Antibacterial Activity of Ginkgo biloba Extracts against Clavibacter michiganensis subsp. michiganensis, Pseudomonas spp., and Xanthomonas vesicatoria

Round 1

Reviewer 1 Report

Title of publication should be improved

Antibacterial effect of fruit extracts was not detected.

Introduction and Abstract should contain clearly described purpose of study.

 

Line 121 Bacterial strain not isolates were used.

Read and Compare:

2.5. In Vitro Antibacterial Activity Assessment (Read: “The bacterial suspensions were then applied to the surface of TSA plates to which the treatments had previously been added at concentrations between 62.5 and 1,500 μg·mL−1”)

And

Table 1 Minimum inhibitory concentrations of G. biloba extracts against the horticultural pathogens, 248 expressed in μg∙mL−1 . (Read: “>1500 for extracts of sarcotesta and sclerostesta”).

And

 Conclusions (Read: “Antibacterial activity was only detected for the leaf extract   ”)

I understood that extracts of sarcotesta and sclerostesta were not effective at concentrations between 62.5 and 1,500 μg·mL−1 and higher concentrations were not tested. The authors of publication guess that the concentrations >1,500 μg·mL−1 must be effective. Table should show their results, not the interpretation of results.

Question: why minimal bactericidal concentrations (MBC) were not determined. MIC shows that the growth of bacteria was inhibited, but it is not clear bacteria were killed or not. Determination of MBC are useful for evaluation of bactericidal concentrations. The publication with such title must contain such data, or the title of publication must be changed. Antibacterial, bacteriostatic and bactericidic are not synonyms.

Author Response

Comments and Suggestions for Authors

Q1. Title of publication should be improved. Antibacterial effect of fruit extracts was not detected.

Response: The mention of leaf and fruit extracts has been removed from the title, given that -as noted by the Reviewer- fruit extracts did not show antibacterial activity and this could be misleading to the reader.

Q2. Introduction and Abstract should contain clearly described purpose of study.

Response: The second sentence in the abstract has been rewritten, removing the details about the methodology used, which should make the aim of the study clearer to the reader. It now reads: “[…] The aim of this study was to characterize the contents of bioactive constituents in extracts from Ginkgo biloba L. leaves and fruits and test their activity against six phytopathogenic bacteria that affect horticultural crops. […]”. Concerning the introduction, a new paragraph has been added at the end of the section to state the aim of the study more explicitly: “[…] The purpose of this study was twofold: (i) to characterize the phytoconstituents of Ginkgo biloba L. leaf and fruit extracts; and (ii) to investigate their activity against the aforementioned six phytopathogenic bacteria that affect horticultural crops.”

Q3. Line 121 Bacterial strain not isolates were used.

Response: Corrected.

Q4. Read and Compare:

2.5. In Vitro Antibacterial Activity Assessment (Read: “The bacterial suspensions were then applied to the surface of TSA plates to which the treatments had previously been added at concentrations between 62.5 and 1,500 μg·mL−1”)

And

Table 1 Minimum inhibitory concentrations of G. biloba extracts against the horticultural pathogens, 248 expressed in μg∙mL−1. (Read: “>1500 for extracts of sarcotesta and sclerostesta”).

And

Conclusions (Read: “Antibacterial activity was only detected for the leaf extract   ”)

I understood that extracts of sarcotesta and sclerostesta were not effective at concentrations between 62.5 and 1,500 μg·mL−1 and higher concentrations were not tested. The authors of publication guess that the concentrations >1,500 μg·mL−1 must be effective. Table should show their results, not the interpretation of results.

Response: We agree with the point made by the Reviewer. We had not considered that indicating ‘>1500’ could mislead the reader by making him/her think that there could be antibacterial activity at higher doses (something that we have not checked), given that the intended meaning was that activity had not been observed even at the highest dose tested. Hence, we have replaced ‘>1500’ with ‘n.a.’, indicating in the table footnote that it stands for ‘no activity detected at the highest assayed dose’.

Q5. Question: why minimal bactericidal concentrations (MBC) were not determined. MIC shows that the growth of bacteria was inhibited, but it is not clear bacteria were killed or not. Determination of MBC are useful for evaluation of bactericidal concentrations. The publication with such title must contain such data, or the title of publication must be changed. Antibacterial, bacteriostatic and bactericidic are not synonyms.

Response: MIC values were chosen to allow comparison with other works reported in the literature, given that MIC values are more commonly determined than MBC values because they are easier and faster to perform, and they provide sufficient information for most ‘clinical’ purposes. The decision not to determine MBC values was not due to the laboriousness of the additional step of subculturing the clear MIC tubes onto a growth medium and assessing for bacterial growth, but rather because MBC values may not precisely reflect the actual bactericidal activity of an antibiotic in vivo, as factors such as host immunity, tissue penetration, and other interactions may affect the outcome.

In our view, the use of ‘antibacterial’ in the title is suitable, given that it is a general term for any agent that kills or inhibits bacteria. Nonetheless, we have replaced the term ‘antibacterial’ with the more accurate ‘bacteriostatic’ at various points throughout the text, to avoid confusion to the reader (in the abstract, in subsection 2.5, in subsection 3.3, and in the conclusions).

Reviewer 2 Report

ABSTRACT:

The abstract must have a rationale, an objective, materials and methods, results, and conclusions. The first sentence must be a rationale. Thus, the authors should mention the treatments and experimental design to explain the main findings.

 

 INTRODUCTION:
The introduction section is relatively general information about the Ginkgo biloba; however, there are some spaces for authors to enhance its quality further, which are as follows:

1-Lines 37–64: There is no need to insert information about the description Ginkgo biloba; it has been discussed in many studies before. Therefore, the critical point is to insert the specific details on the updated role of the bioactive compounds such as dihydro-4-hydroxy-2(3H)- furanone, 2,4-dimethyl-3-hexanol, catechol, 3-O-methyl-D-fructose, 4,6-di-O-methyl-α-D-galactose, methyl 2-O-methyl-α-D-xylofuranoside, and 3-methyl mannoside for against  the phytopathogenic bacteria affecting horticultural crops such as the high value crops such as tomato, cucumber and others.

 

Materials and methods

Lines 113-114:  Please mention if the Ginkgo biloba leaf and fruit samples were collected from 20 female trees; they are grown under the same environmental conditions, soil type and agronomic factors.

After collecting the sample, the second point is, what are the methods for dried samples? For example, was it dried at room temperature in the dark? Or what conditions?

Line 176:Please insert the methods used for cultivating crops, such as soilless culture,  and other information, such as fertilizer, irrigation interval, etc.

 Please insert the environmental conditions under the greenhouse for the cultivation of tomato and pea such as air temperature, humidity,

What are experimental layout and experimental design?

What about replication?

Lines 180-181: For tomato,the G. biloba leaf extract was applied through spraying (3 mL per plant) at two concentrations (MIC and MIC×2, i.e., 500 and 1,000 μg·mL−1, respectively). What is the control treatment? And why use 500 and 1000 concentrations, it is recommended?

Lines 187-188: for Pea, the G. biloba leaf extract was sprayed at two concentrations (MIC and MIC×2, i.e., 750 and 1,500 μg·mL−1, respectively.  What is the control treatment?

The experiment began on the day, month, and year and ended on the same day, month, and year.

What is the program used in this experiment for the statistical analyses of antibacterial activity data? For example, the SAS program

Results and discussion

The results and discussion section must be presented under specific subtitles.

The discussion section must be presented under certain subtitles, as the authors did for the results. This means that as the authors presented their results under certain subtitles in the Results section, are they also suggesting developing subtitles under the Discussion section?

The main concern is that the authors mostly only compare their results with the literature's results.

 

5 Conclusion

The conclusion should have the main findings only.

 

 

Author Response

Comments and Suggestions for Authors

Abstract:

 Q1. The abstract must have a rationale, an objective, materials and methods, results, and conclusions. The first sentence must be a rationale. Thus, the authors should mention the treatments and experimental design to explain the main findings.

Response: Please kindly note that we have started with a ‘background’ sentence to comply with the journal’s guidelines: according to the journal’s instructions for authors (available at https://www.mdpi.com/journal/horticulturae/instructions), “[…] The abstract should be a single paragraph and should follow the style of structured abstracts, but without headings: 1) Background: Place the question addressed in a broad context and highlight the purpose of the study; 2) Methods: Describe briefly the main methods or treatments applied. Include any relevant preregistration numbers, and species and strains of any animals used. 3) Results: Summarize the article's main findings; and 4) Conclusion: Indicate the main conclusions or interpretations. […]”. The rest of the abstract has been rewritten to make the four parts of the structured abstract more clearly distinguishable.

It now reads: “Phytopathogenic bacteria represent a risk to global food production by impacting a variety of crops. The aim of this study was to characterize the contents of bioactive constituents in extracts from Ginkgo biloba L. leaves and fruits and test their activity against six phytopathogenic bacteria that affect horticultural crops. Gas chromatography–mass spectrometry (GC-MS) was used for the chemical profiling of the aqueous methanol extracts; their bacteriostatic activity against Clavibacter michiganensis subsp. michiganensis, Pseudomonas cichorii, Pseudomonas syringae pv. pisi, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. tomato, and Xanthomonas vesicatoria (formerly Xanthomonas campestris pv. vesicatoria) was determined in vitro through the agar dilution method; and the protective effect of the leaf extract was tested in vivo against the most relevant bacterial pathogens based on their scientific/economic importance, i.e., C. michiganensis subsp. michiganensis and P. syringae pv. pisi, in tomato (Solanum lycopersicum L.) and pea (Pisum sativum L.) plants, respectively, under greenhouse conditions. The GC-MS characterization of G. biloba extracts revealed the presence of dihydro-4-hydroxy-2(3H)-furanone, 2,4-dimethyl-3-hexanol, catechol, 3-O-methyl-D-fructose, 4,6-di-O-methyl-α-D-galactose, methyl 2-O-methyl-α-D-xylofuranoside, and 3-methyl mannoside. In vitro growth inhibition tests showed that, while the fruit extract had no activity, the leaf extract exhibited minimum inhibitory concentrations between 500 and 1,000 μg mL⁻¹, which may be attributed to the presence of 2,4-dimethyl-3-hexanol and catechol. In vivo tests of the leaf extract demonstrated full protection in tomato and pea plants at 1,000 and 1,500 μg∙mL⁻¹, respectively. The results indicate that G. biloba leaves may be employed as a biorational source for integrated pest management in horticulture.”

Introduction:

The introduction section is relatively general information about the Ginkgo biloba; however, there are some spaces for authors to enhance its quality further, which are as follows:

Q2. Lines 37–64: There is no need to insert information about the description Ginkgo biloba; it has been discussed in many studies before. Therefore, the critical point is to insert the specific details on the updated role of the bioactive compounds such as dihydro-4-hydroxy-2(3H)- furanone, 2,4-dimethyl-3-hexanol, catechol, 3-O-methyl-D-fructose, 4,6-di-O-methyl-α-D-galactose, methyl 2-O-methyl-α-D-xylofuranoside, and 3-methyl mannoside for against the phytopathogenic bacteria affecting horticultural crops such as the high value crops such as tomato, cucumber and others.

Response: Lines 37-64 have been summarized (from 298 to 136 words), as suggested by the Reviewer. In addition, Figure 1 and Figure 2 have been merged into a single figure. Concerning the second request, please kindly note that the referred compounds were identified as part of the GC-MS chemical profiling conducted in this study, i.e., they are part of the results. In our view, including results in the introduction section is not advisable for several reasons: (i) it can confuse or spoil the reader by revealing the outcome before explaining how it was obtained; (ii) it can reduce the impact or significance of the results by presenting them without proper discussion or interpretation; (iii) it can create redundancy or inconsistency by repeating or contradicting information in different sections; and (iv) it can violate the logical structure or flow of the article by disrupting the progression from problem to solution. Hence, we have chosen to reserve the results for their own section and comment on them in the discussion section.

Materials and methods:

Q3. Lines 113-114:  Please mention if the Ginkgo biloba leaf and fruit samples were collected from 20 female trees; they are grown under the same environmental conditions, soil type and agronomic factors.

Response: We have included the requested clarification. The sentence now reads: “Ginkgo biloba leaf and fruit samples were collected from 20 female trees –grown under the same environmental conditions, soil type, and agronomic factors– along the Avenida de los Monegros in Huesca City, Aragon, Northeastern Spain (42° 7' 46.9" N 0° 24' 6.3" W) in the late days of September 2021. […]”

Q4. After collecting the sample, the second point is, what are the methods for dried samples? For example, was it dried at room temperature in the dark? Or what conditions?

Response: We apologize for the oversight. Considering previous studies on this specific matter [J Sci Food Agric 2021;101: 3290–3297, https://doi.org/10.1002/jsfa.10958], in which freeze drying, infrared drying, hot-air drying, and pulsed-vacuum drying were tested for the drying of G. biloba seeds, we opted for freeze-drying to remove moisture as the optimum method. A clarification has been included in subsection 2.1: “[…] For drying the samples, the freeze-drying method was chosen, in agreement with Boateng and Yang [21]: samples were initially frozen at −20 °C for 24 h, and then freeze-dried at −50 °C.”

Q5. Line 176: Please insert the methods used for cultivating crops, such as soilless culture,  and other information, such as fertilizer, irrigation interval, etc.

Response: The information on the substrate (peat vs. peat+sand for tomato and pea plants, respectively) was already provided in subsection 2.6. Nonetheless, additional details have been included concerning irrigation, as suggested by the Reviewer. Please note that the plants were not fertilized during the experiment (which was one of the modifications in comparison with the experiment with tomatoes conducted by de León et al., who fertilized plants twice a week with 2.5 mL/L of Actigil AA 8-8-6 (Bayer CropScience) solution).

Q6. Please insert the environmental conditions under the greenhouse for the cultivation of tomato and pea such as air temperature, humidity,

Response: Details on day/night temperatures, relative humidity, and use of supplementary lighting have been provided.

Q7. What are experimental layout and experimental design?

Response: The experimental designs were identical to those detailed in the two cited references (de León et al. and Martin-Sanz for the tomato and pea tests, respectively; please note that the latter was based on the procedure reported in [European Journal of Plant Pathology, 2003, 109:555–564, 2003]). Concerning the experimental layout, we chose a completely randomized design, where each experimental unit (plant) was randomly assigned to one of the treatments (negative control, positive control, MIC, or MICx2). We chose this layout due to its simplicity and flexibility and because we did not need to account for sources of variation other than the treatments.

Q8. What about replication?

Response: As noted at the end of subsection 2.6, “In both cases, five plants per treatment were used, with two independent replicates.”

Q9. Lines 180-181: For tomato, the G. biloba leaf extract was applied through spraying (3 mL per plant) at two concentrations (MIC and MIC×2, i.e., 500 and 1,000 μg·mL−1, respectively). What is the control treatment? And why use 500 and 1000 concentrations, it is recommended?

Response: Thank you for bringing the missing information about the controls to our attention. A clarification has been included to point out that control plants were sprayed with water. Concerning the assayed concentrations, we tested a higher dose as our prior experience with other biorationals had demonstrated that an equal concentration to the in vitro-determined MIC is often insufficient in in vivo tests. It should be noted that some of the treatment may be lost due to evaporation, runoff, degradation, etc., thus necessitating a higher concentration of the treatment to achieve the same effect as in vitro. We have clarified this point next to the concentrations: “The G. biloba leaf extract was applied through spraying (3 mL per plant) at two concentrations (MIC and MIC×2, i.e., 500 and 1,000 μg·mL⁻¹, respectively, taking into consideration that some of the treatment may be lost due to evaporation, runoff, degradation, etc., thus necessitating a higher concentration to achieve the same effect as in vitro); control plants were sprayed with water.

Q10. Lines 187-188: for Pea, the G. biloba leaf extract was sprayed at two concentrations (MIC and MIC×2, i.e., 750 and 1,500 μg·mL−1, respectively.  What is the control treatment?

Response: As in Q9, a brief comment has been included to specify that: “[…], and control plants were sprayed with water.”

Q11. The experiment began on the day, month, and year and ended on the same day, month, and year.

Response: The requested information has been included in subsection 2.6: “In regards to the tomato plant trials, the first replicate began on January 31, 2023, and concluded on February 10, 2023; while the second replicate began on February 3, 2023, ending on February 13, 2023. Regarding the pea plant trials, the first replicate started on January 27, 2023, concluding on February 6, 2023. The second replicate started on January 30, 2023, ending on February 9, 2023.”

Q12. What is the program used in this experiment for the statistical analyses of antibacterial activity data? For example, the SAS program

Response: The software used has been specified at the end of subsection 2.5: “[…] Statistical analyses of antibacterial activity data were conducted using IBM (Armonk, NY, USA) SPSS Statistics v.25.”

Results and discussion

Q13. The results and discussion section must be presented under specific subtitles. The discussion section must be presented under certain subtitles, as the authors did for the results. This means that as the authors presented their results under certain subtitles in the Results section, are they also suggesting developing subtitles under the Discussion section?

Response: The phrasing of the Reviewer’s comment makes it difficult to understand the requested modifications. Please kindly note that we already made use of three specific subtitles in the discussion  “4.1. On the Identified Phytochemicals and Their Antimicrobial Activity”, “4.2. On the Antimicrobial Activity of G. biloba Extracts” and “4.3. Comparison with Conventional Antibiotics”. Given that the discussion is lengthy and covers different aspects that are not directly linked to each other, we would rather keep the subsections’ headings. In our view, the use of these subheadings helps to categorize our interpretations into themes. Moreover, it is not against the journal’s instructions for authors or the journal’s template.

Q14. The main concern is that the authors mostly only compare their results with the literature's results.

Response: Please kindly note that we have also compared the antimicrobial activity of the extract with that of the two main constituents and with those of conventional antibiotics. Nonetheless, comparing our results with the literature’s results is the usual procedure for commenting on the results, and complies with the journal’s instructions for authors, “Authors should 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 context possible […]”.

Conclusion

Q15. The conclusion should have the main findings only.

Response: The first sentence on methodology has been deleted, as well as several of the phytoconstituents (the ones that were not tested separately) and content percentages. The remaining information, in our view, is essential and strictly accounts for the main findings only.

Reviewer 3 Report

Dear Authors,

The presented study is exciting and presents original results. At the same time, I consider the text to be quite clear, and the results clearly presented and discussed.

 

However, several comments about the text need to be resolved.

1/ L159 - "...Serial dilutions of cell suspension were..."

2/ L171-172 - information on tetracycline must be written in a sentence and better specified. Please avoid the hyphen (the second one in the same sentence).

3/ L255-256 (yellow part) - it is not possible to comment in this way in the text; please put the information in sentence form and definitely without "equals".

4/ Was MBC also verified?

5/ In the names of tables 1-3, please state better "expressed in ul.mL-1" in brackets immediately after "Minimum inhibitory concentration..." (not at the end of the second line.

6/ It will be more appropriate to indicate the full generic name for all microorganisms in tables 1-3 (in addition, it is not even uniform in its current form - see "C." in table 1 and the full name in table 2).

7/ In tab 1-3, please do not use "=" in the legend, better e.g., a dash, etc.

Author Response

Dear Authors,

The presented study is exciting and presents original results. At the same time, I consider the text to be quite clear, and the results clearly presented and discussed.

However, several comments about the text need to be resolved.

Q1. L159 - "...Serial dilutions of cell suspension were..."

Response: Corrected according to the reviewer’s suggestion.

 

Q2. L171-172 - information on tetracycline must be written in a sentence and better specified. Please avoid the hyphen (the second one in the same sentence).

Response: The two em-dashes were used to set off extra information (in this case, to specify the five antibiotics), provided that commas were being used to separate the elements of the list and that parentheses were also being used to indicate the abbreviations. Nonetheless, to avoid potential confusion to the reader, they have been replaced with commas, which now delimit the explanatory phrase.

 

Q3. L255-256 (yellow part) - it is not possible to comment in this way in the text; please put the information in sentence form and definitely without "equals".

Response: Thank you for pointing out this problem with the cross-reference to Table 2. The extra text coming from the footnote of Table 1 (i.e., “; n.a. = no activity detected at the highest assayed dose.”) had not shown up until all changes made to the manuscript had been accepted (given that we were using MS Word’s track changes tool) and all cross-references had been updated. It has been fixed and now only “Table 2” appears.

 

Q4. Was MBC also verified?

Response: No, only MIC values. MIC values were chosen to allow comparison with other works reported in the literature, given that MIC values are more commonly determined than MBC values because they are easier and faster to perform, and they provide sufficient information for most ‘clinical’ purposes. The decision not to determine MBC values was not due to the laboriousness of the additional step of subculturing the clear MIC tubes onto a growth medium and assessing for bacterial growth, but rather because MBC values may not precisely reflect the actual bactericidal activity of an antibiotic in vivo, as factors such as host immunity, tissue penetration, and other interactions may affect the outcome.

 

Q5. In the names of tables 1-3, please state better "expressed in ul.mL-1" in brackets immediately after "Minimum inhibitory concentration..." (not at the end of the second line.

Response: The three table captions have been modified following the Reviewer’s instructions.

 

Q6. It will be more appropriate to indicate the full generic name for all microorganisms in tables 1-3 (in addition, it is not even uniform in its current form - see "C." in table 1 and the full name in table 2).

Response: Corrected. Full names are now indicated for the six bacteria under study in Tables 1 to 3, as suggested by the Reviewer.

 

Q7. In tab 1-3, please do not use "=" in the legend, better e.g., a dash, etc.

Response: Fixed. The footnote now reads: “PG: penicillin G; AM: ampicillin; GM: gentamicin; CI: ciprofloxacin; TC: tetracycline.” Table 1 footnote has also been updated accordingly.

Reviewer 4 Report

The review of «Antibacterial Activity of Ginkgo biloba Extracts against Clavibacter michiganensis subsp. michiganensis, Pseudomonas spp., and Xanthomonas vesicatoria» for Horticulturae MDPI.

 

The topic of manuscript fits within the scope of the journal Horticulturae MDPI. The manuscript is devoted to the study to contents of bioactive constituents in extracts from Ginkgo biloba leaves and fruits and test their activity against six phytopathogenic bacteria that affect horticultural crops. The results indicate that Ginkgo biloba leaves may be employed as a biorational source for the integrated pest management of phytopathogenic bacteria that affect horticultural crops. What is very relevant at the present time due to the development of resistance by bacteria to traditional means of protection, such as antibiotics.

 

I have just a few remarks, which I give under author’s consideration:

 

2. Materials and Methods

2.3. Extract Preparation

 

1.1. L 125-129: Please specify the percentage of methanol in the extraction solution.

 

3. Results

3.3. Antibacterial Activity of the Extracts

 

1.2. L 262: Please check the need for the notes in Table 1: «Fruit = sarcotesta; core = sclerotesta».

Author Response

The review of «Antibacterial Activity of Ginkgo biloba Extracts against Clavibacter michiganensis subsp. michiganensis, Pseudomonas spp., and Xanthomonas vesicatoria» for Horticulturae MDPI.

The topic of manuscript fits within the scope of the journal Horticulturae MDPI. The manuscript is devoted to the study to contents of bioactive constituents in extracts from Ginkgo biloba leaves and fruits and test their activity against six phytopathogenic bacteria that affect horticultural crops. The results indicate that Ginkgo biloba leaves may be employed as a biorational source for the integrated pest management of phytopathogenic bacteria that affect horticultural crops. What is very relevant at the present time due to the development of resistance by bacteria to traditional means of protection, such as antibiotics.

I have just a few remarks, which I give under author’s consideration:

2. Materials and Methods

2.3. Extract Preparation

 

Q1. 1.1. L 125-129: Please specify the percentage of methanol in the extraction solution.

Response: Corrected. It is now specified that the powdered sample was digested in a methanol:water (1:1, v/v) solution.

 

3. Results

3.3. Antibacterial Activity of the Extracts

 

Q2. 1.2. L 262: Please check the need for the notes in Table 1: «Fruit = sarcotesta; core = sclerotesta».

Response: The information was not essential, and has been deleted from the footnote of Table 1, keeping only “n.a.: no activity detected at the highest assayed dose.”

 

Round 2

Reviewer 2 Report

 

 

Author Response

No files or comments are provided, so we assume that the reviewer agrees with the modifications made. 

Reviewer 3 Report

Dear Authors,

Thank you for corrections. According to my opinion, the manuscript is ready for publication in MDPI journal. 

Best Regards!