Antifungal Activity of Native Trichoderma afroharzianum and Arcopilus cupreus Against Neopestalotiopsis rosae Causing Strawberry Root and Crown Rot in Mexico

Round 1

Reviewer 1 Report

This manuscript investigated the occurrence of Neopestalotiopsis rosae associated with strawberry root and crown rot in Mexico and evaluated the antagonistic activity of native Trichoderma afroharzianum isolates and one Arcopilus cupreus isolate against the pathogen. The study is well organized and presents a relatively comprehensive experimental workflow including pathogen isolation, multilocus phylogenetic identification, pathogenicity assays, and in vitro biocontrol evaluation. The topic is relevant and timely, particularly considering the increasing importance of sustainable disease management strategies in strawberry production systems.

The manuscript provides useful information regarding the diversity and aggressiveness of N. rosae isolates in major strawberry-producing regions of Mexico. In addition, the identification of native T. afroharzianum isolates with strong antagonistic activity represents a potentially valuable contribution for future biological control applications.

Overall, the manuscript is suitable for publication after minor to moderate revision. The following points should be considered to further improve the quality and clarity of the study.

Comments

1. The current study mainly focuses on in vitro antagonistic assays. Although the results are promising, the manuscript would benefit from a brief discussion of the limitations of dual-culture assays and the need for future greenhouse or field validation experiments.
2. The Discussion section could be strengthened by including more mechanistic interpretation regarding the possible modes of action of Trichoderma afroharzianum (e.g., antibiosis, competition, mycoparasitism, or secondary metabolite production).
3. Some phylogenetic nodes in Figure 3 show relatively moderate support values. The authors may briefly discuss the robustness of species identification and whether additional loci could improve phylogenetic resolution.
4. Please carefully revise the manuscript for minor grammatical and stylistic issues. Some sentences in the Introduction and Results sections could be simplified for better readability.

1. In Materials and Methods, genomic DNA was reported to be stored at “−4°C”. Please verify whether this is a typographical error.
2. The formula formatting in the pathogenicity section appears abnormal in the current manuscript version and should be carefully checked before publication.
3. In Table 3, “Percentage of infection (PI)” may be better described as disease severity or disease index for improved clarity.
4. Some figures, particularly the phylogenetic trees, would benefit from higher resolution and larger font sizes to improve readability.
5. Please carefully check the manuscript for minor typographical inconsistencies and formatting issues, including spacing, italics of species names, and unit formatting.
6. Abbreviations should be checked for consistency throughout the manuscript (e.g., PRGI/PRMG).

Author Response

Reviewer 1

Major comments

This manuscript investigated the occurrence of Neopestalotiopsis rosae associated with strawberry root and crown rot in Mexico and evaluated the antagonistic activity of native Trichoderma afroharzianum isolates and one Arcopilus cupreus isolate against the pathogen. The study is well organized and presents a relatively comprehensive experimental workflow including pathogen isolation, multilocus phylogenetic identification, pathogenicity assays, and in vitro biocontrol evaluation. The topic is relevant and timely, particularly considering the increasing importance of sustainable disease management strategies in strawberry production systems. The manuscript provides useful information regarding the diversity and aggressiveness of N. rosae isolates in major strawberry-producing regions of Mexico. In addition, the identification of native T. afroharzianum isolates with strong antagonistic activity represents a potentially valuable contribution for future biological control applications.

Response: Thank you very much for taking the time to review this manuscript. Please find the detailed responses below, along with the corresponding revisions/corrections highlighted in the re-submitted files.

Overall, the manuscript is suitable for publication after minor to moderate revision. The following points should be considered to further improve the quality and clarity of the study.

Comments

1. The current study mainly focuses on in vitro antagonistic assays. Although the results are promising, the manuscript would benefit from a brief discussion of the limitations of dual-culture assays and the need for future greenhouse or field validation experiments.

Response: Thank you for your feedback. We have included a paragraph in the discussion section.

2. The Discussion section could be strengthened by including more mechanistic interpretation regarding the possible modes of action of Trichoderma afroharzianum (e.g., antibiosis, competition, mycoparasitism, or secondary metabolite production).

Response: Thank you for your feedback. We have included a paragraph in the discussion section.

3. Some phylogenetic nodes in Figure 3 show relatively moderate support values. The authors may briefly discuss the robustness of species identification and whether additional loci could improve phylogenetic resolution.

Response: Thank you for your feedback. We have included a paragraph in the discussion section.

4. Please carefully revise the manuscript for minor grammatical and stylistic issues. Some sentences in the Introduction and Results sections could be simplified for better readability.

Response: Thank you for your feedback. We have reviewed the document and made the necessary changes.

Detailed comments

In Materials and Methods, genomic DNA was reported to be stored at “−4°C”. Please verify whether this is a typographical error.

Response: Thank you for your observation. The DNA was stored at -20 °C; we have corrected this in the document.

The formula formatting in the pathogenicity section appears abnormal in the current manuscript version and should be carefully checked before publication.

Response: The formula was reviewed by Dr. Lauro Soto Rojas, a statistician, who verified its correctness.

In Table 3, “Percentage of infection (PI)” may be better described as disease severity or disease index for improved clarity.

Response: Thank you for your suggestion. We have made the change.

Some figures, particularly the phylogenetic trees, would benefit from higher resolution and larger font sizes to improve readability.

Response: Thank you for the observation. We have increased the size of figures 3 and 5 to make them easier to read.

Please carefully check the manuscript for minor typographical inconsistencies and formatting issues, including spacing, italics of species names, and unit formatting.

Response: Thank you for your feedback. We have reviewed the document and made the necessary changes.

Abbreviations should be checked for consistency throughout the manuscript (e.g., PRGI/PRMG).

Response: Thank you for the observation. We have standardized the abbreviations

Reviewer 2 Report

The manuscript addresses a relevant and increasingly important issue related to strawberry root and crown rot caused by Neopestalotiopsis rosae. Considering the growing number of reports involving this pathogen in strawberry-producing areas, the study provides useful information for both plant pathology and biological control research.

A major strength of the work is the combination of morphological observations, molecular identification, pathogenicity tests, and antagonism assays, which together provide consistent support for the conclusions presented. The use of native Trichoderma afroharzianum isolates is particularly interesting, since locally adapted strains may offer advantages under regional production conditions and could represent promising candidates for sustainable disease management strategies.

The results obtained from the antagonism assays demonstrate good potential for the use of these isolates as biological control agents against N. rosae. This is especially relevant given current concerns regarding the excessive use of chemical fungicides, environmental impact, and the need for more sustainable alternatives in strawberry production systems.

The manuscript is generally well organized and clearly written. However, some sections could be strengthened. In particular, the discussion would benefit from a deeper exploration of the possible mechanisms involved in fungal antagonism, including competition, mycoparasitism, and production of antifungal metabolites. It would also be important to discuss the limitations of in vitro assays and the need for future validation under greenhouse and field conditions.

In addition, some methodological details could be described more clearly, especially regarding statistical analyses and experimental replication. Expanding the discussion with comparisons to recent international studies on Neopestalotiopsis spp. would also improve the broader scientific context of the work.

Overall, the study presents good scientific merit and practical relevance. With minor revisions focused mainly on clarity and discussion depth, the manuscript has good potential for publication.

The manuscript presents relevant results and a generally sound experimental approach. The combination of morphological, molecular, pathogenicity, and antagonism analyses provides good support for the conclusions. However, several points should be clarified or strengthened to improve the scientific robustness of the study.

Necessary corrections:

1. Materials and Methods – Sampling strategy
   The manuscript does not clearly indicate the number of sampling sites evaluated in each production region or the criteria used for selecting symptomatic plants. This information is important to better understand the representativeness of the isolates analyzed.
2. Pathogenicity assays
   The description of the pathogenicity tests requires additional detail. The manuscript should clearly specify the number of plants used per treatment, the number of experimental replicates, inoculum concentration, environmental conditions during the assays, and the evaluation criteria used for disease severity assessment. These details are necessary to ensure reproducibility.
3. Statistical analysis
   The statistical methodology is insufficiently described. The manuscript should specify whether assumptions of normality and homogeneity of variance were evaluated before analysis. In addition, the authors should clearly indicate the statistical model used, the software version, and the post-hoc tests applied. Some results are described as significantly different, but the statistical support is not always fully explained in the text or tables.
4. Antagonism assays
   The methodology for calculating mycelial growth inhibition should be more clearly described. It is also unclear whether biological and technical replicates were included. Since antagonism assays are central to the manuscript conclusions, experimental replication should be explicitly detailed.
5. Discussion of in vitro limitations
   The discussion would benefit from a clearer acknowledgment that antagonistic activity observed under in vitro conditions may not necessarily reflect performance under greenhouse or field conditions. This limitation should be explicitly discussed.

Desirable improvements:

1. Introduction and Discussion
   The manuscript could provide a broader discussion of the global emergence of Neopestalotiopsis spp. in strawberry production systems and compare the present findings with recent international reports.
2. Mechanisms of antagonism
   The discussion could be strengthened by including more information regarding potential mechanisms involved in Trichoderma-mediated antagonism, such as mycoparasitism, hydrolytic enzymes, competition for nutrients, and production of antifungal metabolites.
3. Figures and phylogenetic analyses
   Some phylogenetic descriptions in the Results section are excessively detailed and could be summarized more objectively. In addition, improving the clarity of some figure labels and scale bars would facilitate interpretation.
4. Presentation of results
   In some sections, the text repeats information already shown in tables. The Results section would benefit from a more interpretative approach emphasizing the most relevant biological findings rather than reproducing numerical values.
5. Native isolates
   The manuscript highlights the use of native isolates, which is an important aspect of the study. However, the ecological and practical implications of using locally adapted biocontrol agents could be explored more thoroughly in the Discussion.

Statistical review:

At the current stage, a full statistical review does not appear strictly necessary. However, clarification of the statistical procedures is required before publication. If the authors performed more complex analyses beyond basic ANOVA and mean comparisons, or if assumptions were not properly verified, consultation with a statistician may become necessary to confirm the validity of the analyses and interpretation of significance.

Author Response

Reviewer 2

Major comments

The manuscript addresses a relevant and increasingly important issue related to strawberry root and crown rot caused by Neopestalotiopsis rosae. Considering the growing number of reports involving this pathogen in strawberry-producing areas, the study provides useful information for both plant pathology and biological control research.

A major strength of the work is the combination of morphological observations, molecular identification, pathogenicity tests, and antagonism assays, which together provide consistent support for the conclusions presented. The use of native Trichoderma afroharzianum isolates is particularly interesting, since locally adapted strains may offer advantages under regional production conditions and could represent promising candidates for sustainable disease management strategies.

The results obtained from the antagonism assays demonstrate good potential for the use of these isolates as biological control agents against N. rosae. This is especially relevant given current concerns regarding the excessive use of chemical fungicides, environmental impact, and the need for more sustainable alternatives in strawberry production systems.

The manuscript is generally well organized and clearly written. However, some sections could be strengthened. In particular, the discussion would benefit from a deeper exploration of the possible mechanisms involved in fungal antagonism, including competition, mycoparasitism, and production of antifungal metabolites. It would also be important to discuss the limitations of in vitro assays and the need for future validation under greenhouse and field conditions.

In addition, some methodological details could be described more clearly, especially regarding statistical analyses and experimental replication. Expanding the discussion with comparisons to recent international studies on Neopestalotiopsis spp. would also improve the broader scientific context of the work.

Overall, the study presents good scientific merit and practical relevance. With minor revisions focused mainly on clarity and discussion depth, the manuscript has good potential for publication.

Response: Thank you very much for taking the time to review this manuscript. Please find the detailed responses below, along with the corresponding revisions/corrections highlighted in the re-submitted files.

Detailed comments

The manuscript presents relevant results and a generally sound experimental approach. The combination of morphological, molecular, pathogenicity, and antagonism analyses provides good support for the conclusions. However, several points should be clarified or strengthened to improve the scientific robustness of the study.

Necessary corrections:

1. Materials and Methods – Sampling strategy
The manuscript does not clearly indicate the number of sampling sites evaluated in each production region or the criteria used for selecting symptomatic plants. This information is important to better understand the representativeness of the isolates analyzed.

Response:

Table 1 shows the origin of each isolate. As can be seen, they come from three states of the Mexican Republic. The state of Baja California borders the southern United States (Northwest Region), while the states of Michoacán and Guanajuato are located in the central region of Mexico (Central-Western Region). Lines 101-104 of the document list the regions to which the states where the diseased plants were collected belong.

2. Pathogenicity assays
The description of the pathogenicity tests requires additional detail. The manuscript should clearly specify the number of plants used per treatment, the number of experimental replicates, inoculum concentration, environmental conditions during the assays, and the evaluation criteria used for disease severity assessment. These details are necessary to ensure reproducibility.

Response: Thank you for your comment. The document clearly states the requested information. The number of experimental replicates has been added. Line 210 -251.

3. Statistical analysis
The statistical methodology is insufficiently described. The manuscript should specify whether assumptions of normality and homogeneity of variance were evaluated before analysis. In addition, the authors should clearly indicate the statistical model used, the software version, and the post-hoc tests applied. Some results are described as significantly different, but the statistical support is not always fully explained in the text or tables.

Response: Thank you for your comment. The software and its version used for the statistical analyses have been added. We have also included the experimental designs used to design and analyze the pathogenicity and antagonism bioassays. Finally, we have added the missing information on the evaluation of normality and homogeneity of variance before analysis.

4. Antagonism assays
The methodology for calculating mycelial growth inhibition should be more clearly described. It is also unclear whether biological and technical replicates were included. Since antagonism assays are central to the manuscript conclusions, experimental replication should be explicitly detailed.

Response: Thank you for your comment. The daily growth of the isolates was marked on the base of the Petri dish using a permanent marker. The growth was then measured with a caliper. The data were entered into an Excel file and the formula indicated in the document was applied. Because the results were consistent, the antagonism test was performed only once.

5. Discussion of in vitro limitations
The discussion would benefit from a clearer acknowledgment that antagonistic activity observed under in vitro conditions may not necessarily reflect performance under greenhouse or field conditions. This limitation should be explicitly discussed.

Response: Thank you for pointing this out. We agree with your comment. We have included this information in the further research.

Desirable improvements:

1. Introduction and Discussion
The manuscript could provide a broader discussion of the global emergence of Neopestalotiopsis spp. in strawberry production systems and compare the present findings with recent international reports.

Response: Thank you for your comment. A short paragraph has been added to the introduction with information on the first report of the pathogen in strawberries, as well as chronological reports from different strawberry-growing countries. Our results coincide with those reported by Rebollar-Alviter et al. (2020), who made the first report of N. rosae in Mexico.

2. Mechanisms of antagonism
The discussion could be strengthened by including more information regarding potential mechanisms involved in Trichoderma-mediated antagonism, such as mycoparasitism, hydrolytic enzymes, competition for nutrients, and production of antifungal metabolites.

Response: Thank you for your feedback. We have included a paragraph in the discussion section.

3. Figures and phylogenetic analyses
Some phylogenetic descriptions in the Results section are excessively detailed and could be summarized more objectively. In addition, improving the clarity of some figure labels and scale bars would facilitate interpretation.

Response: Thank you for your comment. The description of the phylogeny results only includes the bare minimum necessary to explain the figures. Likewise, the captions and labels were prepared in accordance with internationally established guidelines for publication in mycology journals (including nomenclature and bars). In this way, each figure clearly indicates what is observed in each image.

4. Presentation of results
In some sections, the text repeats information already shown in tables. The Results section would benefit from a more interpretative approach emphasizing the most relevant biological findings rather than reproducing numerical values.

Response: Thank you for your comment. It is true that the results section includes some data from the tables, but only the most relevant data are included, without a complete description of the tables within the text. If this were not the case, the tables would have to be removed. The tables are included so that the reader has access to all the treatment results. Furthermore, the interpretation of the results obtained can be found in the discussion section.

5. Native isolates
The manuscript highlights the use of native isolates, which is an important aspect of the study. However, the ecological and practical implications of using locally adapted biocontrol agents could be explored more greater depth in the Discussion.

Response: Thank you for your comment. We have added a paragraph discussing the practical and ecological implications of using native biocontrol agents.

Statistical review:

At the current stage, a full statistical review does not appear strictly necessary. However, clarification of the statistical procedures is required before publication. If the authors performed more complex analyses beyond basic ANOVA and mean comparisons, or if assumptions were not properly verified, consultation with a statistician may become necessary to confirm the validity of the analyses and interpretation of significance.

Response: Thank you for your comment. We understand your concern on this point. The statistical analyses were performed by the first author under the guidance of Dr. Alfredo Jimenez (co-author) and reviewed by statistician Lauro Soto. Therefore, the authors are confident in the relevance and scientific rigor of the analyses performed.

Reviewer 3 Report

Manuscript entitled “Antifungal Activity of Native Trichoderma afroharzianum and Arcopilus cupreus Against Neopestalotiopsis rosae Causing Strawberry Root and Crown Rot in Mexico”. This study investigated the antifungal activity of several native Trichoderma afroharzianum strains and Arcopilus cupreus strain to isolated Neopestalotiopsis rosae strains. The manuscript is well designed, several points need to be addressed before it can be accepted.

1. Keywords: Neopestalotiopsis, Trichoderma, Arcopilus, strawberry diseases should be replaced by Neopestalotiopsis rosae, Trichoderma afroharzianum, Arcopilus cupreus, Strawberry Root and Crown Rot
2. Line 52. The author didn’t introduce root and crown rot in Line 47-51. Therefore, “Among these” is not suitable.
3. Figure 1 should appear below Line 116.
4. Line 122. Did the author check the effect of surface-disinfected of recovered tissue?
5. Line 163-164. Molecular identification of Neopestalotiopsis using ITS, β-tubulin and translation elongation factor 1-alpha genes for amplification, and identification of Trichoderma using ITS, rpb2 and tef1-α (Line 270). What is the full name of rpb2? Why identification of Neopestalotiopsis and Trichoderma using different genes (β-tub in Neopestalotiopsis, rpb2 in Trichoderma)? Is β-tub specific in Neopestalotiopsis and rpb2 specific in Trichoderma?
6. Line 241, 316, 428. p < 0.05 should be italic.
7. Line 297. “Biocontrol tests” should be replaced by “Antagonism tests”. Because there are any greenhouse or field assays were conducted in the current study, therefore, biocontrol tests are not appropriate,
8. Line 298-299. The sentence should be rewrite. Trichoderma sp. isolates (MBP1–MBP4) and one isolate of Arcopilus cupreus against sixteen Neopestalotiopsis, but not Neopestalotiopsis against Trichoderma and Arcopilus cupreus.
9. Line 664. Greenhouse or field assays to evaluate the control efficacy of Trichoderma sp. isolates (MBP1–MBP4) and one isolate of Arcopilus cupreus against Neopestalotiopsis should be added in the further research.

Manuscript entitled “Antifungal Activity of Native Trichoderma afroharzianum and Arcopilus cupreus Against Neopestalotiopsis rosae Causing Strawberry Root and Crown Rot in Mexico”. This study investigated the antifungal activity of several native Trichoderma afroharzianum strains and Arcopilus cupreus strain to isolated Neopestalotiopsis rosae strains. The manuscript is well designed, several points need to be addressed before it can be accepted.

1. Keywords: Neopestalotiopsis, Trichoderma, Arcopilus, strawberry diseases should be replaced by Neopestalotiopsis rosae, Trichoderma afroharzianum, Arcopilus cupreus, Strawberry Root and Crown Rot
2. Line 52. The author didn’t introduce root and crown rot in Line 47-51. Therefore, “Among these” is not suitable.
3. Figure 1 should appear below Line 116.
4. Line 122. Did the author check the effect of surface-disinfected of recovered tissue?
5. Line 163-164. Molecular identification of Neopestalotiopsis using ITS, β-tubulin and translation elongation factor 1-alpha genes for amplification, and identification of Trichoderma using ITS, rpb2 and tef1-α (Line 270). What is the full name of rpb2? Why identification of Neopestalotiopsis and Trichoderma using different genes (β-tub in Neopestalotiopsis, rpb2 in Trichoderma)? Is β-tub specific in Neopestalotiopsis and rpb2 specific in Trichoderma?
6. Line 241, 316, 428. p < 0.05 should be italic.
7. Line 297. “Biocontrol tests” should be replaced by “Antagonism tests”. Because there are any greenhouse or field assays were conducted in the current study, therefore, biocontrol tests are not appropriate,
8. Line 298-299. The sentence should be rewrite. Trichoderma sp. isolates (MBP1–MBP4) and one isolate of Arcopilus cupreus against sixteen Neopestalotiopsis, but not Neopestalotiopsis against Trichoderma and Arcopilus cupreus.
9. Line 664. Greenhouse or field assays to evaluate the control efficacy of Trichoderma sp. isolates (MBP1–MBP4) and one isolate of Arcopilus cupreus against Neopestalotiopsis should be added in the further research.

Author Response

Reviewer 3

Manuscript entitled “Antifungal Activity of Native Trichoderma afroharzianum and Arcopilus cupreus Against Neopestalotiopsis rosae Causing Strawberry Root and Crown Rot in Mexico”. This study investigated the antifungal activity of several native Trichoderma afroharzianum strains and Arcopilus cupreus strain to isolated Neopestalotiopsis rosae strains.

Response: Thank you very much for taking the time to review this manuscript. Please find the detailed responses below, along with the corresponding revisions/corrections highlighted in the re-submitted files.

The manuscript is well designed, several points need to be addressed before it can be accepted.

1. Keywords: Neopestalotiopsis, Trichoderma, Arcopilus, strawberry diseases should be replaced by Neopestalotiopsis rosae, Trichoderma afroharzianum, Arcopilus cupreus, Strawberry Root and Crown Rot

Response: Thank you for pointing this out. We agree with your comment. We have modified the keywords.

2. Line 52. The author didn’t introduce root and crown rot in Line 47-51. Therefore, “Among these” is not suitable.

Response: Thank you for your observation. We have changed it to “however”.

3. Figure 1 should appear below Line 116.

Response: Thank you for your observation. We have relocated the image as close as possible to the section “Recollection of diseased strawberry plants”

4. Line 122. Did the author check the effect of surface-disinfected of recovered tissue?

Response: Thank you for your comment. The efficiency of the disinfection process is evidenced by the absence of bacterial contamination and the fact that only fungal colonies from within the crown tissue are permitted to develop. The disinfection process has proven to be the most efficient, allowing us to disinfect tissue surfaces without bacterial proliferation.

5. Line 163-164. Molecular identification of Neopestalotiopsis using ITS, β-tubulin and translation elongation factor 1-alpha genes for amplification, and identification of Trichoderma using ITS, rpb2 and tef1-α (Line 270). What is the full name of rpb2? Why identification of Neopestalotiopsis and Trichoderma using different genes (β-tub in Neopestalotiopsis, rpb2 in Trichoderma)? Is β-tub specific in Neopestalotiopsis and rpb2 specific in Trichoderma?

Response: Thank you for your observation. We have incorporated the full name of the rpb2 gene. The genes amplified for multigene phylogenetic identification vary by genus. For Neopestalotiopsis, the work of Maharachchikumbura et al. (2014) was followed, while for the genus Trichoderma, the work of Chaverri et al. (2015) was used as a reference. In both cases, the genes with the highest phylogenetic resolution were amplified to discriminate between species.

6. Line 241, 316, 428. p < 0.05 should be italic.

Response: Thank you for your comment. We have switched to italics.

7. Line 297. “Biocontrol tests” should be replaced by “Antagonism tests”. Because there are any greenhouse or field assays were conducted in the current study, therefore, biocontrol tests are not appropriate,

Response: Thank you for pointing this out. We agree with your comment. We have changed the sentence.

8. Line 298-299. The sentence should be rewrite. Trichoderma sp. isolates (MBP1–MBP4) and one isolate of Arcopilus cupreus against sixteen Neopestalotiopsis, but not Neopestalotiopsis against Trichoderma and Arcopilus cupreus.

Response: Thank you for pointing this out. We agree with your comment. We have rewritten this sentence.

9. Line 664. Greenhouse or field assays to evaluate the control efficacy of Trichoderma sp. isolates (MBP1–MBP4) and one isolate of Arcopilus cupreus against Neopestalotiopsis should be added in the further research.

Response: Thank you for pointing this out. We agree with your comment. We have included this information in the further research.

Reviewer 4 Report

the paper is well focussed and sends a message

some sticky notes with comments have been made     for suggested changes

see the sticky notes for comment  suggestions  changes in sentence structure etc

Comments for author File: Comments.pdf

Author Response

Reviewer 4

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below, along with the corresponding revisions and corrections highlighted in the resubmitted files.

Abstract

Ln 20: were these fields disease free  or showing symptoms

Response: Thank you for your comment. The sampled fields are selected based on the presence of plants showing disease symptoms.

Ln 29: 38%

Response: Thank you for your comment. We have left only 38%.

Introduction

Ln 40: productivity in field, post harvest   and shipping, how about  quality too?

Response: Thank you for pointing this out. We have included that the quality of the fruit is also affected.

Ln 44: how do you define propagules, i do not understand

Response: Thank you for pointing this out. It was an editing error, and we have removed this word from the text.

Ln 61: images?

Response: Images of all these symptoms have already been documented; we have included the citations in the paragraph. Although the pathogen can affect virtually all tissues of the strawberry plant, in this case, we focus only on root and crown rot, for which we provide images.

Ln 74: what of the pathogens you cite become endophytic?

Response: The genus Neopestalotiopsis includes fungi that live on plants in various ways. They can behave as endophytes, pathogens, or saprobes (decomposers), depending on the species, the host, and the environmental conditions.

Ln 78: another soil fungus

Response: Yes, A. cupreus is a fungus present in the soil.

Materials and Methods 

Ln 108: Are these anthocyanins due to water problems

Response: Excellent question. The reddish-purple coloration on the leaves of diseased plants has been observed in the field as a symptom associated with Neopestalotiopsis infection; however, to date, it has not been experimentally demonstrated that this coloration results from anthocyanin production induced by water deficiency in the plant.

Ln 136: at what temperature   please add

Response: Done, we have added the temperature.

Ln 212: these conditions allowed sporulation under all conditions

could the pionotal mass be due to bacterial colonization of mycelial surfaces     

this gives a flattened wet growth

Response: Sporulation of the 16 isolates was achieved under the described conditions. Some colonies exhibiting characteristics distinct from those of Neopestalotiopsis were discarded. No bacteria were observed under the microscope during inoculum quantification.

Ln 213: why did you select the pionotal mass

i understand this is sometimes thought of as an mutation   

so it would not be typical wild type growth

Response: Our isolates produce pionotal masses filled with abundant conidia. Recovering the conidia from these masses allowed us to count them and inoculate at the same concentration from each isolate, which is more complicated to do if only the mycelium is used.

Ln 218: seems  like  a very high inoculum difficult to maintain in a commercial product

Response: This data refers to the pathogen concentration. Although all isolates differ in conidia production, we were able to achieve the target concentration in all cases because multiple plates were cultured for each isolate, based on a rapid estimate made prior to plating for the 16 isolates.

Ln 220: was inoculum tested for bacteria?

Response: No bacteria were observed under the microscope during quantification of the inoculum concentration.

Ln 257: but explain your evaluation method

Response: All Trichoderma isolates recovered from soil were tested in dual culture against several pathogens, including Neopestalotiopsis. We selected only the 4 isolates that, on average, showed the highest inhibition percentages, discarding the rest of the isolates.

Ln 274: good to have multilocucus data sets

Response: Thank you for your comment.

Ln 294: what about biocontrol  in other cases  give references

Response: Thank you for pointing this out. This information can be found in the discussion section.

Ln 305: what is the poisoned food technique not heard of it, dual culture ?

Response: The poisoned food technique is a laboratory method used primarily in microbiology and plant pathology to assess the antifungal or antibacterial activity of specific substances. It works by incorporating a test substance (the "poison") directly into a growth medium to evaluate how effectively it inhibits microorganism growth.

Results

Ln 330: discuss if Macrophima  is also a known pathogen

what about complexes of pathogens

Response: Thank you for your comment. The discussion addresses the coexistence of Neopestalotiopsis and Macrophomina in the samples, highlighting Macrophomina as an emerging pathogen in strawberry production in Mexico.

Ln 379: are there known alternate host plants for M rosea?  discuss even if not published

Response: Macrophomina phaseolina is a highly polyphagous (generalist) soil fungus that is important in warm, dry climates. It is estimated to affect at least 500 plant species across more than 100 botanical families. We decided not to elaborate on this pathogen because it falls outside the scope of this research; however, we are working on another document specifically about Macrophomina, which we hope to submit for review in the near future.

Ln 427: any data on runner contamination? seems essential for strawberries

Response: Thank you for pointing this out. This is precisely what happens during plant production in nurseries; however, in this case, the evaluation was conducted before the plants produced runners.

Ln 558: could combine with Table 4  as the control looks the same

what about medium effects though

on a different growth medium  may act differently

Response: Thank you for your comment.

We considered combining the tables; however, the analysis was conducted separately because the two biocontrol agents have different mechanisms of action. We would prefer that they remain separate.

Indeed, the culture medium used can influence the response of the biocontrol agent; however, PDA medium is the most commonly used for evaluating biocontrol agents. This study is a first step, so we used only PDA in the experiments.

Discussion

Ln 567: good  global significance is important

Response: Thank you for your comment.

Ln 584:

could become

think you should look at synergism with the current main fungal pathogen

Response: done, we have changed the word.

Yes, mixed infections involving Macrophomina and Neopestalotiopsis are increasingly common; in fact, the proportion of Macrophomina isolates associated with plant death increases with each cycle.

Ln 631: could be very media spcific

I would call them secreted products

Response:

Indeed, the culture medium used can influence the production of metabolites. We have added this concept to the text.