Comparison of Three Biological Control Models of Pycnoporus sanguineus on Phytopathogenic Fungi

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Reading this new version, a significant improvement of the manuscript was observed. I recommend your publication.

Author Response

Thank you for your comments and suggestions above, as they greatly enriched the final version of the work.

Author Response File: Author Response.docx

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Title:      Comparison of three biological control models of Pycnoporus sanguineus on phytopathogenic fungi

Authors:

Ricardo Irving Pérez-López, Omar Romero-Arenas, Conrado Parraguirre Lezama, Anabel Romero López, Lilia Cedillo Ramírez, Antonio Rivera

 

 

The manuscript aimed to evaluate three models of Pycnoporus sanguineus for the inhibition growth of the phytopathogens Botrytis cinerea and Fusarium oxysporum. Model 1 involves dual tests of the antagonistic activity of P. sanguineus vs phytopathogens, Model 2 involves antifungal effectiveness tests of Cinnabarin, and  Model 3 involves antifungal effectiveness tests of P. sanguineus extract. Models 2 and 3 were contrasted with products containing Benomyl and Captan.

 

1.      “To determine the presence and optical characterization of Cinnabarin, 0.5 mL of the ex tract was analyzed using spectrophotometry performed with a spectrometer Cary 100 UV- Vis System, Agilent, at 25 °C and quartz cells with an optical path of 1 cm. UV/Vis (C₄H₈O₂): λmax (log ε) = 251 (1.09), 427 (0.19), and 446 (0.18) nm (Fig 1a.)”

 

Reference substance group should be designed to add to experiments.

 

2.       “HPLC (CH₃CN/H₂O): the test showed a maximum peak at 1.5 minutes (Fig 1b.).”

 

Experimental design lacks standard reference materials group too.

 

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Observations 1 and 2

We fully appreciate and understand your observations on this matter. Here are our detailed response:

It is acknowledged that the use of a reference compound, such as cinabarin, would facilitate precise detection and facilitate a comprehensive discussion of the results obtained in our study. Nevertheless, various research groups have focused their studies on the characterization of cinabarina using diverse spectroscopic methods, including UV-VIS and nuclear magnetic resonance (NMR), as well as analytical techniques that enable the separation of complex mixtures of diverse substances, with the objective of identifying, quantifying, and purifying them, as exemplified by high-performance liquid chromatography (HPLC). A comparison of the results obtained with the information derived from the literature confirms the presence of cinabarina, a compound that currently exhibits significant microbial activity.

We trust that the absence of the reference compound does not detract from the rigor of our work. We have employed rigorous methods and have validated our findings by comparing them with existing literature. If deemed necessary, we are prepared to obtain the pure compound for contrast in future studies to ensure the highest level of accuracy and rigor.

We hope that these comments address your concerns satisfactorily. We appreciate your valuable comments.

Author Response File: Author Response.docx

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

1.     In introduction section, the literatures and research on antifungal activity of the genus Pycnoporus should be described. Please explain and describe the criteria of three different modes?

2.     In result section, the experiment of characteristics of P. sanguineus should be performed.

3.     The  extracts inside or outside the cell should be analyzed. Are there any negative or positive P. sanguineus stain?

4. The qulity of images should be improved.

Comments on the Quality of English Language

The quality of language should be improved. 

Author Response

Comments and Suggestions for Authors

1. In introduction section, the literatures and research on antifungal activity of the genus Pycnoporus should be described. Please explain and describe the criteria of three different modes?

Adding this information at the end of the introductory section indeed provides better continuity to the text. Therefore, you will now find this additional paragraph in the revised version. We greatly appreciate your contribution.

2. In result section, the experiment of characteristics of P. sanguineus should be performed

We appreciate the feedback and wish to address this consideration. However, we have doubts about what the experiment on the “characteristics of P. sanguineus“ refers to. We have reviewed the section carefully but could not identify any missing relevant information. We consider that the results section includes all the information provided by the statistical analyses conducted on the experiments of the three restriction models evaluated. However, if you still believe that we have overlooked any relevant information about the experiments that should be included in that section, we would greatly appreciate it so that we can correct it appropriately. Thanks.

3. The extracts inside or outside the cell should be analyzed. Are there any negative or positive P. sanguineus stain?

As such, the pigments of P. sanguineus (in this case, cinnabarine) are embedded in the tissue of the fungus, and the restriction they impose on other microorganisms occurs directly through contact with the fungal mycelial tissue. Few studies have addressed the possibility that P. sanguineus releases these pigments into the medium outside of its cellular body (at least for the pigments), although it could occur for other compounds present in the fungus, making it an important topic for future research. We are also unaware of any staining techniques specific to this species. We hope we have adequately addressed your concerns on this matter.

4. The quality of images should be improved.

We have already improved the quality of the images in the text. If you need to send the images separately, we will gladly do so and you can send them to the editor.

 Comments on the Quality of English Language

The quality of language should be improved.

We are pleased to inform you that before uploading the document to the platform, it was reviewed by the language translation and style company (MDPI) with the certificate English-Editing-Certificate-81351, a second opinion was requested and some grammatical changes were made to the text. It is now believed that the level of language used in this file is appropriate. This is expected to be the case and comments would be greatly appreciated if there are still specific parts of the text that are not expressed correctly. We hope you agree with this, and we would greatly appreciate your feedback if there are still specific parts of the text that are not expressed correctly.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

Accept after minor revisions (corrections to minor methodological errors and text editing

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Hello
Thank you for the comments and suggestions.

The changes were made at the reviewer's suggestion, marked in blue in the new document.

In addition, the English language edition was reviewed and corrected again.

Best regards

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, three models of biological control were adopted to assess the potential of Pycnoporus sanguineus on inhibition of Botrytis cinerea and Fusarium oxysporum, two phytopathogenic fungi isolated from strawberry crop. And the model 3 was the most effective method for showing the potential of P. sanguineus. However, the method operation of model 3 was unreasonable, and I propose that the article be not for publishing in its present form.

 

Materials and methods

 

2.2 Model 1: Dual tests of antagonistic activity

Point 1. L95: Delete “Potato Dextrose Agar”.

 

Point 2. L102：Change to GR= (FR-IR)/number of days in growth; the same error found in L105.

 

Point 3. L105：Change “*” to “×”

 

2.3 Model 2: Antifungal effectiveness tests of Cinnabarin

Point 4. L143-146: The extract (Cinnabarin) of P. sanguineus was dissolved into water ?

 

Point 5. L153-154: Please add reference(s) for the “the impregnated disc diffusion technique with the previously described compounds”.

 

2.4. Model 3: Antifungal effectiveness tests of full P. sanguineus extract

Point 6. In this paper, the authors described “a complete extract of P. sanguineus mycelia” as “P. sanguineus extract” directly, I am not agreement with it.

 

Point 7. L 197-200: “A total of 6.2 grams of P. sanguineus dehydrated mycelium was obtained, which was subsequently pulverized using a Nutribullet®. The powder obtained was added to the PDA at a concentration of 24.8 mg/mL, which was sterilized by filtration through 0.22 μm Millipore filters.’ Bacteria can be filtered out by 0.22 μm Millipore filters, I do not think that the PDA containing P. sanguineus powder sterilized by it is a wise method. please make sure that the sterilization method.

 

2.5. Statistical analysis

Point 8. L216, L223: “using the statistical software SPSS Statistics version 17 for the Windows operating environment” is repeated. The description of this section is confuse and complicated, authors should rephrase. 

 

3. Results

Point 9. L237-240: “The ANOVA results for PIRG values among the different treatments evaluated against F. oxysporum indicate that in Model 1, P. sanguineus has a moderate antagonistic effect, similar to the action of Cinnabarin and Benomyl in Model 2 (9.54%, 17.79%, and 15.98%, respectively, p < 0.05).” The sentence may confuse the readers. I suggest author delete the content in parenthese.

 

Point 10. Table 1 and table 2，p ≤ 0.05 or p < 0.05, please check.

 

Point 11. Figure 2，the resolution of picture (fungal colonies) was very low and were very difficult to read.

 

Discussion.

Point 12. L286-287: “various pathogenic fungal”

 

Point 13. L306: “ a lower GR”

 

Point 14. Please check the Latin writing of fungal species in this manuscript. For example, L338:“… Colletotrichum fragariae, C. gloeosporioides, and B. cinerea, …”

 

References

Point 15. Please unify the format of references in this manuscript.

Author Response

Thanks for the comments and suggestions.

The changes were made and are shown in green in the document.

Materials and methods

2.2 Model 1: Dual tests of antagonistic activity

Point 1. L95: Delete “Potato Dextrose Agar”.   Done

Point 2. L102：Change to GR= (FR-IR)/number of days in growth; the same error found in L105.  Done

 Point 3. L105：Change “*” to “×” Done

 2.3 Model 2: Antifungal effectiveness tests of Cinnabarin

Point 4. L143-146: The extract (Cinnabarin) of P. sanguineus was dissolved into water? Was dissolved into ethyl acetate.

Point 5. L153-154: Please add reference(s) for the “the impregnated disc diffusion technique with the previously described compounds”.

We apologize for the confusion generated by this fragment of text. What we meant to express was the classic method used, which is the paper disc diffusion method. We now describe it at the beginning of this section, and it reads as follows: “disc diffusion method in which the pathogenic fungi were grown in the center of Petri dishes containing 6mm diameter paper discs impregnated with the different treatments. These discs were placed one centimeter away from the edge of the Petri dish and arranged at the four cardinal points”.

 2.4. Model 3: Antifungal effectiveness tests of full P. sanguineus extract

Point 6. In this paper, the authors described “a complete extract of P. sanguineus mycelia” as “P. sanguineus extract” directly, I am not agreement with it.

We understand your concern in this regard. We use the term complete extract because we mix the pigmented mycelium of P. sanguineus directly into the medium. On the other hand, we also consider that the ideal way to carry out this procedure would be to use the fruiting bodies. The problem is that The procedure of fructifying the fungus did not give the expected results, perhaps due to inexperience with said species. After we improve our P. sanguineus production techniques, we will work with the fructifications instead of pigmented mycelium because we consider that the active ingredients found in higher concentration in these.

Point 7. L 197-200: “A total of 6.2 grams of P. sanguineus dehydrated mycelium was obtained, which was subsequently pulverized using a Nutribullet®. The powder obtained was added to the PDA at a concentration of 24.8 mg/mL, which was sterilized by filtration through 0.22 μm Millipore filters.’ Bacteria can be filtered out by 0.22 μm Millipore filters, I do not think that the PDA containing P. sanguineus powder sterilized by it is a wise method. please make sure that the sterilization method.

We regret not having been able to express this to you correctly, at first the solution was filtered using the 0.22 micron filters. After this, the medium was sterilized at 120 degrees for 15 min according to the standard technique. 

2.5. Statistical analysis

Point 8. L216, L223: “using the statistical software SPSS Statistics version 17 for the Windows operating environment” is repeated. The description of this section is confuse and complicated, authors should rephrase. 

The description of the program used for the statistical analysis is now included at the end of the statistical analysis paragraph for clarity.

3 Results

Point 9. L237-240: “The ANOVA results for PIRG values among the different treatments evaluated against F. oxysporum indicate that in Model 1, P. sanguineus has a moderate antagonistic effect, similar to the action of Cinnabarin and Benomyl in Model 2 (9.54%, 17.79%, and 15.98%, respectively, p < 0.05).” The sentence may confuse the readers. I suggest author delete the content in parenthese.  Done

 Point 10. Table 1 and table 2，p ≤ 0.05 or p < 0.05, please check. Done

Point 11. Figure 2，the resolution of picture (fungal colonies) was very low and were very difficult to read.

We appreciate your comment on the image resolution. The image already presents the characteristics indicated by the journal; however, we have inserted a larger image in this corrected version of the text, hoping that the journal does not encounter complications due to its size and weight. We do not emphasize the part of the photographs of the fungal colony growth within the figure since we have only included them as a visual reference. Therefore, if it is a problem, we could eliminate them without much difficulty.

 Discussion.

Point 12. L286-287: “various pathogenic fungal”

In this section of the discussion, we try to argue the diversity of fungi from different aspects, both medicinal and the food industry with which comparative studies can be recognized.

 Point 13. L306: “a lower GR” Done

 Point 14. Please check the Latin writing of fungal species in this manuscript. For example, L338:“… Colletotrichum fragariae, C. gloeosporioides, and B. cinerea, …” Done

 References

Point 15. Please unify the format of references in this manuscript. Done

Reviewer 2 Report

Comments and Suggestions for Authors

The authors developed a study with the fungus Pycnoporus sanguineus in vitro and managed to achieve the proposed objectives.

It has been demonstrated that P. sanguineus produces bioactive substances against the fungi Botrytis cinerea and Fusarium oxysporum, especially when its extract was added to the PDA culture medium. However, its inhibitory effect was lower than the effects of the fungicides Benomyl and Captan.

Questions about this study

1. Explain in detail why the fungi B. cinerea and F.oxysporum were chosen for this type of study? The first is an aerial pathogen and the second is a root pathogen and and how the active ingredients of P. sanguineus extract could control these phytopathogens.

2. Explain in detail and references about the choice of the fungicides Benomyl (systemic action on aerial parts) and Captan (protective action on plants and seeds) for this type of study as a comparison of antibiosis against P. sanguineus. Benomyl is not an active ingredient suitable for controlling B. cinerea, due to the known induction of resistance (see literature) in strains of this fungus. Also, it is not a suitable active ingredient against F. oxysporum, as it is a soil pathogen. Captan is an active ingredient for protecting plants and seeds and is perhaps better than Benomyl for this type of study. For Botrytis, another active ingredient would be better, such as mancozeb, iprodione, chlorothalonil…

3. Was the experimental design appropriate for the study? The use of 5 replications for each treatment in the experimental design should be reviewed. A greater number of repetitions (perhaps up to 10) could have been used to reduce experimental error, with more satisfactory results in statistical analysis and statistical difference between means.

4. The method of incorporating active ingredients into PDA medium to evaluate the inhibition of pathogen development may not have been efficient. The inhibition of the P. sanguineus extract may have been masked, because the PDA medium is rich in nutrients and this can affect the inhibition of mycelial growth. The use of minimal medium plus carbohydrates would be closer to what happens during parasitism of the pathogens in plants, revealing marked effects of P. sanguineus extracts in vitro. As these pathogens produce a series of enzymes, they can detoxify the cultivation medium, reducing the effect of the active ingredients present in the P. sanguineus extract.

5. I would consider the choice of other foliar pathogens which cause powdery mildew and rust, as well as post-harvest fruit diseases, perhaps more important diseases. Botrytis cinerea causes gray mold on shoots, young leaves and fruits, in addition to being a saprophyte, while F. oxysporum is a root pathogen. In the latter case, the extract or active ingredient would end up being difficult to use for its control.

Author Response

Questions about this study

1. Explain in detail why the fungi B. cinereaand F.oxysporumwere chosen for this type of study? The first is an aerial pathogen and the second is a root pathogen and and how the active ingredients of P. sanguineus extract could control these phytopathogens.

The choice of the pathogens B. cinerea and F. oxysporum was precisely because we wanted to test the effectiveness of P. sanguineus against pathogens with different characteristics affecting plants. At the same time, these species cause significant damage to crops in our region but also globally, so the results of our work could be replicated in other regions of the world. By testing them on pathogens with different infection characteristics, it's assumed a broader range of action. Although cinnabarin is one of the compounds present in the extract of P. sanguineus that has been most studied, especially for studied in the control of bacterial pathogens (Smânia et al., 1995 and 1997) and even viral ones (Smânia et al., 2003), little research has been done on this compound against fungal pathogens, although it has been considered that the nonspecific extracts of P. sanguineus may have greater potential in controlling fungal pathogens due to the presence of other components with antifungal activity such as polyporin (Henrique Rosa et al., 2003), ergosterol (Dias & Urban, 2009), and 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl (Teoh et al., 2011), as well as enzymes and other compounds involved in antioxidant processes (Borderes et al., 2011). Therefore, one of the principal purposes of this study was precisely to recognize the contrast of the action of cinnabarin as a fungal controller compared to a solution of complete extract from P. sanguineus.

2. Explain in detail and references about the choice of the fungicides Benomyl (systemic action on aerial parts) and Captan (protective action on plants and seeds) for this type of study as a comparison of antibiosis against P. sanguineus. Benomyl is not an active ingredient suitable for controlling B. cinerea, due to the known induction of resistance (see literature) in strains of this fungus. Also, it is not a suitable active ingredient against F. oxysporum, as it is a soil pathogen. Captan is an active ingredient for protecting plants and seeds and is perhaps better than Benomyl for this type of study. For Botrytis, another active ingredient would be better, such as mancozeb, iprodione, chlorothalonil…

Dear Reviewer, you are right that the fungicide Benomyl is not a suitable active ingredient to control B. cinerea, since this pathogen is resistant as mentioned in several investigations. However, for this study it was decided to use it as a control, since in Mexico there is no culture of selecting the best active ingredients for a specific pathogen. It was used because in Mexico it is the one with the highest sales and distribution in various agricultural crops, as mentioned in the following publications, in addition to being also used in various studies as a control group:

Alejandro, M. N. M., Guadalupe, B. E., Omar, T. S. F., & Patricia, R. R. (2022). Temporal and spatial analysis of benomyl/carbendazim in water and its possible impact on Nile tilapia (Oreochromis niloticus) from Tenango dam, Puebla, Mexico. Environmental Monitoring and Assessment, 194(1), 23.

Núñez-Rios, T., Leyva-Mir, S. G., Rodríguez-Pérez, J. E., & Mariscal-Amaro, L. A. (2013). Etiología y control de la necrosis de flores y pudrición de frutos de pepino en Morelos, México. Revista Chapingo. Serie horticultura, 19(2), 255-255.

Espinoza-Altamirano, D., Silva-Rojas, H. V., Leyva-Mir, S. G., Marbán-Mendoza, N., & Rebollar-Alviter, Á. (2017). Sensitivity of Colletotrichum acutatum isolates obtained from strawberry to tiophanate-methyl and azoxystrobin fungicides. Revista mexicana de fitopatología, 35(2), 186-203.

Sánchez-Alarcón, J., Milić, M., Bonassi, S., Gómez-Arroyo, S., Cortés-Eslava, J., Flores-Márquez, A. R., ... & Valencia-Quintana, R. (2023). Occupational exposure to pesticides: DNA damage in horticulturist from Nativitas, Tlaxcala in Mexico. Environmental Toxicology and Pharmacology, 100, 104141.

Moo-Muñoz, A., Azorín-Vega, E., Ramírez-Durán, N., & Moreno-Pérez, P. (2020). State of the production and consumption of pesticides in Mexico. Tropical and Subtropical Agroecosystems, 23(2).

Ramírez-Salcedo, H. E., Barrientos-Ramírez, L., Vargas-Radillo, J. J., Rodríguez-Macías, R., Ruíz-López, M. A., & Virgen-Calleros, G. (2019). Inhibición de Colletotrichum gloeosporioides y Botrytis cinerea con extractos de Guazuma ulmifolia Lam. Revista mexicana de fitopatología, 37(2), 330-344.

Aguilar-Barragan, A., García-Torres, A. E., Odriozola-Casas, O., Macedo-Raygoza, G., Ogura, T., Manzo-Sánchez, G., ... & Beltrán-García, M. J. (2014). Chemical management in fungicide sensivity of Mycosphaerella fijiensis collected from banana fields in México. Brazilian Journal of Microbiology, 45, 359-364.

Another reason for using it is the fact of reporting Benomyl-resistant fungi and regions where they are found, in this case the MA-BC20 strain of B. cinerea isolated from roots and fruits of a strawberry crop in the municipality of Puebla, There is no record of its presence in the producers' fields, thanks to this study it will be revealed that the strain is resistant and the corresponding report will be given to the health authorities to avoid the use of Benomyl in this region, this will give the opportunity to apply another fungicide like the ones you mention and thus avoid the spread or appearance of other strains resistant to Benomyl, such as Colletotrichum truncatum in bell pepper fields in Trinidad as a result of the continuous use of the benzimidazole fungicide.

Ramdial, H., Hosein, F. N., & Rampersad, S. N. (2016). Detection and molecular characterization of benzimidazole resistance among Colletotrichum truncatum isolates infecting bell pepper in Trinidad. Plant disease, 100(6), 1146-1152.

Álvarez-Medina, A., Silva-Rojas, H. V., Leyva-Mir, S. G., Marbán-Mendoza, N., & Rebollar-Alviter, Á. (2017). Resistencia de Botrytis cinerea de fresa (Fragaria x ananassa Duch.) a fungicidas en Michoacan México. Agrociencia, 51(7), 783-798.

3. Was the experimental design appropriate for the study? The use of 5 replications for each treatment in the experimental design should be reviewed. A greater number of repetitions (perhaps up to 10) could have been used to reduce experimental error, with more satisfactory results in statistical analysis and statistical difference between means.

We agree that a higher number of replications could emphasize the results obtained. However, we regret that this was mainly limited to the amount of P. sanguineus available growing at the time. We hope to be able to carry out more complete experiments once we have more appropriate techniques for the growth of the P. sanguineus fungus at the laboratory level.

4. The method of incorporating active ingredients into PDA medium to evaluate the inhibition of pathogen development may not have been efficient. The inhibition of the P. sanguineusextract may have been masked, because the PDA medium is rich in nutrients and this can affect the inhibition of mycelial growth. The use of minimal medium plus carbohydrates would be closer to what happens during parasitism of the pathogens in plants, revealing marked effects of P. sanguineusextracts in vitro. As these pathogens produce a series of enzymes, they can detoxify the cultivation medium, reducing the effect of the active ingredients present in the P. sanguineus extract.

We very much appreciate this observation. In fact, initially the contrast we expected was the reduction in growth compared to PDA without additives, perhaps assuming that a medium low in nutrients could mask the normal growth of the pathogen especially in cases where benomyl and captan compounds were used, which already reduced and completely halted their growth, perhaps in a nutrient-poor medium, no growth would have been observed, but it can also be read inversely. Thanks to your suggestion, we will surely include this variant in subsequent tests to rule out this situation.

5. I would consider the choice of other foliar pathogens which cause powdery mildew and rust, as well as post-harvest fruit diseases, perhaps more important diseases. Botrytis cinereacauses gray mold on shoots, young leaves and fruits, in addition to being a saprophyte, while F. oxysporumis a root pathogen. In the latter case, the extract or active ingredient would end up being difficult to use for its control.

Indeed, the application methods of these models in the field is one of the questions we have raised for the near future, in fact, during the final phase of the experiment, we asked ourselves about the possibility of applying both cinnabarin and P. sanguineus extract and one of the answers was the application of post-harvest fruit conservation. This is probably the line to follow in experiments derived from this work, in the same way we have planned its application in the field for both the aerial part and the root part of plants through dilutions by spraying (for aerial parts) and irrigation (for underground parts).

Reviewer 3 Report

Comments and Suggestions for Authors

Title:  Comparison of three models of biological control of fungal dis eases in plants using the fungus Pycnoporus sanguineus

Authors:

Ricardo Irving Pérez-López, Omar Romero-Arenas, Conrado Parraguirre Lezama, Anabel Romero López, Lilia Cedillo Ramírez, Antonio Rivera

 

The objective of this study is to evaluate the effect of three action models of Pycnoporus sanguineus on the growth of two phytopathogenic fungi in vitro that have a significant economic impact on vegetable cultivation. These will be compared with two commercial products intended for the same purpose, allowing the proposal of models for the generation of high-quality biotechnological products with a direct application in the management and control of diseases in the main commercial crops.

 

1. Line 102: “the formula GR = FR-IR/number of days” is wrong.

It should be “the formula GR = (FR-IR)/number of days”.

 

2. Line 105: “the formula PIRG = R1-R2/R1*100” is wrong.

It should be “the formula PIRG = (R1-R2)/R1*100”.

 

3. Line 158: “…..as well as water sensidiscs vs B. cinerea and F. oxysporum,……”

Water should be replaced by culture medium.

 

4. In Table 2: p < 0.05

But * Media followed by the same letter does not present statistically significant differences (p ≤ 0.05) according to Tukey’s test.

p value should keep consistent.

 

5. There are many grammar and tense errors.

Such as Line 360-361: “The results of this study indicate a trend in the control of the phytopathogens B.  cinerea and F. oxysporum.”

The past tense should be used.

Please revise them carefully after checking all the manuscript.

Comments on the Quality of English Language

Extensive editing of English language required

Author Response

Thanks for the comments and suggestions.

The changes were made and are shown in blue in the document.

1. Line 102: “the formula GR = FR-IR/number of days” is wrong. Done

It should be “the formula GR = (FR-IR)/number of days”.

2. Line 105: “the formula PIRG = R1-R2/R1*100” is wrong. Done

It should be “the formula PIRG = (R1-R2)/R1*100”.

3. Line 158: “…..as well as water sensidiscs vs B. cinerea and F. oxysporum,……”

Water should be replaced by culture medium. Done

4. In Table 2: p < 0.05

But * Media followed by the same letter does not present statistically significant differences (p ≤ 0.05) according to Tukey’s test.

p value should keep consistent.   Done

5. There are many grammar and tense errors.

Such as Line 360-361: “The results of this study indicated a trend in the control of the phytopathogens B.  cinerea and F. oxysporum.”

The past tense should be used.  Done

Please revise them carefully after checking all the manuscript. 

Comments on the Quality of English Language

Extensive editing of English language required

We greatly appreciate your concern for the quality of language presented in our work. We have considered that, since the group that prepared this work lacks a native English speaker, we have decided to send our work for professional review. Therefore, the updated and corrected version of the work has already undergone this review (the certificate is attached: english-81351). We hope that this will contribute to a significant improvement in the quality of our work. Thank you very much.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised did not resolve my previous concerns and i still suggest to reject it at the present form.