Review Reports - Fungicidal Effect of Guava Wood Vinegar against <i>Colletotrichum coccodes</i> Causing Black Dot Disease of Potatoes

Round 1

Reviewer 1 Report

I think the article provides a new use for wood vinegar on a new species of fungus and confirms its antifungal capacity. However, I believe that more information should be given about the results, the replicates of each experiment and the deviations from the means. Also some clarification on the composition of the wood vinegar, since when working with areas of the chromatogram, it does not mean that they correspond directly with the real concentrations in the wood vinegar. In the pdf I do some appreciations and comments.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer

We would like to thank the reviewers for reviewing the manuscript and highlighting some important points. Thanks for your suggestion we have formatted the article according to Journal format All comments in PDF fill were covered here you can see it with different color

Reviewer I

I suggest that you review this work, where the chemical composition of the product is specified: http://dx.doi.org/10.3390/en1310241

Response: Thank you for your suggestion but this link is not active: http://dx.doi.org/10.3390/en1310241

I dont agree, why is a recycled substance?

Response: Wood vinegar is a compound produced during the charcoal production process and is not a recycled material.

More modern sources are the last paper recommended and in this one: DOI: 10.1002/fes3.253

Response: We added the reference in the search and used it in the work.

I cannot guarantee, since I cannot access that work, only its abstract, that this study says this. Please reference studies that speak of the safety of this product, if possible in an open format

Response: This compound is safe for the environment, and there are references that mention it, such as:

Aguirre J.L.; Baena, J.; Martin, M.T.; Nozal, L.; Gonzalez, S.; Manjon, J.L.; Peinado, M. Composition, ageing and herbicidal properties of wood vinegar obtained through fast biomass pyrolysis. Energies 2020, 13, 2418.https://doi.org/10.3390/en13102418.

It is necessary a more detailed explanation of the obtaining of wood vinegar, which part of the plants were used, obtaining temperatures or mechanism of condensation of the product.

Response: Wood is the plant part of the guava tree used in this study.

Response: Source of guava wood vinegar

WV is a secondary product produced when smoke from charcoal production is cooled by outside air while passing through a chimney or flue pipe. The cooling effect causes condensation of WV, particularly when the temperature of the smoke produced by carbonization ranges between 80 and 180 oC. The guava WV used in this study was derived from the charcoal production of guava trees wood (Psidium guajava) in Egypt's El-Kalyobia governorate. The obtained guava WV was stored at room temperature for further studies.

In all the results it is necessary to indicate the exact number of replicates obtained and indicate the standard deviations or errors in all the tables or graphs. It is the only way to see if the results are statistically robust

Response: All experiments were carried out in three replicates and all data are presented as mean ± standard deviation (SD).

The area percentage does not have to reflect the amount of the molecule, since different molecules respond with different intensity to the GCMS methodology. This is important to indicate, and results from other studies that relate area and quantification can be compared. For example, Table 4 of this study DOI: 10.3390/en13102418

Response: Paragraph has been modified

The main compounds present in guava WV were quantified using GC-MS. Data in Figure 2 show that the chromatogram obtained shows the major peaks corresponding to the more abundant compounds present in the sample. The most abundant compound (16.12%) was identified as phenol, 2,6-dimethoxy-, which was associated with peak 3, followed by phenol, 2-methoxy- (12.82%) associated with peak 1 (Fig. 2), and 1,2,4-trimethoxybenzene (6.39%) associated with peak 4. Table 1 describes the overall chemical composition, retention times of each compound, and percentages of each compound present in guava WV. The presence of several bioactive compounds in guava WV, including phenols, esters, alcohols, antioxidants, and organic acids (Table 1 and Figure 3). GC-MS analyses revealed the presence in guava WV of phenols, esters, organic acids, antioxidants and alcohols.

How can it be that there is no acetic acid in the majority composition?, if precisely the compound is called wood vinegar due to the presence of that compound. Is there any other example of the absence of wood vinegar in the major compounds in the bibliography? Further information about wood vinegar compounds is needed. A more comprehensive list of the most abundant compounds, grouped into families, is reccomended. Several important compounds such as acetic acid and light oxygenates are missing."

Response: There are some references that indicate that wood vinegar does not necessarily have acetic acid for example, Molecules 2016, 21(9), 1150; https://doi.org/10.3390/molecules21091150

Include standar deviation or standar error

Response: data are presented as mean ± standard deviation (SD).

Include number of replicates and standar deviation or standar error

Response: all experiments were carried out in three replicates

Include number of plants , standar error or standar deviation

why??

Response: all experiments were carried out in three replicates and data are presented as mean ± standard deviation (SD). It has been modified in the article

Its not abundance, its area of the chromatogram

Response: It has been modified in the article

This sentence is better in the next paragraph

Response: this sentence was transferred in the next Paragraph

This cannot be indicated unless it is justified with publications that refer to the product and have studied its effects on the environment.

Response: A reference was used to prove this

Yatagai, M.; Nishimoto, M.; Hori, K.; Ohira, T.; Shibata, A. Termiticidal activity of wood vinegar, its components and their homologues. J. Wood Sci. 2002, 48, 338–342. https://doi.org/10.1007/BF0083135

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors

I read with pleasure the manuscript entitled: Fungicidal effect of wood vinegar on Colletotrichum coccodes

which causes black spot disease in potato'. For me the article is good, although it needs to be edited in some parts, the comments are in the PDF, I kindly ask the authors to respond point by point to each comment in the text. The introduction needs to be fixed, while the results and discussions are really well done. I suggest adding the recommended references because they are the latest published work on this product. All references in the text and in the references section need to be edited following the style of the journal horticulturae.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Dear Reviewer

I would like to thank the reviewers for reviewing the manuscript and highlighting some. All comments in PDF fill were covered here you can see it with different color

Dear Authors

I read with pleasure the manuscript entitled: Fungicidal effect of wood vinegar on Colletotrichum coccodes which causes black spot disease in potato'. For me the article is good, although it needs to be edited in some parts, the comments are in the PDF, I kindly ask the authors to respond point by point to each comment in the text. The introduction needs to be fixed, while the results and discussions are really well done. I suggest adding the recommended references because they are the latest published work on this product. All references in the text and in the references section need to be edited following the style of the journal horticulturae.

please follow the journal guidelines, indicating with the number

Response: Thank you for the suggestions; we formatted the article according to journal format and sent it in English; the certificate can be found in the supplement material. All comments in the PDF file were covered here, and you can see them in different colour.

add the corresponding author

Response:* Correspondence: ka@kau.edu.sa or kaaboelyousr@agr.au.edu.eg

please add a reference

all the references in the text have to be mentioned like [1], following the journal guidelines, please change everywhere

Response: all references have been formatted according to the journal format.

i suggest the authors to connect this two sentences

for living organisms the authors means animal? i suggest to extend this part including also arable plants that are sprayed during the application on the crops.

Response: It is a low-cost, all-natural product that has no negative effects on living organisms or the environment [18]. It has been demonstrated to suppress a number of fungal plant diseases [19].

add this reference:

https://doi.org/10.3390/d14080669

Response: Fanfarillo, E.; Fedeli, R.; Fiaschi, T.; de Simone, L.; Vannini, A.; Angiolini, C.; Loppi, S.; Maccherini, S. Effects of wood distillate on seedling emergence and first-stage growth in five threatened arable plants. Diversity 2022, 14, 669. https://doi.org/10.3390/d14080669

please indicate the coordinates

Response: El-Beheira Governorate, Egypt; 30° 91125″ N, 29° 97119″ E.

What the authors mean "when required"??

Response: were irrigated when required= when it was needed

please indicate the characteristics of the extracion procedure

Response: WV is a secondary product produced when smoke from charcoal production is cooled by outside air while passing through a chimney or flue pipe. The cooling effect causes condensation of WV, particularly when the temperature of the smoke produced by carbonization ranges between 80 and 180 oC. The guava WV used in this study was derived from the charcoal production of guava trees wood (Psidium guajava L.) in Egypt's El-Kalyobia governorate. The obtained guava WV was stored at room temperature for further studies.

This paragraph is not a result, it's just an alysisi of the WV. for me this part have to stay in the M&M because this is not the aim of the work but just the analysis of the WV used

Response: this paragraph was removed from the results and added to the M&M

this is mean? Median? where are the error associated?

Response: all experiments were carried out in three replicates and data are presented as mean ± standard deviation (SD). It has been modified in the article

legnht? i whink the author make a mistake, reformulate all the sentence

reformulate

Response: It was modified to plant height.

follow the journal guidelines

Response: the journal format was followed to coordinate the research and all references have been formatted according to the journal format.

Author Response File: Author Response.docx

Reviewer 3 Report

If I correctly read, the manuscript ID horticulturae-2355568 “Fungicidal effect of wood vinegar on Colletotrichum coccodes causing black dot disease in potato” reports on the application of Wood Vinegar (WV) from guava (Psidium guajava) to control, both in vitro and under greenhouse conditions, Colletotrichum coccodes, the aetiological agent of potato black dot disease. The in vitro mycelial growth of C. coccodes was completely inhibited by guava WV at 3% (v/v). The presence of guava WV in the PDA medium at 0.25, 0.50, 1, 2, and 3% (v/v), significantly (P ≤ 0.05) reduced the C. coccodes mycelial growth in the range of 40-70% compared to the control. In greenhouse experiments, guava WV applied as foliar spray treatment considerably reduced the severity of potato black dot disease, including stem colonization, root surface covered with sclerotia, and wilted plants %, in both, 2021 and 2022, potato growing seasons. Guava WV also increased plant height, stem diameter and tuber yield of treated potato plants. Gas chromatography-mass spectrometry (GC-MS) analyses revealed the presence in guava WV of phenols, esters, organic acids, antioxidants and alcohols, e.g., 2-methoxyphenol, 2-Methoxy-5-methylphenol, 2,6-dimethoxyphenol, 1,2,4-Trimethoxybenzene, 2-phenyl-1,3-dioxan-5-yl-ester-9,12,15-Octadecatrienoic acid, and Ethyl iso-allocholate. This by-product of charcoal production is considered one of the most promising alternatives to synthetic pesticide and fertilizer applications, especially for organic production.

Notwithstanding the scientific sound of this work, the presentation in the form of a manuscript is confusing, unclear, repetitive, and in some parts unreadable and requires adjustments.

The title is not fully pertinent to the topics of the manuscript. A suggestion: “Fungicidal effect of guava wood vinegar against Colletotrichum coccodes causing black dot disease of potato”.

Follow the journal template to re-write the whole manuscript, including line numeration to make possible point-by-point efficient suggestions.

Incorporate all the sections provided by the template, including “Conclusions”, “Author Contributions”, “Funding”, “Data Availability Statement”, “Acknowledgments”, and “Conflicts of Interest” if necessary.

Also, “references must be numbered in order of appearance in the text (including citations in tables and legends) and listed individually at the end of the manuscript. Include the digital object identifier (DOI) for all references where available”.

The abstract is a very important part of the manuscript; I suggest rewriting it.

The keyword descriptors are not fully representative of the manuscript. For instance, insert the name of guava WV components.

Introduction

The Introduction section is pertinent, but it is not clear. Some concepts are repeated. Eliminate unnecessary repetitions. Re-write.

It is recommended to insert the Latin binomial of the species at the first mention in the abstract and introduction sections. Insert “Psidium guajava” at the first mention of guava in the abstract and introduction.

Use “2009-2010 potato growing season” instead of “2009/2010”.

What does “daughter” mean?

What does “WV” mean? Is it referring to all wood vinegar or guava wood vinegar?

What does “synthetic fungicides” mean? Is it referring to all synthetic fungicides?

What does “opposed” mean?

What does “prohibited” mean? I suggest “revoked”.

What does “giving plants” mean?

What does “infections” mean?

The hypothesis of the work is not clearly defined. The objectives are not clear and concrete.

Materials and methods

This section is not clear, and difficult to read. Improve.

Use: “2.1. Isolation and identification of pathogen”

What does “acquired” mean?

Where “pure cultures sub-cultured slants at 4°C”? Do Colletotrichum coccodes grow well at 4°C? I suggest: “Pure cultures were sub-cultured on PDA slants and stored at 4°C until use.”.

Defined soil volumes used in the 30 cm in diameter pots.

Associate a year to 1^st February.

Is “20/20/20” a mixture of commercial fertilizer or a specific product?

Section “2.2.3. Inoculation of plants” is not clear. Re-write.

Insert the diameter of the discs used as inoculum.

How was the conidia suspension used?

What does “injected” mean?

What does “:” mean at the end of “2.3. subtitle”? Delete.

The section “Disease assessment” is not clear. Re-write.

Verify DSI calculation. The correct formula should be: DSI (%) = 100 × [2× (0 or 1) + 6× (0 or 1) +10× (0 or 1) + 14× (0 or 1)]/32

Explain the significance of the number in the DSI formula.

What does “:” mean at the end of “2.4. subtitle”? Delete.

Improve section “2.4. Source of wood vinegar from guava trees”.

What does “:” mean at the end of “2.5. subtitle”? Delete.

Improve section “2.5. Analysis of the chemical composition of guava wood vinegar by GC–MS”.

What does “:” mean at the end of “2.6. subtitle”? Delete.

Improve section “2.6. Antifungal activity of guava WV against C. coccodes in vitro”.

Use the formula “R = [(C –B)/C] × 100”.

What does “:” mean at the end of “2.7. subtitle”? Delete.

Improve section “2.8. Statistical analysis”.

This section reports that means were compared using the least significant difference (LSD). Data in Figure 4, Table 2 and Table 3 show the significance by the Duncan test. Please uniform.

The Materials and Methods section lacks information on the effects of guava wood vinegar on plant growth characteristics and tuber yield.

A possible arrangement of the Materials and Methods section:

2.1. Fungal isolation and identification

2.2. Pathogenicity test

2.2.1. Plant material

2.2.2. Inoculum preparation

2.2.3. Inoculation of plants

2.2.4. Disease assessment

2.3. Antifungal activity

2.3.1. Guava wood vinegar: production and chemical composition

2.3.2. Plate assays

2.3.3 Greenhouse experiments

2.4. Effects of guava wood vinegar on plant growth and tuber yield

2.5. Statistical analysis

Results

The results are presented confusingly. Usually, the result section provides a concise and precise description of the experimental results. Avoid information pertinent to material and methods.

Follow the proposed Materials and Methods subsubsections to rewrite the results.

Pay attention to subsections numeration. Two “3.5.” sections are present. As well as two “Figure 2”

Figures and tables must be self-explanatory, clear, and easy to understand without needing any extra explanation.

Label the figure on page 5 as Figure 1. Rewrite the legend. I propose the picture sequence:

Symptoms of black dot disease on naturally infected potato tuber,

Colony morphology of Colletotrichum coccodes on Potato Dextrose Agar (PDA) medium,

C. coccodes conidiomata on PDA medium;

Conidia formed on PDA (insert a magnification bar).

Section 3.2. is not clear. Rewrite.

Section 3.3. is not clear. Rewrite.

Table 1: verify the correct IUPAC name of the listed compound. Avoid the division between two pages.

Figure 2:

Enlarge following the “Horticulturae-template”.

What does “numbers” mean? Gas chromatogram peaks are associated with the Retention Time.

You define 6 peaks only. And the other?

The legend of Figure 2 is confusing. Rewrite.

Figure 3:

If it is possible, insert the formula of the other compound listed in Table 1.

Section 3.4. is not clear. Rewrite.

Figure 4:

use “Growth inhibition (%)” instead of “% of growth inhibition”

delete all the “%” presents in abscissa.

use “guava wood vinegar concentration (% v/v)” instead of “WV concentration (v/v)”.

The legend of Figure 4 is confusing. Rewrite.

Figure 5: enlarge the picture.

Section “3.5. Effects of guava WV on potato black dot disease in greenhouse conditions during the 2021 and 2022 growing seasons” is not clear. Rewrite.

Table 2

What does “DI % = Disease incidence percentage.” mean?

Table 2 title: use “Table 2. Disease severity index (%) referred to stem colonization (SC), root covered with sclerotia (RCS), and wilt of potato plants inoculated with Colletotrichum coccodes and exposed to different treatments under greenhouse conditions during 2021 and 2022 growing seasons”.

Insert data standard deviation or standard error.

Use “Disease severity index (%)*” instead of “Disease severity index (%)”

Use “Inoculate and untreated control” instead of “Infected control”.

Delete “SC %*= Stem colonization %, RCS %**= Root covering with sclerotia %. Values in a column followed by the same letter do not differ significantly according to Duncan test (p ≤ 0.5). DI % = Disease incidence percentage.”

Use “*Values are the mean of ??? replicates ± ??. In each column, data followed by the same letter do not differ significantly according to the Duncan test (p ≤ 0.5).”

Section “3.5. Effect of WV treatment on plant height, stem diameter and tuber yield of potato under greenhouse conditions” is not clear. Rewrite.

Table 3

Table 3 title: use “Table 3. Effect of different treatments on plant height, stem diameter and tuber yield/plant of potato plants cropped under greenhouse conditions during 2021 and 2022 growing seasons.*”.

Use a capital letter for “plant”.

Insert standard deviation or standard error of data.

Table 3 title report “tuber yield/plant”, while in the table is present “tuber yield/pot”. Uniform.

What does “gm” mean?

What does “Infected control” mean?

What does “Healthy control” mean?

Use “*Data are the mean of ??? replicates ± ??. In each column, data followed by the same letter do not differ significantly according to Duncan test (p ≤ 0.5).”

The legend of Figure 6 is not clear. Re-write.

Discussion

This section is presented in a confusing manner. Improve.

Authors should discuss the results and how they can be interpreted from the perspective of previous studies and the working hypotheses. The findings and their implications should be discussed in the broadest context possible. Future research directions may also be highlighted.

Kind regards

Author Response

Reviewer 3

I would like to thank the reviewers for reviewing the manuscript and highlighting some important points thanks for your suggestion we have formatted the article according to Journal format.

If I correctly read, the manuscript ID horticulturae-2355568

“Fungicidal effect of wood vinegar on Colletotrichum coccodes causing black dot disease in potato”

reports on the application of Wood Vinegar (WV) from guava (Psidium guajava) to control, both in vitro and under greenhouse conditions, Colletotrichum coccodes, the aetiological agent of potato black dot disease. The in vitro mycelial growth of C. coccodes was completely inhibited by guava WV at 3% (v/v). The presence of guava WV in the PDA medium at 0.25, 0.50, 1, 2, and 3% (v/v), significantly (P ≤ 0.05) reduced the C. coccodes mycelial growth in the range of 40-70% compared to the control. In greenhouse experiments, guava WV applied as foliar spray treatment considerably reduced the severity of potato black dot disease, including stem colonization, root surface covered with sclerotia, and wilted plants %, in both, 2021 and 2022, potato growing seasons. Guava WV also increased plant height, stem diameter and tuber yield of treated potato plants. Gas chromatography-mass spectrometry (GC-MS) analyses revealed the presence in guava WV of phenols, esters, organic acids, antioxidants and alcohols, e.g., 2-methoxyphenol, 2-Methoxy-5-methylphenol, 2,6-dimethoxyphenol, 1,2,4-Trimethoxybenzene, 2-phenyl-1,3-dioxan-5-yl-ester-9,12,15-Octadecatrienoic acid, and Ethyl iso-allocholate. This by-product of charcoal production is considered one of the most promising alternatives to synthetic pesticide and fertilizer applications, especially for organic production.

Notwithstanding the scientific sound of this work, the presentation in the form of a manuscript is confusing, unclear, repetitive, and in some parts unreadable and requires adjustments.

Response: Thank you for the suggestions; we formatted the article according to journal format and sent it in English.

The title is not fully pertinent to the topics of the manuscript. A suggestion: “Fungicidal effect of guava wood vinegar against Colletotrichum coccodes causing black dot disease of potato”.

Response: Fungicidal effect of guava wood vinegar against Colletotrichum coccodes causing black dot disease of potato

Follow the journal template to re-write the whole manuscript, including line numeration to make possible point-by-point efficient suggestions.

Response: We formatted the article according to journal format

Response: These parts have been added to the article

Response: all references have been numbered in order of appearance in the text according to the journal format. Include the digital object identifier (DOI) for all references.

The abstract is a very important part of the manuscript; I suggest rewriting it.

Response: the summary has been rewritten

The keyword descriptors are not fully representative of the manuscript. For instance, insert the name of guava WV components.

Response: Keywords: potato; Colletotrichum coccodes; potato; guava wood vinegar components; GC-MS technique

Introduction

The Introduction section is pertinent, but it is not clear. Some concepts are repeated. Eliminate unnecessary repetitions. Re-write.

Response: Unnecessary repetition has been eliminated in some sentences as shown in the article

Response: Scientific names have been added to the article, such as Psidium guajava L., Colletotrichum coccodes

Use “2009-2010 potato growing season” instead of “2009/2010”.

Response: corrected

What does “daughter” mean?

Response: It was deleted because it was an extra word

What does “WV” mean? Is it referring to all wood vinegar or guava wood vinegar?

Response: It has been corrected in the text to become guava wood vinegar and WV refers to wood vinegar in general.

What does “synthetic fungicides” mean? Is it referring to all synthetic fungicides?

Response: yes this is in general.

What does “opposed” mean?

Response: The sentence has been omitted.

What does “prohibited” mean? I suggest “revoked”.

Response: The sentence has been omitted.

What does “giving plants” mean?

Response: It was modified to "WV ester compounds can increase photosynthesis, chlorophyll and the production of sugar and amino acids, which give plants more resistance to infections and pests".

What does “infections” mean?

Response: Infections caused by fungi, bacteria and viruses it was replaced by diseases

The hypothesis of the work is not clearly defined. The objectives are not clear and concrete.

Response: It was modified to "The objectives of this study were to evaluate the efficacy of guava wood vinegar against Colletotrichum coccodes which causes black dot disease, and investigate its effect on potato plant growth as well. Determine and identify the bioactive components that are most prevalent in guava WV using the GC-MS technique"

Materials and methods

This section is not clear, and difficult to read. Improve.

Use: “2.1. Isolation and identification of pathogen”

What does “acquired” mean?

Response: sorry for this mistake it was acquired

Where “pure cultures sub-cultured slants at 4°C”? Do Colletotrichum coccodes grow well at 4°C? I suggest: “Pure cultures were sub-cultured on PDA slants and stored at 4°C until use.”.

Response: Pure cultures were sub-cultured on PDA slants and stored at 4°C until use

Defined soil volumes used in the 30 cm in diameter pots.

Response: plastic pot of 30 cm diameter (2.4 kg soil), filled with a sterilized mixture of clay and sand soil (4:1 w/w).

Associate a year to 1^st February.

Response: Sowing was done on February 1st in season 2020.

Is “20/20/20” a mixture of commercial fertilizer or a specific product?

Response: NPK (20/20/20) is a commercial fertilizer of nitrogen, phosphorus and potassium.

Section “2.2.3. Inoculation of plants” is not clear. Re-write.

Response: It was modified

Insert the diameter of the discs used as inoculum.

Response: 6 mm

How was the conidia suspension used?

Response: by using hand sprayer

What does “injected” mean?

Response: injected means inoculated plants in wounds

What does “:” mean at the end of “2.3. subtitle”? Delete.

Response: delete :"at the end of "

The section “Disease assessment” is not clear. Re-write.

Verify DSI calculation. The correct formula should be: DSI (%) = 100 × [2× (0 or 1) + 6× (0 or 1) +10× (0 or 1) + 14× (0 or 1)]/32

Explain the significance of the number in the DSI formula.

Response: the fungal colonization of stems was recorded as a binary outcome with 1 = colonized, and 0 = noncolonized.

What does “:” mean at the end of “2.4. subtitle”? Delete.

Response: delete "at the end of" it's mean at the end of growing

Improve section “2.4. Source of wood vinegar from guava trees”.

Response: 2.4. Source of wood vinegar from guava trees

What does “:” mean at the end of “2.5. subtitle”? Delete.

Response: delete "at the end of" it's mean at the end of growing

Improve section “2.5. Analysis of the chemical composition of guava wood vinegar by GC–MS”.

Response: 2.5. Analysis of the chemical composition of guava wood vinegar by GC–MS

What does “:” mean at the end of “2.6. subtitle”? Delete.

Improve section “2.6. Antifungal activity of guava WV against C. coccodes in vitro”.

Response: 2.6. Antifungal activity of guava WV against C. coccodes in vitro

Use the formula “R = [(C –B)/C] × 100”.

Response: R = [(C –B)/C] × 100

What does “:” mean at the end of “2.7. subtitle”? Delete.

Response: delete "at the end of "

Improve section “2.8. Statistical analysis”.

Response: It is improved

This section reports that means were compared using the least significant difference (LSD). Data in Figure 4, Table 2 and Table 3 show the significance by the Duncan test. Please uniform.

Response: Values in the column followed by the same letter within a column are not significantly different as determined by the LSD test p = 0.05.

The Materials and Methods section lacks information on the effects of guava wood vinegar on plant growth characteristics and tuber yield.

Response: Plant height, stem diameter and tuber yield of potato per pot were measured at the end of the growing season.

A possible arrangement of the Materials and Methods section:

2.1. Fungal isolation and identification

2.2. Pathogenicity test

2.2.1. Plant material

2.2.2. Inoculum preparation

2.2.3. Inoculation of plants

2.2.4. Disease assessment

2.3. Antifungal activity

2.3.1. Guava wood vinegar: production and chemical composition

2.3.2. Plate assays

2.3.3 Greenhouse experiments

2.4. Effects of guava wood vinegar on plant growth and tuber yield

2.5. Statistical analysis

Response: We rearrange it according your suggestion

Results

The results are presented confusingly. Usually, the result section provides a concise and precise description of the experimental results. Avoid information pertinent to material and methods.

Response: We avoided information related to the M & M

Follow the proposed Materials and Methods subsubsections to rewrite the results.

Response: We followed the proposed Materials and Methods sub subsections to rewrite the results.

Pay attention to subsections numeration. Two “3.5.” sections are present. As well as two “Figure 2”

Response: It was modified

Figures and tables must be self-explanatory, clear, and easy to understand without needing any extra explanation.

Response: Figures and tables are clear and easy to be understood without any extra explanation.

Label the figure on page 5 as Figure 1. Rewrite the legend. I propose the picture sequence: Symptoms of black dot disease on naturally infected potato tuber, colony morphology of Colletotrichum coccodes on Potato Dextrose Agar (PDA) medium, C. coccodes conidiomata on PDA medium, conidia formed on PDA (insert a magnification bar).

Response: Figure 1. (A) Symptoms of black dot disease on naturally infected potato tuber, (B) colony morphology of C. coccodes on PDA medium, (C) C. coccodes conidiomata on PDA medium and (D) C. coccodes produce conidia on PDA medium at 40X magnification.

Section 3.2. is not clear. Rewrite.

Response: It was modified

Section 3.3. is not clear. Rewrite.

Response: It was modified

Table 1: verify the correct IUPAC name of the listed compound. Avoid the division between two pages.

Response: Phenol, 2-methoxy-

2-Methoxy-5-methylphenol

Phenol, 2,6-dimethoxy-

1, 2, 4-Trimethoxybenzene

9, 12, 15-Octadecatrienoic acid,

2-phenyl-1, 3-dioxan-5-yl ester

Ethyl iso-allocholate

Figure 2:

Enlarge following the “Horticulturae-template”.

Response: has been modified to "Figure 2 has been enlarged according to Horticulturae-template"

What does “numbers” mean? Gas chromatogram peaks are associated with the Retention Time.

You define 6 peaks only. And the other?

Response: Numbers have been removed to Figure 2. Gas chromatogram from the GC-MS analysis of guava WV.

The legend of Figure 2 is confusing. Rewrite.

Response: Figure 2. Gas chromatogram from the GC-MS analysis of guava WV.

Figure 3:

If it is possible, insert the formula of the other compound listed in Table 1.

Section 3.4. is not clear. Rewrite.

Response: we rewrite it again hope it is clear

Figure 4:

use “Growth inhibition (%)” instead of “% of growth inhibition”

Response: Growth inhibition (%)

delete all the “%” presents in abscissa.

Response: delete % from presents in abscissa

use “guava wood vinegar concentration (% v/v)” instead of “WV concentration (v/v)”.

Response: guava wood vinegar concentration (% v/v)

The legend of Figure 4 is confusing. Rewrite.

Response: Figure 4. Effect of different guava WV concentrations (% v/v) on C. coccodes in vitro. Values are the mean of three replicates ± standard deviation (SD). In each column, data followed by the same letter do not differ significantly as determined by the LSD test p = 0.05.

Figure 5: enlarge the picture.

Response: Figure 5 has been enlarged

Section “3.5. Effects of guava WV on potato black dot disease in greenhouse conditions during the 2021 and 2022 growing seasons” is not clear. Rewrite.

Table 2

What does “DI % = Disease incidence percentage.” mean?

Response: It was modified to Disease severity index (%).

Response: Table 2. Disease severity index (%) referred to stem colonization (SC), root covered with sclerotia (RCS), and wilt of potato plants inoculated with C. coccodes and exposed to different treatments under greenhouse conditions during 2021 and 2022 growing seasons.

Insert data standard deviation or standard error.

Response: data are presented as mean ± standard deviation (SD).

Use “Disease severity index (%)*” instead of “Disease severity index (%)”

Response: Disease severity index (%)*

Use “Inoculate and untreated control” instead of “Infected control”.

Response: Uninoculated and untreated control

Use “*Values are the mean of ??? replicates ± ??. In each column, data followed by the same letter do not differ significantly according to the Duncan test (p ≤ 0.5).”

Response: Values are the mean of three replicates ± standard deviation (SD). In each column, data followed by the same letter do not differ significantly as determined by the LSD test p = 0.05.

Section “3.5. Effect of WV treatment on plant height, stem diameter and tuber yield of potato under greenhouse conditions” is not clear. Rewrite.

Response: This Section has been rewritten

The data presented in Table 3 show that foliar application of guava WV improved plant growth characteristics such as plant height, stem diameter and tuber yield per pot of potato in both seasons (2021 and 2022). Plant height (38.67 and 38.00 cm), stem diameter (11.00 and 11.33 mm), and tuber yield (274.54 and 281.72 g) were the greatest values in the treatment of 3% guava WV (v/v) in both seasons 2021 and 2022, respectively, followed by WV at 2% (Fig. 6). In comparison to the guava WV treatment, the fungicide-treated infected plants had the lowest plant height and stem diameter. Furthermore, data also showed that using guava WV increased potato tuber yield per pot when compared to untreated plants.

Table 3

Response: Table 3. Effect of different treatments on plant height, stem diameter and tuber yield/pot of potato plants cropped under greenhouse conditions during 2021 and 2022 growing seasons.

Use a capital letter for “plant”.

Response: Plant height (cm)

Insert standard deviation or standard error of data.

Response: data are presented as mean ± standard deviation (SD).

Table 3 title report “tuber yield/plant”, while in the table is present “tuber yield/pot”. Uniform.

Response: tuber yield/pot

What does “gm” mean?

Response: g

What does “Infected control” mean?

Response: Inoculated and untreated control

What does “Healthy control” mean?

Response: Uninoculated and untreated control

Use “*Data are the mean of ??? replicates ± ??. In each column, data followed by the same letter do not differ significantly according to Duncan test (p ≤ 0.5).”

The legend of Figure 6 is not clear. Re-write.

Discussion

This section is presented in a confusing manner. Improve.

Response: In future investigations may shed more light on the possibility of separating some components of guava WV and evaluating their effectiveness in controlling some plant diseases.

Kind regards

Author Response File: Author Response.docx

Reviewer 4 Report

Comments for Authors

The subject of the manuscript fell within the general scope of the journal and the sound of manuscript is scientific. The subject has original contribution about the fungicidal effect of wood vinegar on Colletotrichum coccodes causing black dot disease in potato. This paper is a general interest of readers of Horticulturae. However, the manuscript presents some details that need to be corrected and improved.

Major concerns:

-The Materials and Methods need to be improved. It is important that the Authors in this section describe them with sufficient detail in order to allow others to replicate the published results.

-The Results need to be improved: The Authors should provide a concise and precise description of the experimental results, as well as their interpretation.

-The discussion needs to be improved. The Authors need to discuss the results and how they can be interpreted in perspective of previous studies. The findings and their implications should be discussed in the broadest possible context and limitations of the work highlighted. The discussion section doesn’t mean just to mention the work done by others. The authors must explain what their work is going to add in the frontiers of scientific knowledge. On the other hand, the discussion should focus on the effect of WV on fungi, not bacteria, so I suggest removing the discussion on bacteria.

Minor concerns:

Page 1. Abstract…. the Instructions for Authors indicates a maximum of 200 words. The abstract has 270 words...it is suggested to summarize

Page 1. Abstract, line 17…the Authors say: “Moreover, WA is a good replacement…”…please correct WA by WV

Page 1. Introduction, 2nd paragraph, last line....the Authors mention that synthetic pesticides are prohibited..... What pesticides are used to control this disease? And, which ones are prohibited? please include some examples

Page 2. Introduction, 2^nd paragraph, line 4....the Authors say: Sugars, carboxylic acids, hydroxy aldehydes, hydroxy ketones, and phenolic acids are among the major groups of substances in WV......... in what type of vinegar this composition has been reported? Can the Authors mention the origin of the WV?

Page 2. Introduction. 2^nd paragraph, line 11….the Authors say: At a 1:32 dilution, WV completely inhibited the growth of Alternaria mali the causal agent of apple Alternaria blight, when applied at a 1:32 dilution,...... 1:32 dilution is repeated....please modify the wording and correct

Page 2, Introduction, third paragraph, line 9....the Authors say: WV increases the abiotic stress tolerance (Saberi et al., 2013a) and growth..... please add comma after parenthesis, and delete "and"

Page 2. Materials and methods, first paragraph, last line…the Authors say: Pure cultures were sub-cultured on PDA slants at 4°C until use…… the Authors want to say: … on PDA slants and preserved at 4°C until use? Please clarify

Page 2, section 2.2.1. Plant material, last line….the Authors say:…. and covered with soil… please specify what type of soil was used

Page 3, section 2.2.3. Inoculation of plants….. The Authors should describe this section with sufficient detail to allow others to replicate and build on published results. Please include information about how many plants were inoculated, how many wounds were made on the stem. Also, once in the greenhouse, at what relative humidity were they kept, and how were the plants examined, how often and for how long were they examined…..

In addition, the Authors mention: After three days, every injected stem exhibited necrosis, but control stems exhibited no signs….. This is a result, please move it to results section

Page 3, section 2.3. Disease assessment:…..please delete the colon.

In addition, in line 9 the authors mention: After 10 days, there was visible fungal growth on the agar…..this information correspond to results, please move it to results section.

Page 3, section 2.4. Source of wood vinegar from guava trees:…… please delete the colon.

Please describe how the WV was obtained,…include reference

Page 3, section 2.5. Analysis of the chemical composition of guava wood vinegar by GC–MS….….. The Authors should describe this section with more detail to allow others to replicate and build on published results. Please include information about what type of detector was used, what was the carrier gas, the flow rate, the injector temperature, in general what were the chromatographic conditions used for the detection of the compounds. Also include information about how the identification of the compounds was carried out, if commercial standards were used, what they were, or if they were identified based on the GC-mass library.....how the compounds were quantified, etc…. It is not clear how the identification and quantification of the compounds were carried out, please explain

Page 4, section 2.7. Effect of wood vinegar on potato black dot disease under greenhouse conditions:…… please delete the colon.

Please include information about how many plants were inoculated.

Also, please include information about how the plants were evaluated, what type of determinations were made

Page 4, section 3. Results

3.1. Isolation and identification of the C. coccodes isolates…..please describe the results, what were the characteristics observed, of both the colony and the conidia of this pathogen...

Page 5, Figure 2….please correct the figure number, it should be number 1……..also, please delete spores…C. cocoodes produce conidia not spores

Page 5, section 3.3. Chromatographic analysis of guava WV:…… please delete the colon

The authors say: Retention time (RT), molecular weight, molecular formula, and area% are used to identify these components. The main compounds present in guava WV were quantified using GCMS…..That information is part of the methodology, please move it to M&M section......were commercial standards used for identification of the compounds? or mass library? It is not clear how the identification of the compounds was carried out, please explain

Page 6, Figure 2…..the Authors say: Numbers represent the main bioactive compounds of the sample……the numbers correspond to retention times of the detected compounds? Please check and correct

Figure 4 and Tables 2 and 3…….please include the standard deviations…….the authors mention that: Values in a column followed by the same letter do not differ significantly according to Duncan test (p ≤ 0.5)…Duncan test was not included in M&M section, please check and correct it

Page 10….please write all the fungi names in italics

Page 10…..the reference Andrivon et al., 1998 was not found cited in the text, please delete it or included it in the text

Author Response

Reviewer 4

We would like to thank the reviewers for reviewing the manuscript and highlighting some important points thanks for your suggestion we have formatted the article according to Journal format.

Comments for Authors

Major concerns:

-The Materials and Methods need to be improved. It is important that the Authors in this section describe them with sufficient detail in order to allow others to replicate the published results.

-The Results need to be improved: The Authors should provide a concise and precise description of the experimental results, as well as their interpretation.

Response: Thank you for the suggestions; we formatted the article according to journal format. All comments were covered here, and you can see them in different colour.

Minor concerns:

Page 1. Abstract…. the Instructions for Authors indicates a maximum of 200 words. The abstract has 270 words...it is suggested to summarize

Response: The summary has been shortened as much as possible.

Page 1. Abstract, line 17…the Authors say: “Moreover, WA is a good replacement…”…please correct WA by WV

Response: The word has been corrected to WV

Response: paragraphs have been removed from the introduction

Response:Thanks for your observation , it was added e.g. oak, poplar, pine, pruning litter, forest waste

Response: It was modified to "WV completely inhibited the growth of Alternaria mali the causal agent of apple Alternaria blight, when applied at a 1:32 dilution, where it was as effective as polyoxin-B fungicide at a 2 mg/ml concentration [21]."

Page 2, Introduction, third paragraph, line 9....the Authors say: WV increases the abiotic stress tolerance (Saberi et al., 2013a) and growth..... please add comma after parenthesis, and delete "and"

Response: It was modified to "Additionally, WV increases the abiotic stress tolerance [28], growth, production, and quality of a wide range of crops [29]".

Response: It was modified to "Pure cultures were sub-cultured on PDA slants and stored at 4°C until use"

Page 2, section 2.2.1. Plant material, last line….the Authors say:…. and covered with soil… please specify what type of soil was used

Response: a sterilized mixture of clay and sand soil (4:1 w/w).

In addition, the Authors mention: After three days, every injected stem exhibited necrosis, but control stems exhibited no signs….. This is a result, please move it to results section

Response: one wound was made into the base of the stems (above the soil's surface). Three plants were used for each treatment as replicates. The plants were then examined every day to check for symptoms.

Page 3, section 2.3. Disease assessment:…..please delete the colon.

Response: 2.3. 4. Disease assessment

In addition, in line 9 the authors mention: After 10 days, there was visible fungal growth on the agar…..this information correspond to results, please move it to results section.

Response: This is a reference method for estimating fungal colonization of stems

Page 3, section 2.4. Source of wood vinegar from guava trees:…… please delete the colon.

Please describe how the WV was obtained,…include reference

Response: 9. Anonim. Wood vinegar. Forest Energy Forum, 9, FAO of United Nations, 2001.

Response: In Assiut University Faculty of Science Department of Chemical, components in guava WV were analyzed using a gas chromatography-mass spectrometry (GC-MS) system (GC Trace 1300 Thermo Scientific). GC-MS was used to identify bioactive chemicals using a capillary column TG-5MS, 30 m×0.25 mm×1 m equipped with a GC Trace 1300 Thermo Scientific. A single quadruple mass spectrometer (ISQ 7000 Thermo Scientific) was used. The carrier (He, 99.999%) flow was 1ml min-1, split 10:1, and injected volumes 2 µl. The column temperature was maintained initially at 110 °C and hold for 5 min and at rate of 10°C/min the temperature increased up to 200 °C and hold for 5 min, then the temperature increased up to 250°C at rate of 5°C/min and hold for 5 min. the injector temperature was 250°C and this temperature was held constant during the analysis. The electron impact energy was 70 eV and the ion source temperature was set at 250°C. Electron impact (EI) mass scan (m/s) was recorded in the 40-650 amu range [37].

Page 4, section 2.7. Effect of wood vinegar on potato black dot disease under greenhouse conditions:…… please delete the colon.

Response:2.7. Effect of guava WV on potato black dot disease under greenhouse conditions

Please include information about how many plants were inoculated.

Also, please include information about how the plants were evaluated, what type of determinations were made

Response: Three plants were used for each treatment as replicates. The plants were then examined every day to check for symptoms.

Page 4, section 3. Results

3.1. Isolation and identification of the C. coccodes isolates…..please describe the results, what were the characteristics observed, of both the colony and the conidia of this pathogen..

Response: The colony morphology and conidia of C. coccodes are shown in Figure 1 (B, C and D). The culture of the pathogen was growing in the form of white circular colony at first and becoming grey with age on PDA medium, as shown in Figure 1 (B). The conidia spores were aseptate, hyaline, smooth-walled, and straight (Figure 1. D). Pure cultures were sub-cultured on PDA slants at 4°C until use.

Page 5, Figure 2….please correct the figure number, it should be number 1……..also, please delete spores…C. cocoodes produce conidia not spores

Response: Figure 1. (A) Symptoms of black dot disease on naturally infected potato tuber, (B) colony morphology of C. coccodes on PDA medium, (C) C. coccodes conidiomata on PDA medium and (D) C. coccodes produce conidia on PDA medium at 40X magnification.

Page 5, section 3.3. Chromatographic analysis of guava WV:…… please delete the colon

Response: 3.3. Chromatographic analysis of guava WV

Response: Retention time (RT), molecular weight, molecular formula, and area% were used to identify these components.

Page 6, Figure 2…..the Authors say: Numbers represent the main bioactive compounds of the sample……the numbers correspond to retention times of the detected compounds? Please check and correct

Response: Figure 2. Gas chromatogram from the GC-MS analysis of of guava WV.

Response: all experiments were carried out in three replicates and data are presented as mean ± standard deviation (SD). It has been modified in the article

Response: Values are the mean of three replicates ± standard deviation (SD). In each column, data followed by the same letter do not differ significantly as determined by the LSD test p = 0.05.

Page 10….please write all the fungi names in italics

Response: Plasmopara viticola, Verticillium dahliae, Phytophthora capsici, and Fusarium graminearum.

Page 10…..the reference Andrivon et al., 1998 was not found cited in the text, please delete it or included it in the text

Response: 6. Andrivon, D.; Lucas, J.M.; Guerin, C.; Jouan, B. Colonization of roots, stolons, tubers and stems of various potato (Solanum tuberosum) cultivars by the black dot fungus Colletotrichum coccodes. Plant Pathol. 1998, 47, 440–445. https://doi.org/10.1046/j.1365-3059.1998.00267.x

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I have read both the paper with the changes and the responses to the comments, and I think the quality and clarity of the paper has improved a lot. The only thing I still don't agree with is the issue of composition: if you look at the chromatogram there are a lot of very intense peaks, and they have decided to put the ones they wanted in the table, including one which was 0.04% in area.

However, the main issue is really the fungicidal action, not the composition. Although it is true that perhaps with a more detailed analysis it could be better justified from the chemical point of view.

Author Response

Reviewer 1

Response: We would like to thank the reviewers for reviewing the manuscript and highlighting some important points.

Several compounds were placed in the table by realizing them from the analysis through the intense peaks as shown in Table 1

Reviewer 2 Report

The authors have followed the proposed indications and certainly the work has improved considerably, congratulations. A last suggestion that I feel like giving is to read and include in the manuscript a new manuscript (https://doi.org/10.1111/aab.12837) just published on the biostimulant effects of WV on potatoes which binds very well with the aim of this work that studies the fungicidal effect of WV causing black dots on potatoes. In my opinion, adding these considerations the work would be even more interesting and complete by taking into consideration both the biostimulant effect and the fungicidal effect on the tuber, but at different concentrations.

Congratulations again

Author Response

Response: We inserted this reference in the text.

In a recent study, Fedeli et al. [62] showed that the application of wood distillate at 0.2% significantly increased the content of soluble sugars, starch and total carbohydrates in treated potato tubers.

Fedeli, R.; Vannini, A.; Grattacaso, M.; Loppi, S. Wood distillate (pyroligneous acid) boosts nutritional traits of potato tubers. Ann. Appl. Biol. 2023;1–6. https://doi.org/10.1111/aab.12837

Kind regards

Reviewer 3 Report

The revised version of manuscript ID horticulturae-2355568-peer-review-v2 “Fungicidal effect of wood vinegar against Colletotrichum coccodes causing black dot disease in potato” was improved.

The presentation in the form of a manuscript remains confusing and, in some parts, repetitive.

This version of the manuscript fails to apply the “horticulturae” template, including line numeration.

The template available at the link “https://www.mdpi.com/files/word-templates/horticulturae-template.dot” allows you to format the manuscript with a simple copy and paste.

I hope to be clear in the indications for the necessary integrations.

A further suggestion for the title: use “potatoes” instead of “potato”.

Insert the Authors name to all the cited organisms (see Colletotrichum coccodes in abstract, page 2: Plasmopara viticola, Verticillium dahliae, Phytophthora capsica, Fusarium graminearum, Alternaria mali, Cryptomeria japonica, Pythium splendens, P. capsica, Ralstonica solanacearum…) or delete the Authors indications present for Psidium guajava (abstract), Solanum tuberosum and Colletotrichum coccodes (page 1).

In non-taxonomic papers, it is facultative to indicate the Authors associated with Latin names of plants, fungi, and organisms.

The abstract remains difficult to read.

Are the abbreviations “[(SC %)”, “(RCS %)] necessary?

Use “completely inhibited” instead of “completely inhibited (100%)”

Use “and percentage of wilted plants” instead of “and wilted plants %”

Keywords: delete the second “potato”.

Eventually add “growth inhibition, antifungal activity, plant growth stimulation” and/or the name of some guava WV components.

Introduction

The Introduction section contains unnecessary repetitions. See:

1) “Although it can infect any part of the plant, tubers are where it is most commonly seen” and “C. coccodes can infect any part of a potato plant, such as leaves, tubers, stolons, roots, basal stems”

2) “It is a low-priced product [12]” and “It is a low-cost,”

….

Introduction, page 1, line 1: delete “ thus is”

Introduction, page 1, lines 2-3: delete “Although it can infect any part of the plant, tubers are where it is most commonly seen.”

Introduction, page 1, lines 3-4: Use “Early reports of the disease in potato and tomato date back to the early 19th century, and are described in detail by Dickson [Dickson BT, 1926. The black dot disease of potato. Phytopathology 16, 23– 40.]. In Egypt, the disease was first reported in Salhiya and Abo Swair areas during the 2009-2010 seasons [2].” instead of “This disease was discovered for the first time in Egypt at Salhiya and Abo Swair areas during the 2009-2010 seasons [2].”

Introduction, page 1, line 6: Use “pathogen” instead of “disease”

Introduction, page 2, line 11: delete “content”.

Introduction, page 2, line 12: use “soil-borne plant pathogens” instead of “fungal diseases”. It is well known that several species of Pythium, Phytophthora, Rhizoctonia, and Fusarium causing root rots. Among these, the parasitic oomycetes Pythium and Phytophthora are included in the Kingdom Chromista.

The sentence “WV completely inhibited the growth of Alternaria mali the causal agent of apple Alternaria blight, when applied at a 1:32 dilution, where it was as effective as polyoxin-B fungicide at a 2 mg/ml concentration [21].” is not clear. Improve.

Introduction, page 2, lines 22-23: use “WV ester compounds can increase chlorophyll, photosynthesis, sugar and amino acids production, and stimulate plants resistance to diseases and pests” instead of “WV ester compounds can increase photosynthesis, chlorophyll and production of sugar and amino acids, which give plants more resistant to diseases and pests”.

Materials and methods

This section remains difficult to read.

Section 2.1. Improve.

Section 2.1., line 1: use “strain” instead of “isolate” or “recovered” instead of "isolated”.

Section 2.1., line 4: specify the used sodium hypochlorite ( a specific technical solution or commercial bleach).

Section 2.1., lines 4-5: use “sterile water and dried with sterile filter paper. The surface-sterilized samples were then plated onto PDA medium and incubated at 25°C in the dark.” instead of “sterile water. Using sterilized filter paper for drying, the surface-sterilized samples were then plated onto PDA medium. At 25°C, Petri dishes were incubated.”

Section 2.2.1:

How were potato tubers sterilized?

Where were the potato tuber pieces left?

Provide information on the 20/20/20 fertiliser used (trade name, manufacturer, country).

Section 2.2.3 title: use “Plants inoculation” instead of “Inoculation of plants”

Section 2.2.4.

Insert information on evaluation of stem colonization (SC %), root covering with sclerotia (RCS %), and wilted plants.

Insert information on the re-isolation procedure.

Page 4, Section 2.3.3 title: delete “2.3.3. Analysis of the chemical composition of guava wood vinegar by GC–MS”

Page 4, lines 2-15: Improve.

Page 2, Section 2.3.3 title: Use “2.3.2.” instead of “2.3.3.”

Section 2.3.3 a possible rearrangement:

“2.3.2. Plate assays

The inhibition of C. coccodes mycelial radial growth by the guava WV was tested at concentrations of 0.25, 0.50, 1.00, 2.00 and 3.00%. The guava WV was added directly to the autoclaved (20 min at 121°C) PDA medium cooled to 45°C, poured into sterile Petri dishes [38] and allowed to set. The centre of each test plate was subsequently inoculated with a 6 mm size plug of 15-day-old C. coccodes culture and incubated at 27°C in the dark. As a control, the fungus was cultivated on PDA medium without guava WV. For each treatment, three replications were performed. When maximum growth was observed in the control plates, the colony's radial growth was measured, and the percentage of growth inhibition (R) was calculated using the formula.

R = [(C –B)/C] × 100

Whereas: C = radial growth in control and B = radial growth in treatment.

Insert section 2.3.4. Greenhouse experiments

Page 4, section 2.4.:

improve

associate a year to 1^st February.

Results

The results are presented confusingly.

Avoid information pertinent to material and methods.

Follow the Materials and Methods subsections to rewrite the results.

Some suggestions:

3.1. Fungal isolation and identification

C. coccodes was obtained from tubers of infected potatoes with symptoms of black dot disease (Figure 1A). Grown on PDA medium, C. coccodes formed a circular colony at first white and then grey (Figure 1B) with age. Coniomata (Figure 1C) produced aseptate, hyaline, smooth-walled and straight conidia (Figure 1D).

Figure 1. Colletotrichum coccodes symptoms of black dot disease on naturally infected potato tuber (A), ??-day-old colony (B), conidiomata (C) and conidia at 40X magnification (D) on Potato Dextrose Agar medium.

3.2. Pathogenicity test

Improve

Following Material and methods indications, insert data on stem colonization (SC %), root covering with sclerotia (RCS %), wilted plants and DSI. If it is possible as a table.

Section 3.3: use 3.3. Guava wood vinegar production and chemical composition

Rewrite.

Table 1: verify the correct IUPAC name of the listed compounds.

Figure 2 legend: Rewrite.

Figure 3: insert the formula of all the compounds listed in Table 1 or delete the Figure 3.

Section 3.4: use 3.4. Plate assays

Rewrite.

Figure 4:

If it is possible insert sd as a vertical bar on each histogram.

Use “Growth inhibition (%)” instead of “Growth inhibition %”.

If possible insert a LSD vertical bar.

Figure 4. Effect of different guava wood vinegar concentrations on in vitro Colletotrichum coccodes radial growth inhibition. Values are the mean of three replicates ± SD. In each column, data followed by the same letter do not differ significantly as determined by the LSD test p = 0.05.

Figure 5:

Figure 5. Mycelial growth of Colletotrichum coccodes on Potato Dextrose Agar medium alone (Control) or amended with different guava wood vinegar concentrations.

Section 3.5: use 3.5. Greenhouse experiments

Section 3.6: use 3.6. Effects of guava wood vinegar on plant growth and tuber yield

Discussion

Improve.

Kind regards

Author Response

Reviewer 3

We would like to thank the reviewers for reviewing the manuscript and highlighting some important points. Thanks for your suggestion we have formatted the article according to Journal format. All comments were covered here you can see it with different color.

The revised version of manuscript ID horticulturae-2355568-peer-review-v2 “Fungicidal effect of wood vinegar against Colletotrichum coccodes causing black dot disease in potato” was improved.

The presentation in the form of a manuscript remains confusing and, in some parts, repetitive.

This version of the manuscript fails to apply the “horticulturae” template, including line numeration.

Response: We have formatted the article according to Journal format.

The template available at the link “https://www.mdpi.com/files/word-templates/horticulturae-template.dot” allows you to format the manuscript with a simple copy and paste.

I hope to be clear in the indications for the necessary integrations.

A further suggestion for the title: use “potatoes” instead of “potato”.

Response: Title "Fungicidal effect of guava wood vinegar against Colletotrichum coccodes causing black dot disease of potatoes"

Response: Inserted the Authors name to all the cited organisms in the text "Colletotrichum coccodes [(Wallroth) S.J.Hughes]; Plasmopara viticola (Berk. & M. A. Curtis; Berl. & De Toni); Verticillium dahliae (Klebahn); Phytophthora capsici (Leonian); Fusarium graminearum (Schwabe); Alternaria mali (Roberts); Cryptomeria japonica (Linnaeus) Don; Pythium splendens (H.Braun); Ralstonica solanacearum (Smith & Yabuuchi)"

In non-taxonomic papers, it is facultative to indicate the Authors associated with Latin names of plants, fungi, and organisms.

The abstract remains difficult to read.

Are the abbreviations “[(SC %)”, “(RCS %)] necessary?

Response: It was deleted of abstract and only mentioned in the Materials and Methods

Use “completely inhibited” instead of “completely inhibited (100%)”

Response: it was modified to "completely inhibited"

Use “and percentage of wilted plants” instead of “and wilted plants %”

Response: it was modified to "and percentage of wilted plants"

Keywords: delete the second “potato”.

Response: it was deleted "potato"

Eventually add “growth inhibition, antifungal activity, plant growth stimulation” and/or the name of some guava WV components.

Response: Keywords: Potato; Colletotrichum coccodes; guava wood vinegar components; GC-MS technique; growth inhibition; antifungal activity; plant growth stimulation

Introduction

The Introduction section contains unnecessary repetitions. See:

Response: Thanks for your suggestion it was corrected

2) “It is a low-priced product [12]” and “It is a low-cost,”….

Response: it was modified to “It is a low-cost”

Introduction, page 1, line 1: delete “ thus is”

Response: "thus is" was deleted

Introduction, page 1, lines 2-3: delete “Although it can infect any part of the plant, tubers are where it is most commonly seen.”

Response: the sentence was deleted "Although it can infect any part of the plant, tubers are where it is most commonly seen"

Response: the sentence was added and the reference was added in the text "Early reports of the disease in potato and tomato date back to the early 19^th century, and are described in detail by Dickson [7]."

Introduction, page 1, line 6: Use “pathogen” instead of “disease”

Response: we added "pathogen"

Introduction, page 2, line 11: delete “content”.

Response: we deleted "content"

Response: we added “soil-borne plant pathogens”

Response: The sentence was modified “WV causes complete inhibition of the growth of Alternaria mali the causal agent of apple Alternaria blight, when applied at a 1:32 dilution [21]”

Response: The sentence was added "WV ester compounds can increase chlorophyll, photosynthesis, sugar and amino acids production, and stimulate plants resistance to diseases and pests"

Materials and methods

This section remains difficult to read.

Section 2.1. Improve.

Response: this section improved

Section 2.1., line 1: use “strain” instead of “isolate” or “recovered” instead of "isolated”.

Response: it was added "strain" and "recovered"

Section 2.1., line 4: specify the used sodium hypochlorite (a specific technical solution or commercial bleach).

Response: The sodium hypochlorite used is commercial bleach.

Response: Small sections of the infected tubers were cut up and surface treated for 1 minute with 1% sodium hypochlorite (commercial bleach) before being rinsed in sterile water and dried with sterile filter paper. The surface-sterilized samples were then plated onto PDA medium and incubated at 25°C in the dark. The developed colonies were then purified using the hyphal tip method and identified according to the cultural features, morphological and microscopical characteristics stated by Sutton [33]. Pure cultures were sub-cultured on PDA slants and stored at 4°C until use.

Section 2.2.1:

How were potato tubers sterilized?

Response: Cara cv. potato tubers were sterilized with sodium hypochlorite 1% for 2 minutes.

Where were the potato tuber pieces left?

Response: Tubers were cut into pieces and left for 48 hours before planting in vitro at room temperature to allow for partial wound healing.

Provide information on the 20/20/20 fertiliser used (trade name, manufacturer, country).

Response: The plants were fertilized with NPK (Dotra Fert: 20/20/20) from the Egyptian Dotra Company at a concentration of 2 g/L water.

Section 2.2.3 title: use “Plants inoculation” instead of “Inoculation of plants”

Response: We used "Plants inoculation"

Section 2.2.4.

Insert information on evaluation of stem colonization (SC %), root covering with sclerotia (RCS %), and wilted plants.

Response: stem colonization percentage is about the presence (1) or absence (0) of fungus in the stem. It is determined by cutting the stem into pieces that are 2, 6, 10, and 14 cm in length, sterilizing them, and then placing them on PDA medium and incubating. The equation shown in M&M was used to calculate the colonization percentage following incubation.

-Root covering with sclerotia (RCS %), which represents the percentage of the sclerotia-covered area of the root and has 3 scales (0, 1, 2 and 3) as shown in M&M.

-Wilted plants =The percentage of wilted plants to the total number of inoculated plants

Insert information on the re-isolation procedure.

Response: This process is carried out as mentioned in section 2.1 of Materials and Methods

Page 4, Section 2.3.3 title: delete “2.3.3. Analysis of the chemical composition of guava wood vinegar by GC–MS”

Response: this title was deleted "2.3.3. Analysis of the chemical composition of guava wood vinegar by GC–MS"

Page 4, lines 2-15: Improve.

Response: it was modified

Page 2, Section 2.3.3 title: Use “2.3.2.” instead of “2.3.3.”

Response: this title was modified to

2.3.2. Analysis of the chemical composition of guava wood vinegar by GC–MS

Section 2.3.3 a possible rearrangement:

“2.3.2. Plate assays

Response: this title was modified

The inhibition of C. coccodes mycelial radial growth by the guava WV was tested at concentrations of 0.25, 0.50, 1.00, 2.00 and 3.00%. The guava WV was added directly to the autoclaved (20 min at 121°C) PDA medium cooled to 45°C, poured into sterile Petri dishes [39] and allowed to set. The centre of each test plate was subsequently inoculated with a 6 mm size plug of 15-day-old C. coccodes culture and incubated at 27°C in the dark. As a control, the fungus was cultivated on PDA medium without guava WV. For each treatment, three replications were performed. When maximum growth was observed in the control plates, the colony's radial growth was measured, and the percentage of growth inhibition (R) was calculated using the formula.

R = [(C –B)/C] × 100

Whereas: C = radial growth in control and B = radial growth in treatment.

Insert section 2.3.4. Greenhouse experiments

Response: this title was modified to "2.3.4. Greenhouse experiments"

Page 4, section 2.4.:

improve

Response: the section was modified to "The MSTAT-C software version 2.1 was used to conduct a data analysis of variance (ANOVA) [40]. All experiments were carried out in three replicates and data are presented as mean ± standard deviation (SD). The treatment means were compared using the least significant difference (LSD) (P<0.05), according to Gomez and Gomez [41].

associate a year to 1^st February.

Response: On 1st February at seasons 2021 and 2022,

Results

The results are presented confusingly.

Response: The results have been modified

Avoid information pertinent to material and methods.

Response: We avoided mentioning the Material and Methods information

Follow the Materials and Methods subsections to rewrite the results.

Some suggestions:

3.1. Fungal isolation and identification

coccodeswas obtained from tubers of infected potatoes with symptoms of black dot disease (Figure 1A). Grown on PDA medium, C. coccodes formed a circular colony at first white and then grey (Figure 1B) with age. Coniomata (Figure 1C) produced aseptate, hyaline, smooth-walled and straight conidia (Figure 1D).

Response: We added the paragraph to the results "C. coccodes was obtained from tubers of infected potatoes with symptoms of black dot disease (Figure 1A). Grown on PDA medium, C. coccodes formed a circular colony at first white and then grey (Figure 1B) with age. Conidiomata (Figure 1C) produced aseptate, hyaline, smooth-walled and straight conidia (Figure 1D)."

Response: the title Figure 1 was corrected to: Figure 1. C. coccodes symptoms of black dot disease on naturally infected potato tuber (A), 15-day-old colony (B), conidiomata (C) and conidia at 40X magnification (D) on Potato Dextrose Agar medium.

3.2. Pathogenicity test

Improve

Response: the paragraph was improved:

3.2. Pathogenicity of C. coccodes isolate on potato plants

The symptoms of the disease were observed on potato plants inoculated with the pathogen. Every injected stem showed necrosis after three days, but control stems showed no symptoms. The black dot disease symptoms on inoculated plants were recorded as stem colonization (SC %), root covering with sclerotia (RCS %), and wilted plants. Koch's hypotheses were confirmed when the causal pathogen was successfully isolated from inoculated plants.

Following Material and methods indications, insert data on stem colonization (SC %), root covering with sclerotia (RCS %), wilted plants and DSI. If it is possible as a table.

Response: it was mentioned in section "2.2. 4. Disease assessment"

Section 3.3: use 3.3. Guava wood vinegar production and chemical composition

Rewrite.

Response: the section was rewrite

Table 1: verify the correct IUPAC name of the listed compounds.

Response: We changed some compound names according to IUPAC for example,

Phenol, 2-methoxy- = Guaiacol

Phenol, 2,6-dimethoxy- = 2,6-Dimethoxyphenol

Figure 2 legend: Rewrite.

Response: We changed to ". Figure 2. GC-MS chromatogram analysis of guava wood vinegar"

Figure 3: insert the formula of all the compounds listed in Table 1 or delete the Figure 3.

Section 3.4: use 3.4. Plate assays

Rewrite.

Response: the title was modified

The data in Figures 4 and 5 demonstrate that guava WV treatment significantly (P ≤ 0.05) reduced C. coccodes mycelial growth at all tested concentrations when compared to the control. Higher concentrations of WV, like 2 and 3%, successfully inhibited the pathogen's growth on the PDA medium as compared to the lowest concentrations. The 3% concentration of guava WV had the highest effect, which completely inhibited the pathogen's growth (100%) on PDA medium. The percentage of mycelial growth inhibition increased in general as the concentration of the tested compound increased. Furthermore, the data revealed that there were significant differences in the percentage of pathogen growth inhibition among the tested concentrations.

Figure 4:

If it is possible insert sd as a vertical bar on each histogram.

Response: We inserted SD as a vertical bar on each histogram.

Use “Growth inhibition (%)” instead of “Growth inhibition %”

Response: we added "Growth inhibition (%)

If possible insert a LSD vertical bar.

Response: We inserted LSD as a vertical bar on each histogram.

Response: it was added "Figure 4. Effect of different guava wood vinegar concentrations on in vitro Colletotrichum coccodes radial growth inhibition. Values are the mean of three replicates ± SD. In each column, data followed by the same letter do not differ significantly as determined by the LSD test p = 0.05.

Figure 5:

Figure 5. Mycelial growth of Colletotrichum coccodes on Potato Dextrose Agar medium alone (Control) or amended with different guava wood vinegar concentrations.

Response: it was added "Figure 5. Mycelial growth of Colletotrichum coccodes on Potato Dextrose Agar medium alone (control) or amended with different guava wood vinegar concentrations.

Section 3.5: use 3.5. Greenhouse experiments

Response: 3.5. Greenhouse experiments

Section 3.6: use 3.6. Effects of guava wood vinegar on plant growth and tuber yield

Response: Effects of guava wood vinegar on plant growth and tuber yield

Discussion

Improve.

Response: we improved some sentence in discussion