.

6.Comments: Why treatment II did not use the same methodology?

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. For this question, we have the following answer:

Treatment I is to determine the inhibitory effect of eugenol of different mass concentrations on fruit rot of passion fruit after inoculation. We select the best concentration of eugenol to inhibit fruit rot by observing and calculating the inductive effect of eugenol on the pathogenic fungi of fruit rot.

The treatment II is to treat the Passion Fruit with the selected concentration of eugenol, and determine its impact on the subsequent quality and disease resistance of Passion Fruit, such as total phenol content, flavonoid content, and defense enzyme activity of Passion Fruit.

We used four concentrations (0, 1/2MIC, MIC, 3/2MIC) to screen out the MIC mass concentration of eugenol as the optimal concentration for treating fruit rot disease in Treatment I, and only used MIC concentration of eugenol as the treatment group in Treatment II.

Perhaps our statement is not very clear, and we have made changes in the newly submitted document. The specific modifications are in the reply below and the revised manuscript:

Lines 219-228: The method description in this paragraph has been more carefully rewritten, changing from "Treatment II: Passion fruit were rinsed in sterile water and MIC eugenol solution for 15 min before air-drying. The same method as Treatment I was conducted, using 75% ethanol to disinfect the surface and naturally air-drying the fruit.  Using sterile needles, wounds were created on the equatorial region of the passion fruit. After natural air-drying, 10 μl (10⁶ CFU/mL) of the B-spore suspension was inoculated into each wound. After natural drying, the fruit was stored at 25±1 °C and 90±5% relative humidity for 12 days in an incubator; the diameter of the lesion was measured every 2 days based on the cross-shape formula. A lesion closer to 1 cm was used to determine defence enzyme activity and the concentration of antifungal substances. The induction rate was calculated as follows" to "Select fresh and healthy passion fruits and divide them into two groups. The control group and the treatment group are immersed in distilled water without eugenol and MIC concentration of eugenol for 15 minutes, then air dry and set aside. After air drying, the two sets of fruits are treated using the same method as treatment I. The surface is disinfected with 75% ethanol and the fruits are naturally air dried. Using sterile needles, wounds were created on the equatorial region of the passion fruit. After natural air-drying, 10 μl (10⁶ CFU/mL) of the Lasiodiplodia theobromae spore suspension was inoculated into each wound. After natural drying, the fruit was stored at 25±1 °C and 90±5% relative humidity for 12 days in an incubator; the diameter of the lesion was measured every 2 days based on the cross-shape formula. A lesion closer to 1 cm was used to determine defence enzyme activity and the concentration of antifungal substances. The induction rate was calculated as follows" (Lines 231-242, Page 5-6)

7.Comments: The study of the effect of eugenol treatment on the shriveling of passion fruit used packaging in polyethylene bags. How was the effect of the modified atmosphere created by packaging in polythene bags evaluated or ruled out? In this case, packaging surely caused high relative humidity, thus, shriveling was masked.

Response: Thank you very much for your overall comprehensive and positive

comments on this manuscript, and thank you for giving us the chance to revise.

For this comment, we have the following response:

Previous pre-experiments found that passion fruit fruits would lose water and crumple rapidly when placed directly in the fruit frame, which affected the observation of fruit changes and quality. Therefore, we used polyethylene bags after processing passion fruit to slow down the fruit crumpling for subsequent observations and experiments.

For the effect of modified atmosphere produced by packing passion fruit with polyethylene bags, both treatment and control groups were in the same nature of the bags, and their gas environment was consistent and their effect on the gas environment The effects were consistent.

8.Comments: Data analysis. It is stated that comparisons were made with the support of Duncan's test, but the corresponding statistical design was not explained. For the cases in which statistical tools were used, the comparison of results was made at certain times, but the analysis within each treatment over time seems to be lacking. In this sense, when declarations of increase or decrease of the value of some parameter are made, there is no corresponding endorsement. Without neglecting the fact that time is a random effects factor from the statistical point of view and that the analysis should have been conducted as a mixed model, at least time should have been considered as a variation factor.

Response: Thank you very much for your overall comprehensive and positive

comments on this manuscript, and thank you for giving us the chance to revise.

For this comment, we have the following response:

We performed the comparison with the support of Duncan's test, using a statistical design of the effect of different treatment groups at the same time on experimental indicators, using in the text the same time period (e.g. 0, 2, 4, 6, 8h) for different treatments (0, 1/2 MIC, MIC, 3/2 MIC eugenol) on the same indicator. We used Duncan analysis to investigate the effects of different concentrations of eugenol treatment on the pathogenic fungi of passion fruit or fruit rot disease at the same time, which is a design that changes with eugenol treatment.

We have added analysis of pathogenic fungi and fruit detection indicators for different treatment groups at the same time in the conclusion analysis. The specific modifications are in the reply below and the revised manuscript:

Lines 385-390: We have rephrased this conclusion from "As shown in Figure 3, with an increase in eugenol concentration, the inhibitory effect of eugenol on the pathogenic fruit rot fungi increased. Furthermore, the inhibitory effect of 0.30 mg/mL eugenol was significantly higher than that of 0.15 mg/mL eugenol (p<0.05). Throughout the entire cultivation process, the inhibition rate of filamentous fungi with 0.30 mg/mL, 0.45 mg/mL, and 0.60 mg/mL eugenol was 100%." to "As shown in Figure 3, as the concentration of eugenol increases, the inhibitory effect of eugenol on pathogenic fruit rot fungi increases. After 4 days of treatment with different concentrations of eugenol, the diameter of the pathogen colony in the control group was 44.87mm, which was 1.73 times that of the pathogen colony in the 0.30 mg/mL eugenol treatment. Compared with the control group, the 0.30 mg/mL eugenol treatment significantly inhibited the diameter of the pathogen colony (p<0.05); After 6 days of treatment, the bacterial colony diameter of the control group was 72.64mm, while the bacterial colony diameter of the 0.30 mg/mL eugenol treatment was 37.13mm. The bacterial colony diameter of the control group was 1.95 times that of the 0.30 mg/mL eugenol treatment. Compared with the control group, the 0.30 mg/mL eugenol treatment significantly inhibited the bacterial colony diameter (p<0.05)(Figure 3B)" (Lines 407-416, Page 10).

Lines 391-394: We have rephrased this conclusion from "Furthermore, the inhibitory effect of 0.30 mg/mL eugenol was significantly higher than that of 0.15 mg/mL eugenol (p <0.05). Throughout the entire cultivation process, the inhibition rate of filamentous fungi with 0.30 mg/mL, 0.45 mg/mL, and 0.60 mg/mL eugenol was 100%." to "Furthermore, when treated with eugenol for 4 days, the inhibitory effect of 0.30 mg/mL eugenol on the growth of pathogenic fungi was 66.23%, while the inhibitory effects of 0.05 and 0.15 mg/mL eugenol on the growth of pathogenic fungi were 32.05%, 47.48%, the inhibitory effects of 0.30 mg/mL eugenol were significantly higher than those of 0.05 and 0.15 mg/mL eugenol (p<0.05) (Figure 3C).. Throughout the entire cultivation process, the inhibition rate of filamentous fungi with 0.30 mg/mL, 0.45 mg/mL, and 0.60 mg/mL eugenol was 100%." (Lines 417-421, Page 10).

Lines 424-428: We have rephrased this conclusion from "After 8 h, the relative permeability of L theobromae treated with 1/2 MIC, MIC, and 3/2 MIC of eugenol reached 71.64%, 76.67%, and 79.59%, respectively, while that of the control group reached 61.93% (Figure 5A)." to "After 8 hours, the relative permeability of L theobromae treated with 1/2 MIC, MIC and 3/2 MIC of eugenol reached 71.64%, 76.67% and 79.59%, respectively, while the relative permeability of the control group reached 61.93%, 1.15 times, 1.23 times and 1.28 times higher than that of the control group; Duncan's multi interval test results showed that eugenol treatment had a significant impact on the conductivity of pathogenic fungi (p<0.05) (Figure 5A)." (Lines 463-468, Page 12).

Lines 431-434: We have rephrased this conclusion from "As the treatment time increased, the difference between the nucleic acid release levels of the control and treated groups increased, with the control group exhibiting a gradual decrease in nucleic acid release levels compared to the treated group. " to "As the treatment time increased, the difference between the nucleic acid release levels of the control and treated groups increased, with the control group exhibiting a gradual decrease in nucleic acid release levels compared to the treated group.When treated for 6 and 8 hours, the nucleic acid release of pathogenic fungi treated with 0.30mg/mL eugenol was significantly higher than that of the control group (p<0.05)" (Lines 471-476, Page 13).

Lines 434-478: We have rephrased this conclusion from "Treatment with eugenol significantly increased the amount of protein released into the fungal cells. After 8 h treatment with 1/2 MIC, MIC, or 3/2 MIC eugenol, the released protein in the fungal strain B reached 42.73 mg/mL, 44.56 mg/mL, and 47.53 mg/mL, respectively, whereas that in the control group was 39.20 mg/mL." to "Treatment with eugenol significantly increased the amount of protein released into the fungal cells (p<0.05). After 8 h treatment with 1/2 MIC, MIC, or 3/2 MIC eugenol, the released protein in the fungal strain L theobromae reached 42.73 mg/mL, 44.56 mg/mL, and 47.53 mg/mL, respectively, whereas that in the control group was 39.20 mg/mL. They are 1.09 times, 1.13 times, and 1.16 times higher than the control group, respectively. Among them, compared with the control group, MIC or 3/2 MIC eugenol treatment had a significant impact on protein release(p<0.05). " (Lines 486-492, Page 13).

Lines 452-455: We have rephrased this conclusion from "After treatment with thymol at 3/2 MIC for 6 h, the release of soluble sugar from L theobromae pathogen rapidly increased to 0.43 mg/mL, while that of the control strain was maintained at 0.34 mg/mL. During the whole incubation period, the release of soluble sugar from the control strain was consistently lower than that of the treatment strain." to "After treatment with thymol at 3/2 MIC for 6 h, the release of soluble sugar from L theobromae pathogen rapidly increased to 0.43 mg/mL, while that of the control strain was maintained at 0.34 mg/mL. Compared with the control group, the 3/2 MIC eugenol treatment significantly increased the release of soluble sugars(p<0.05)." (Lines 497-500, Page 13).

Lines 458-460: We have rephrased this conclusion from "The MDA content in L theobromae pathogen in the 6 h, 3/2 MIC thymol treatment group increased rapidly to 1.19 mmol/L, while that in the control group was 0.64 mmol/L. " to "The MDA content in L theobromae pathogen in the 6 h, 3/2 MIC thymol treatment group increased rapidly to 1.19 mmol/L, while that in the control group was 0.64 mmol/L, which was three times higher than the control group. 3/2 MIC thymol treatment significantly increased the MDA content of pathogenic fungi (p<0.05)." (Lines 504-508, Page 13).

Lines 478-481: We have rephrased this conclusion from "On day 6, after inoculation, the surface of the fruits exhibited obvious white woolly fungal mycelia, and by day 8, the incidence of the lesion reached 92.85% (Figure 6A), with observations of the lesions indenting inward, rotten skin, and acidic odours. " to "On the 6th day after inoculation in the control group,the surface of the fruits exhibited obvious white woolly fungal mycelia, and by day 8, the incidence of the lesion reached 92.85% (Figure 6A), with observations of the lesions indenting inward, rotten skin, and acidic odours." (Lines 526-529, Page 14).

Lines 483-487: We have rephrased this conclusion from "The low concentration eugenol treatment groups (1/2 MIC and MIC) also exhibited infection 4 days after inoculation, although the infection rate and symptoms were evidently lower than those of the controls. In the 3/2 MIC eugenol treatment group, infection occurred on the sixth day; however, on day 6, the incidence of lesions was still 46.43% lower than that in the control group." to "The low concentration eugenol treatment groups (1/2 MIC and MIC) also exhibited infection 4 days after inoculation, although the infection rate and symptoms were evidently lower than those of the controls (p<0.05). In the 3/2 MIC eugenol treatment group, infection occurred on the sixth day; however, on day 6, the incidence of lesions was still 46.43% lower than that in the control group. Compared with the control group, the 3/2 MIC eugenol treatment group significantly inhibited the occurrence of diseases (p<0.05)." (Lines 532-537, Page 14).

Lines 495-499: We have rephrased this conclusion from "After passion fruit was soaked in eugenol, the incidence of crown rot decreased significantly; after eight days of treatment, the MIC and 3/2 MIC treatment groups possessed a fruit rot incidence of 60.71% and 42.86%, respectively, while the corresponding incidence in the control group reached up to 92.85% (Figure 6C)." to " After passion fruit was soaked in eugenol, the incidence of crown rot decreased significantly; after eight days of treatment, the MIC and 3/2 MIC treatment groups possessed a fruit rot incidence of 60.71% and 42.86%, respectively, while the corresponding incidence in the control group reached up to 92.85% (Figure 6C). Compared with the control group, MIC and 3/2 MIC eugenol treatments significantly reduced the occurrence of fruit rot (P<0.05)." (Lines 545-550, Page 14-15).

Lines 499-502 We have rephrased this conclusion from "In addition, after passion fruit was soaked in eugenol and inoculated with the pathogen, the lesion diameter decreased; on the 8th day, the lesion diameter of the 3/2 MIC treatment was 4.37 mm, while the lesion diameter of the control group was 33.03 mm (Figure 6B)" to "In addition, after passion fruit was soaked in eugenol and inoculated with the pathogen, the lesion diameter decreased; on the 8th day, the lesion diameter of the 3/2 MIC treatment was 4.37 mm, while the lesion diameter of the control group was 33.03 mm (Figure 6B);The lesion diameter of the control group was 7.5 times that of the 3/2 MIC eugenol treated group, and the 3/2 MIC eugenol treatment significantly inhibited fruit lesion (p<0.05). " (Lines 550-555, Page 15).

Lines 507-509: We have rephrased this conclusion from "The shrinkage index of the control group showed a rapid upward trend during the postharvest storage period; specifically, its shrinkage index was 2.33 on the 8th day of storage, which was 3.11 times that of the 3/2 MIC treatment group." to " The shrinkage index of the control group showed a rapid upward trend during the postharvest storage period; specifically, its shrinkage index was 2.33 on the 8th day of storage, which was 3.11 times that of the 3/2 MIC treatment group. Compared with the control group, 3/2 eugenol treatment can significantly reduce the shrinkage rate of fruits (p<0.05)." (Lines 506-564, Page 15).

9.Comments: In the study of the inhibitory effect of eugenol on the passion fruit rot pathogen, a statistical analysis over time within each treatment seems to be lacking (see Figure 3).

Response:  Thank you very much for your overall comprehensive and positive

comments on this manuscript, and thank you for giving us the chance to revise.

For this comment, we have the following response:

Figure 3A is to screen for the minimum inhibitory concentration (MIC) of eugenol against the pathogenic fungi of passion fruit rot. Therefore, different concentrations (0, 0.15, 0.30, 0.45, 0.60 mg/mL eugenol) were selected for 2 days to screen for MIC. Figure 3B shows the changes in the diameter of pathogenic fungal colonies with increasing cultivation time. We added a significance analysis between the effects of different concentrations of eugenol at the same time on the diameter of pathogenic fungal colonies; Figure 3C shows the inhibitory effect of different concentrations of eugenol on pathogenic fungi with the extension of cultivation time. We have added a significance analysis of the inhibitory effect of different concentrations of eugenol on pathogenic fungi at the same time, and highlighted it in red in the newly submitted "manuscript file".

So, in the whole Figure 3, we show the picture of screening MIC (Figure 3A), the change of pathogenic fungal colony diameter with incubation time (Figure 3-B) and the inhibition of pathogenic fungi by different concentrations of eugenol with incubation time (Figure 3C).

 The specific modifications are in the reply below and the revised manuscript:

Lines 385-390: We have rephrased this conclusion from "As shown in Figure 3, with an increase in eugenol concentration, the inhibitory effect of eugenol on the pathogenic fruit rot fungi increased. Furthermore, the inhibitory effect of 0.30 mg/mL eugenol was significantly higher than that of 0.15 mg/mL eugenol (p<0.05). Throughout the entire cultivation process, the inhibition rate of filamentous fungi with 0.30 mg/mL, 0.45 mg/mL, and 0.60 mg/mL eugenol was 100%." to "As shown in Figure 3, as the concentration of eugenol increases, the inhibitory effect of eugenol on pathogenic fruit rot fungi increases. After 4 days of treatment with different concentrations of eugenol, the diameter of the pathogen colony in the control group was 44.87mm, which was 1.73 times that of the pathogen colony in the 0.30 mg/mL eugenol treatment. Compared with the control group, the 0.30 mg/mL eugenol treatment significantly inhibited the diameter of the pathogen colony (p<0.05); After 6 days of treatment, the bacterial colony diameter of the control group was 72.64mm, while the bacterial colony diameter of the 0.30 mg/mL eugenol treatment was 37.13mm. The bacterial colony diameter of the control group was 1.95 times that of the 0.30 mg/mL eugenol treatment. Compared with the control group, the 0.30 mg/mL eugenol treatment significantly inhibited the bacterial colony diameter (p<0.05)(Figure 3B)" (Lines 407-416, Page 10).

Lines 391-394: We have rephrased this conclusion from "Furthermore, the inhibitory effect of 0.30 mg/mL eugenol was significantly higher than that of 0.15 mg/mL eugenol (p <0.05). Throughout the entire cultivation process, the inhibition rate of filamentous fungi with 0.30 mg/mL, 0.45 mg/mL, and 0.60 mg/mL eugenol was 100%." to "Furthermore, when treated with eugenol for 4 days, the inhibitory effect of 0.30 mg/mL eugenol on the growth of pathogenic fungi was 66.23%, while the inhibitory effects of 0.05 and 0.15 mg/mL eugenol on the growth of pathogenic fungi were 32.05%, 47.48%, the inhibitory effects of 0.30 mg/mL eugenol were significantly higher than those of 0.05 and 0.15 mg/mL eugenol (p<0.05) (Figure 3C).. Throughout the entire cultivation process, the inhibition rate of filamentous fungi with 0.30 mg/mL, 0.45 mg/mL, and 0.60 mg/mL eugenol was 100%." (Lines 417-421, Page 10).

10.Comments: The same situation occurs with other variables (see Figures 5 and 6).

Response:  Thank you very much for your overall comprehensive and positive

comments on this manuscript, and thank you for giving us the chance to revise.

For this comment, we have the following response:

In Figure 5, we used four concentrations of eugenol (0, 1/2MIC, MIC, 3/2MIC) to treat pathogenic fungi. We tested the effects of different concentrations on the conductivity, nucleic acid release, intracellular protein release, soluble sugar release, and MDA content of pathogenic fungi. Therefore, we used the same time period to analyze the effects of different concentrations of eugenol on pathogenic fungi.

Figure 6-A, B, C and D respectively show the effects of different concentrations of eugenol on the fruit rot of passion fruit, the spot diameter of fruit rot, the incidence rate of fruit rot, and the shrinkage of passion fruit. We analyze the effects of different concentrations of eugenol on the fruit rot of passion fruit at the same time.

The specific modifications are in the reply below and the revised manuscript:

Lines 424-428: We have rephrased this conclusion from "After 8 h, the relative permeability of L theobromae treated with 1/2 MIC, MIC, and 3/2 MIC of eugenol reached 71.64%, 76.67%, and 79.59%, respectively, while that of the control group reached 61.93% (Figure 5A)." to "After 8 hours, the relative permeability of L theobromae treated with 1/2 MIC, MIC and 3/2 MIC of eugenol reached 71.64%, 76.67% and 79.59%, respectively, while the relative permeability of the control group reached 61.93%, 1.15 times, 1.23 times and 1.28 times higher than that of the control group; Duncan's multi interval test results showed that eugenol treatment had a significant impact on the conductivity of pathogenic fungi (p<0.05) (Figure 5A)." (Lines 463-468, Page 12).

Lines 431-434: We have rephrased this conclusion from "As the treatment time increased, the difference between the nucleic acid release levels of the control and treated groups increased, with the control group exhibiting a gradual decrease in nucleic acid release levels compared to the treated group. " to "As the treatment time increased, the difference between the nucleic acid release levels of the control and treated groups increased, with the control group exhibiting a gradual decrease in nucleic acid release levels compared to the treated group.When treated for 6 and 8 hours, the nucleic acid release of pathogenic fungi treated with 0.30mg/mL eugenol was significantly higher than that of the control group (p<0.05)" (Lines 471-476, Page 13).

Lines 434-478: We have rephrased this conclusion from "Treatment with eugenol significantly increased the amount of protein released into the fungal cells. After 8 h treatment with 1/2 MIC, MIC, or 3/2 MIC eugenol, the released protein in the fungal strain B reached 42.73 mg/mL, 44.56 mg/mL, and 47.53 mg/mL, respectively, whereas that in the control group was 39.20 mg/mL." to "Treatment with eugenol significantly increased the amount of protein released into the fungal cells (p<0.05). After 8 h treatment with 1/2 MIC, MIC, or 3/2 MIC eugenol, the released protein in the fungal strain L theobromae reached 42.73 mg/mL, 44.56 mg/mL, and 47.53 mg/mL, respectively, whereas that in the control group was 39.20 mg/mL. They are 1.09 times, 1.13 times, and 1.16 times higher than the control group, respectively. Among them, compared with the control group, MIC or 3/2 MIC eugenol treatment had a significant impact on protein release(p<0.05). " (Lines 486-492, Page 13).

Lines 452-455: We have rephrased this conclusion from "After treatment with thymol at 3/2 MIC for 6 h, the release of soluble sugar from L theobromae pathogen rapidly increased to 0.43 mg/mL, while that of the control strain was maintained at 0.34 mg/mL. During the whole incubation period, the release of soluble sugar from the control strain was consistently lower than that of the treatment strain." to "After treatment with thymol at 3/2 MIC for 6 h, the release of soluble sugar from L theobromae pathogen rapidly increased to 0.43 mg/mL, while that of the control strain was maintained at 0.34 mg/mL. Compared with the control group, the 3/2 MIC eugenol treatment significantly increased the release of soluble sugars(p<0.05)." (Lines 497-500, Page 13).

Lines 458-460: We have rephrased this conclusion from "The MDA content in L theobromae pathogen in the 6 h, 3/2 MIC thymol treatment group increased rapidly to 1.19 mmol/L, while that in the control group was 0.64 mmol/L. " to "The MDA content in L theobromae pathogen in the 6 h, 3/2 MIC thymol treatment group increased rapidly to 1.19 mmol/L, while that in the control group was 0.64 mmol/L, which was three times higher than the control group. 3/2 MIC thymol treatment significantly increased the MDA content of pathogenic fungi (p<0.05)." (Lines 504-508, Page 13).

Lines 478-481: We have rephrased this conclusion from "On day 6, after inoculation, the surface of the fruits exhibited obvious white woolly fungal mycelia, and by day 8, the incidence of the lesion reached 92.85% (Figure 6A), with observations of the lesions indenting inward, rotten skin, and acidic odours. " to "On the 6th day after inoculation in the control group,the surface of the fruits exhibited obvious white woolly fungal mycelia, and by day 8, the incidence of the lesion reached 92.85% (Figure 6A), with observations of the lesions indenting inward, rotten skin, and acidic odours." (Lines 526-529, Page 14).

Lines 483-487: We have rephrased this conclusion from "The low concentration eugenol treatment groups (1/2 MIC and MIC) also exhibited infection 4 days after inoculation, although the infection rate and symptoms were evidently lower than those of the controls. In the 3/2 MIC eugenol treatment group, infection occurred on the sixth day; however, on day 6, the incidence of lesions was still 46.43% lower than that in the control group." to "The low concentration eugenol treatment groups (1/2 MIC and MIC) also exhibited infection 4 days after inoculation, although the infection rate and symptoms were evidently lower than those of the controls (p<0.05). In the 3/2 MIC eugenol treatment group, infection occurred on the sixth day; however, on day 6, the incidence of lesions was still 46.43% lower than that in the control group. Compared with the control group, the 3/2 MIC eugenol treatment group significantly inhibited the occurrence of diseases (p<0.05)." (Lines 532-537, Page 14).

Lines 495-499: We have rephrased this conclusion from "After passion fruit was soaked in eugenol, the incidence of crown rot decreased significantly; after eight days of treatment, the MIC and 3/2 MIC treatment groups possessed a fruit rot incidence of 60.71% and 42.86%, respectively, while the corresponding incidence in the control group reached up to 92.85% (Figure 6C)." to " After passion fruit was soaked in eugenol, the incidence of crown rot decreased significantly; after eight days of treatment, the MIC and 3/2 MIC treatment groups possessed a fruit rot incidence of 60.71% and 42.86%, respectively, while the corresponding incidence in the control group reached up to 92.85% (Figure 6C). Compared with the control group, MIC and 3/2 MIC eugenol treatments significantly reduced the occurrence of fruit rot (P<0.05)." (Lines 545-550, Page 14-15).

Lines 499-502 We have rephrased this conclusion from "In addition, after passion fruit was soaked in eugenol and inoculated with the pathogen, the lesion diameter decreased; on the 8th day, the lesion diameter of the 3/2 MIC treatment was 4.37 mm, while the lesion diameter of the control group was 33.03 mm (Figure 6B)" to "In addition, after passion fruit was soaked in eugenol and inoculated with the pathogen, the lesion diameter decreased; on the 8th day, the lesion diameter of the 3/2 MIC treatment was 4.37 mm, while the lesion diameter of the control group was 33.03 mm (Figure 6B);The lesion diameter of the control group was 7.5 times that of the 3/2 MIC eugenol treated group, and the 3/2 MIC eugenol treatment significantly inhibited fruit lesion (p<0.05). " (Lines 550-555, Page 15).

Lines 507-509: We have rephrased this conclusion from "The shrinkage index of the control group showed a rapid upward trend during the postharvest storage period; specifically, its shrinkage index was 2.33 on the 8th day of storage, which was 3.11 times that of the 3/2 MIC treatment group." to " The shrinkage index of the control group showed a rapid upward trend during the postharvest storage period; specifically, its shrinkage index was 2.33 on the 8th day of storage, which was 3.11 times that of the 3/2 MIC treatment group. Compared with the control group, 3/2 eugenol treatment can significantly reduce the shrinkage rate of fruits (p<0.05)." (Lines 506-564, Page 15).

11.Comments: In Figure 7, the meaning of one or two asterisks is not clear.

Response: Thank you very much for your overall comprehensive and positive

comments on this manuscript, and thank you for giving us the chance to revise.

For this comment, we have the following response:

The meaning of an asterisk is that there is a significant difference between different treatment groups at the same time (p<0.05). The meaning of two asterisks is that there is a very significant difference between different treatment groups at the same time (p<0.01)

12.Comments: It is necessary to explain the meaning of the abbreviations the first time they are mentioned: MIC, PDA, MDA, PI, etc.

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have made changes to the noun that first appeared, written its full name, and highlighted it in red in the submitted 'manuscript file'.

The specific modifications are as follows:

Lines 121-122 : This sentence provides a comprehensive explanation, replacing "Mahdi's method was used to determine the MIC " with "Mahdi's method was used to determine the minimal inhibitory concentration (MIC)" (Lines 121-122, Page 3)

Lines 79 : This sentence provides a comprehensive explanation, replacing "this tissue was used for pathogenic isolation, and was transferred to a PDA plate." with "The tissue was used for pathogen isolation and transferred to potato dextrose agar (PDA) plates." (Lines 79, Page 2)

Lines 171-173 : This sentence provides a comprehensive explanation, replacing "To measure cell membrane permeability, relative conductivity was measured according to Liu’s method [20], intracellular nucleic acid leakage was determined using Tao’s method [21], intracellular protein release and soluble sugar release were determined using Chen’s method [22], and MDA content was determined using Wei’s method [23]. " with "To measure cell membrane permeability, relative conductivity was measured according to Liu’s method [20], intracellular nucleic acid leakage was determined using Tao’s method [21], intracellular protein release and soluble sugar release were determined using Chen’s method [22], and malondialdehyde (MDA) content was determined using Wei’s method [23]." (Lines 171-173, Page 4)

Lines 212 : This sentence provides a comprehensive explanation, replacing "PI staining to Observe the Effect of Eugenol on the DNA of Passion Fruit Rot–Causing Fungi " with " Propidium iodide (PI) staining to Observe the Effect of Eugenol on the DNA of Passion Fruit Rot–Causing Fungi " (Line 212, Page 5)

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors, thanks a lot for your effort to enhance the manuscript, however further improvements are still required, and a check of English language and style is suggested. The detailed comments are listed below:

Lines 68-71. To make the period more easily understandable, I suggest this modification: "Therefore, this study investigated the effects of different concentrations of eugenol on Lasiodiplodia theobromae. Mycelial growth, cell membrane, spore germination, and fruit rot of passion fruit; additionally, the potential mechanism by which eugenol inhibits the fruit rot pathogen and its effect on fruit rot resistance were established."

Line 82. Please rewrite the following sentence in the passive form "We used a total of 50 PDA plates for the isolation and 82 purification of different pathogens".

Lines 90-95. Please rewrite the sentence in a more suitable passive form.

Lines 161-165. Please rewrite the sentence in the passive form.

Line 218. Please rewrite the sentence in the passive form.

Lines 237-241. Please rewrite the period in a more suitable passive form.

Lines 365-366. I am not sure if this sentence is the result of a mistranslation from another language, but the pathogen studied in this paper is a fungus, so it is incorrect to refer to bacteria. Rewrite the sentence in an appropriate passive form and correctly. Also, check the entire text carefully because there are similar errors.

Line 286. Pathogenic bacteria is not correct!

Lines 413 and 415 Bacterial colony is not correct!

Author Response

Dear Editor and Reviewers,

First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. Once again, we appreciate for your warm work earnestly, and hope that this revised manuscript will be approved by the editors and reviewers. Notably, all my responses are marked in blue, the original contents are marked in green, and the modified part is represented in red and highlighted in yellow. Please find my itemized responses below and my corrections in the “Manuscript File”. We sincerely hope that you find our responses and modifications satisfactory and that the manuscript is now acceptable for publication.

Thanks again!

Responds to the Editor’s comments:

1.Comments: Lines 68-71. To make the period more easily understandable, I suggest this modification: "Therefore, this study investigated the effects of different concentrations of eugenol on Lasiodiplodia theobromae. Mycelial growth, cell membrane, spore germination, and fruit rot of passion fruit; additionally, the potential mechanism by which eugenol inhibits the fruit rot pathogen and its effect on fruit rot resistance were established."

Response: First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. We have revised the manuscript file and marked it in red. Please review the file. The specific modifications are in the reply below and the revised manuscript:

We have modified the original inappropriate expression from "Therefore, this study investigated the effects of different concentrations of eugenol on mycelial growth, cell membrane, spore germination, and fruit rot of passion fruit; additionally, the potential mechanism by which eugenol inhibits the fruit rot pathogen and its effect on fruit rot resistance were established." to "Therefore, this study investigated the effects of different concentrations of eugenol on Lasiodiplodia theobromae. Mycelial growth, cell membrane, spore germination, and fruit rot of passion fruit; additionally, the potential mechanism by which eugenol inhibits the fruit rot pathogen and its effect on fruit rot resistance were established." (Lines 67-70, Page 2)

2.Comments:Line 82. Please rewrite the following sentence in the passive form "We used a total of 50 PDA plates for the isolation and 82 purification of different pathogens".

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. We have made modifications to the inappropriate descriptions in the manuscript file and highlighted them in red. The specific modifications are in the reply below and the revised manuscript:

We have modified the original inappropriate expression from "We used a total of 50 PDA plates for the isolation and purification of different pathogens." to "50 PDA plates were used for isolation and purification of different pathogenic fungi." (Lines 80-81, Page 2)

3.Comments:Lines 90-95. Please rewrite the sentence in a more suitable passive form.

Response: First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. We have made modifications to the inappropriate descriptions in the newly submitted 'manuscript file'.The specific modifications are in the reply below and the revised manuscript:

We have modified the original inappropriate expression from "Take ninety fresh healthy passion fruit, scrub the surface with 75% alcohol to disinfect, rinse the alcohol with distilled water and dry, traumatize the center of the passion fruit surface with a hole punch, receive the pathogenic bacteriophage cake to the skin trauma site and make the side with mycelium touch the fruit wound part;" to "Ninety fresh and healthy passion fruits were used in the experiment. The surface was wiped with 75% alcohol for disinfection, washed with distilled water for alcohol, dried, and the center of the passion fruit surface was punctured with a double punch. Pathogenic fungal cake was inoculated to the center of the passion fruit surface damage area, allowing the mycelium side to contact the wound area of the passion fruit;" (Lines 88-93, Page 2)

4.Comments:Lines 161-165. Please rewrite the sentence in the passive form.

Response: First, thank you very much for your overall approval and comprehensive comments on this manuscript. We have made modifications to the inappropriate descriptions in the newly submitted 'manuscript file'.The specific modifications are in the reply below and the revised manuscript:

We have modified the original inappropriate expression from "Filter the obtained solution through four layers of gauze to remove hyphae. Centrifuge the filtrate at 10000 r/min for 10 minutes to remove precipitates. Resuspend the spores of fruit rot pathogens in a phosphate buffer salt (PBS) solution. Use a blood cell counting plate to adjust the spore concentration to 10⁶ CFU/mL, and prepare a spore suspension for later use." to "Four layers of gauze are used to filter the solution to remove hyphae and obtain the filtrate. The filtrate is used to centrifuge at 10000r/min for 10 minutes to remove sediment. Resuspended the spores of the pathogenic fungi causing passion fruit rot in a phosphate buffered salt (PBS) solution. The blood cell counting board was used to regulate the spore concentration to 10⁶CFU/mL and prepare spore suspensions for future use." (Lines 159-164, Page 4)

5.Comments:Line 218. Please rewrite the sentence in the passive form.

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. We have rewritten the sentences in the newly submitted 'manuscript file' and marked them in red. The specific modifications are in the reply below and the revised manuscript:

We have changed the sentence from ''Collect the cultured mycelium, dry it, and store it for future use.'' to ''Collect, dry, and store the cultivated fungal hyphae for future use''.(Lines 215, Page 5)

6.Comments:Lines 237-241. Please rewrite the period in a more suitable passive form.

Response: We have rewritten the sentences in the newly submitted 'manuscript file' and marked them in red. The specific modifications are in the reply below and the revised manuscript:

We have changed the sentence from ''Select fresh and healthy passion fruits and divide them into two groups. The control group and the treatment group are immersed in distilled water without eugenol and MIC concentration of eugenol for 15 minutes, then air dry and set aside. After air drying, the two sets of fruits are treated using the same method as treatment I. The surface is disinfected with 75% ethanol and the fruits are naturally air dried. Using sterile needles, wounds were created on the equatorial region of the passion fruit. After natural air-drying, 10 μl (106 CFU/mL) of the Lasiodiplodia theobromae spore suspension was inoculated into each wound. After natural drying, the fruit was stored at 25±1 °C and 90±5% relative humidity for 12 days in an incubator;'' to ''Fresh and healthy passion fruit was used in the experiment and divided into two groups. The control group and the treatment group are immersed in distilled water without eugenol and MIC concentration of eugenol for 15 minutes, then air dry and set aside. After air drying, the two sets of fruits are treated using the same method as treatment I. Passion fruit surfaces were disinfected with 75% ethanol, and sterile inoculation needles were used to create wounds in the equatorial region of the passion fruit. After natural drying, 10 μl (106 CFU/mL) of the Lasiodiplodia theobromae spore suspension was inoculated into each wound. After natural drying, the fruits were stored in an incubator at 25 ± 1°C and 90 ± 5% relative humidity for 12 days;''.(Lines 234-242, Page 5-6)

7.Comments:Lines 365-366. I am not sure if this sentence is the result of a mistranslation from another language, but the pathogen studied in this paper is a fungus, so it is incorrect to refer to bacteria. Rewrite the sentence in an appropriate passive form and correctly. Also, check the entire text carefully because there are similar errors.

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. It is inappropriate for us to use bacterial cake as a description here, and we have made modifications in the newly submitted 'manuscript file'. The specific modifications are in the reply below and the revised manuscript:

We have changed the sentence from ''Inoculate blank PDA bacterial cake as a control, and inoculate PDA bacterial cake with purified protofungal bacteria onto healthy fruits as the treatment group;'' to ''Inoculate blank PDA pathogenic fungal cake as the control, and inoculate PDA fungal cake containing purified pathogenic true fungi onto healthy fruits as the treatment group;''.(Lines 361-364, Page 8)

We have changed the sentence from ''However, the mechanism by which eugenol inhibits the growth of the Pathogen that causes passion fruit rot remains unclear; ultimately, this greatly limits the practical application of eugenol'' to ''However, the mechanism by which eugenol inhibits the growth of the Pathogenic fungi that causes passion fruit rot remains unclear; ultimately, this greatly limits the practical application of eugenol.''.(Lines 63-64, Page 2)

We have changed the sentence from ''Isolation and Identification of Pathogenic Bacteria and Detection of Pathogenicity;'' to ''Isolation and Identification of Pathogenic Fungi and Detection of Pathogenicity''.(Lines 74, Page 2)

8.Comments:Line 286. Pathogenic bacteria is not correct!

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. It is inappropriate for us to use Pathogenic bacteria for description here. We have made modifications in the newly submitted 'manuscript file'. The specific modifications are in the reply below and the revised manuscript:

We have changed the sentence from ''Identification of The Pathogenic Bacteria of Fruit rot of Passion Fruit'' to ''Identification of Pathogenic Fungi of Passion Fruit Rot Disease''.(Lines 383, Page 9)

9.Comments:Lines 413 and 415 Bacterial colony is not correct!

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. It is inappropriate for us to use the 'Bacterial Colony' as a description here. We have made modifications in the newly submitted 'manuscript file'.. The specific modifications are in the reply below and the revised manuscript:

We have changed the sentence from '' After 4 days of treatment with different concentrations of eugenol, the diameter of the pathogen colony in the control group was 44.87mm, which was 1.73 times that of the pathogen colony in the 0.30 mg/mL eugenol treatment. Compared with the control group, the 0.30 mg/mL eugenol treatment significantly inhibited the diameter of the pathogen colony (p<0.05); After 6 days of treatment, the bacterial colony diameter of the control group was 72.64mm, while the bacterial colony diameter of the 0.30 mg/mL eugenol treatment was 37.13mm. The bacterial colony diameter of the control group was 1.95 times that of the 0.30 mg/mL eugenol treatment. Compared with the control group, the 0.30 mg/mL eugenol treatment significantly inhibited the bacterial colony diameter (p<0.05)'' to '' After 4 days of treatment with different concentrations of eugenol, the pathogenic fungal colony diameter of the control group was 44.87 mm, which was 1.73 times larger than that of the pathogenic fungal colony diameter of 0.30 mg/mL eugenol treatment. Compared with the control group, 0.30 mg/mL eugenol treatment had a significant inhibitory effect on the pathogenic fungal colony diameter (P<0.05); after 6 days of treatment, the pathogenic fungal colony diameter of the control group was 72.64 mm, while that of the 0.30 mg/mL eugenol treatment was 37.13 mm. the pathogenic fungal colony diameter of the control group was 0.30 mg/mL 1.95 times of the eugenol treatment. The 0.30 mg/mL eugenol treatment significantly inhibited the pathogenic fungal colony diameter compared to the control group (P<0.05).''(Lines 407-416, Page 10)

Author Response File: Author Response.docx