Characterization, Biocontrol, and Fungicide Sensitivity of Macrophomina phaseolina Isolates Associated with Charcoal Rot of Sesame in Northern Sinaloa, Mexico

Round 1

Reviewer 1 Report

Just need to some improvements  

1. The authors must add a separate section for a statistical analysis in the materials and methods 

2. Line 181-183:"Two isolates of Trichoderma asperellum (FAVF675 and FAVF676) and three isolates of T. harzianum (H7, H21, and H27),":
What are the resource of  five isolates?

3. The section:"3.2. Cultural and morphological characterization":
Do you find "Pycnidia" in the mycelial growth? What about conidia? you don't show any photos about conidia or Pycnidiospores.

4,. The sections: "3.2. Cultural and morphological characterization" and   "3.3. Molecular identification": 
The authors must add a table including isolates with characterizes morphology (Macro + micro) and similarity with other isolates in NCBI

5. The section:"3.6. In vitro fungicide sensitivity":
You need to add photos for this test

6. Line 349-350:"(ITS, act, ßt, cal, and tef1-a).":
You already used ITS and tef-1, why are you mentioned here, if you think costly , why used them.

7. Line 398-401: From "found, under greenhouse" to end: 
You don't work in greenhouse or use plant extract or combination. You can not consider in this study about using combination or individual method.

8. What about the morphological characterizes, you don't mention about that in conclusions.

Author Response

Dear reviewer, we appreciate all the observations made for the improvement of this document; the responses can be found in the attached document.

Author Response File: Author Response.docx

Reviewer 2 Report

1. Objectives are present but it seems hypotheses is not testable. What specific hypotheses were you testing? For example: M. phaseolina isolates from sesame form a monophyletic clade distinct from those on other hosts or Trichoderma asperellum shows greater inhibition than T. harzianum. Without hypotheses, the study is descriptive, not hypothesis‑driven. Justify why a purely descriptive study merits publication.
2. Isolates were collected from only 6 municipalities over two years, yet you generalize to all of Sinaloa. How do you know your six isolates represent the genetic and phenotypic diversity of M. phaseolina across the state? What is the sampling intensity (fields/isolates per hectare)? Provide a power analysis or justify sample size.
3. Only two loci (ITS and tef1‑α). Several recent Macrophomina phylogenies employ 5 loci (ITS, tef1‑α, tub2, cal, act). How do you defend this minimal approach against claims that your tree is under‑resolved? Show bootstrap/posterior probability values for every node, not just selected ones. If your tree fails to resolve relationships among your 6 isolates, state that explicitly.
4. Authors claimed all isolates were pathogenic but provide no disease severity index, no AUDPC, no statistical comparison of aggressiveness among isolates, and no comparison between the two cultivars. How can a reader judge whether isolate CCLF674 is more aggressive than CCLF670? Why did you not measure lesion length, wilting percentage, or plant survival? Without these, the pathogenicity test is merely a qualitative demonstration insufficient. 
5. Sterile toothpicks used as controls. Did you also include a wound‑only control (toothpick inserted without any inoculum) and a non‑wounded control? How do you rule out that wounding alone contributed to symptoms? Provide data on control plant health.
6. 5 Trichoderma isolates were tested without a negative control (e.g., sterile agar plug instead of Trichoderma). How do you know the observed inhibition is not due to nutrient competition from any fast‑growing fungus?
7. PMGI values with letter groupings within each column (Table 1). However, the critical question is there a significant Trichoderma isolate × M. phaseolina isolate interaction? If yes, some Trichoderma strains are specifically effective against certain pathogen strains a key finding. You did not report a two‑way ANOVA with interaction term. Why?
8. Antagonism class (1–5) but never integrate it with PMGI. For example, could a class 2 interaction have a higher PMGI than a class 1 interaction? If the scale adds no information beyond PMGI, why include it? Show a correlation analysis or drop it.
9. Pyraclostrobin, you added SHAM to block alternative respiration, but you did not test whether SHAM alone inhibits M. phaseolina growth. If SHAM itself reduces growth, your EC₅₀ values for pyraclostrobin are artificially lowered. Provide a SHAM‑only control for each isolate. Without it, the pyraclostrobin data are uninterpretable.
10. Each assay was conducted twice. Are these independent biological repeats (different culture batches, different days) or mere technical replicates? Were the two runs statistically combined or analyzed separately? If there was a significant run‑to‑run variation, pooling is invalid. Provide run‑effect analysis.
11. You mention Shapiro‑Wilk and Levene’s tests but report none of the results. For which variables? What were the W and F values and p‑values? If data violated normality, what transformation did you apply? If assumptions were met, state so explicitly. Current omission suggests either incomplete analysis or problematic data.
12. No molecular identification (e.g., ITS or tef1‑α sequences) for your 5 Trichoderma isolates. How do you know they are truly T. asperellum and T. harzianum? Morphology alone is insufficient. Provide GenBank accession numbers or withdraw species‑level claims.
13. Your discussion and conclusion recommend Trichoderma and fungicides for integrated management. Yet you performed zero in planta or field trials. In vitro inhibition does not guarantee disease control in soil. Defend why readers should believe these in vitro results would translate to the field without any validation data. If you cannot, remove such claims.
14. You do not report any failed isolations, non‑pathogenic isolates, or coisolated fungi. Given the high disease incidence (30–100%), it is unlikely that M. phaseolina was the sole organism. Did you isolate other fungi (Fusarium, Rhizoctonia, etc.)? If yes, why omit them? If no, explain why you only targeted Macrophomina.
15. The original contributions are included in the article. However, raw data (colony diameters, individual EC₅₀ curves, pathogenicity scores) are not provided in a supplementary file. Journal strongly encourages open data. Will you deposit all raw data in a public repository (e.g., Figshare, Dryad)?

1. Abstract: The sentence in 2024, a total of 64,318.08 hectares… the precision (0.08 ha) is unnecessary. Round to 64,318 ha.
2. Line 2 (page 2): direct‑harvest varieties define or rephrase.
3. Section 2.1: spring‑summer cycles specify months or temperature conditions.
4. Section 2.5: five days old specify from what (plating/transfer?).
5. Results 3.2: Mycelial growth rate ranged from 18.90 to 22.55 mm day⁻¹ include standard deviations.
6. Discussion (page 11): CCLF671 showed the highest mycelial growth rate (22.0 mm day⁻¹) this is not the highest according to your own data (CCLF670 = 22.55). Check consistency.
7. References: Several references are incomplete (e.g., Ref. 57 has no volume/page numbers). Update all
8. Data availability: The statement says included in the article but sequences are in GenBank specify accession numbers in the text (already done, but also list in a dedicated data statement).

Author Response

Dear reviewer, we appreciate all the observations made for the improvement of this document; the responses can be found in the attached document.

Author Response File: Author Response.docx

Reviewer 3 Report

In this study, the authors isolated and characterized the fungal pathogen Macrophomina phaseolina associated with charcoal rot in sesame. They also evaluated the antagonistic activity of multiple Trichoderma isolates against M. phaseolina using in vitro plate assay. The efficacy of three fungicides against M. phaseolina was also assessed under in vitro conditions to compare the chemical control options. This is a relevant study addressing an important plant disease management problem. The authors may consider extending this work to greenhouse or field trials in future studies to validate the efficacy of biocontrol agents and fungicides under in planta conditions.

The limitation of the study is the experimental design of the pathogenicity assay. The pathogenicity assay sections (both methods and results) need to be revised to support the conclusions. The authors tested the virulence of six M. phaseolina isolates on two sesame cultivars. In the conclusion section, the authors reported that "All isolates were pathogenic, with varying aggressiveness" (Line 405). However, the manuscript does not include any details regarding how disease severity or aggressiveness between different isolates was measured. The methods or results section does not show that if pathogen re-isolation has been performed, which is important to confirm Koch’s postulate. Also, the statistical methods used for analyzing the pathogenicity assay is missing.

Major:

Line 172: The manuscript lacks description of how disease severity was measured and compared between different M. phaseolina isolates. The disease scoring criteria and assessment methods should be included in the methods section. Also, provide details of the statistical method used for pathogenicity assay analysis. I recommend  including a graph or table presenting the quantitative assessment of virulence of different M. phaseolina isolates (use lesion size or the incidence of disease).

For figure 4, it would be good to include the picture of the uninoculated controls.

Lines 369-379: Were any differences in aggressiveness observed for CCLF671 compared to other isolates? Add details in the discussion.

 

Minor:

Line 25: Change "It also assessed the in vitro..." to "The study also evaluated the in vitro..."

Line 177: Please specify the method used for inoculating controls.

Line 233: Please refer to specific panels rather than only citing Figure 2.

Author Response

Dear reviewer, we appreciate all the observations made for the improvement of this document. All the requested information is included in the new version of the document.

Reviewer 4 Report

Necessary Rectifications
Units of  EC₅₀ values reported in the abstract (0.04–1.39, 0.002–0.123, 0.029–0.539 µg/mL) are not the same as in the Results section.  Ranges are not exactly the same and do not match the values reported (lines 323–324: 0.044–0.123, 0.049–1.397, 0.029–0.539 mg L⁻¹). Also check Figure 6.
Line  370 … suggesting these factors may influence isolate aggressiveness.
Line 405… All isolates were pathogenic, with varying aggressiveness.
Lines 266–270 / Figure 4: Pathogenicity is confirmed qualitatively but no quantitative data are presented — no disease severity scale, no incidence percentage, no statistical comparison between isolates or cultivars. Either quantitative pathogenicity data must be added or the claim of differential aggressiveness worked out. No correlation analysis between morphological variables and pathogenicity data was performed

Lines 172–178 Pathogenicity method state that four plants per pot were used with four replicates, and the experiment was conducted twice, but no mention is made of how disease severity was scored or measured.
Only two isolates are shown (CCLF673 on cv. Paraguayo and CCLF675 on cv. Dormilón), with no quantitative measurement, no disease severity scale applied, and no statistical comparison between isolates or cultivars.

Lines 229–234 / Table 1: The mycelial growth rate data and microsclerotia diameter data are described in the text but no statistical summary table is presented for these morphological variables. The text refers to significant differences but the supporting statistics (e.g. F values, degrees of freedom, p-values) are absent.
Lines 181–182: Five Trichoderma isolates are used (FAVF675, FAVF676, H7, H21, H27) but their origin and prior identification are not described. References? . This information is necessary to evaluate the representativeness of the biocontrol results.

 

Minor Improvements
Lines 86–88: The reported disease incidence of 30–100% in commercial fields during 2020–2021 is a central justification for the study but is not supported by any citation or description of how this was assessed. A reference should be provided.
Lines 176–178 / inoculation method: The toothpick inoculation method is used without discussion of how well it replicates natural infection conditions. A brief justification comparing it to soil inoculation methods would strengthen the methodological rationale, particularly since M. phaseolina is a soilborne pathogen that typically infects through the root system rather than stem wounds.
Lines 215–218 SHAM was added to pyraclostrobin plates and also to control plates without fungicide. It is not explicitly stated whether SHAM at 100 µg mL⁻¹ affected mycelial growth of M. phaseolina on its own. If not verified, this could confound EC₅₀ calculations for pyraclostrobin.
Figure 3: The phylogenetic tree includes only five described Macrophomina species. It would be desirable to confirm whether additional recently described species (e.g., M. pseudophaseolina, already included, plus any described after 2020) were considered, since the genus has undergone recent taxonomic revision.
A statistician review is not required given the statistical methods used (Shapiro-Wilk, Levene, Tukey, logistic-log regression in R) are standard and well-established for this type of study, provided the missing pathogenicity quantitative data issue mentioned above is resolved.

Author Response

Dear reviewer, we appreciate all the observations made for the improvement of this document; the responses can be found in the attached document.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Just need to some improvements  

1. Line 194:"Trichoderma sp. (FAVF675, FAVF676)":

Who identified these two isolates? if the authors are identified, must mention write about how identified and add photos for isolates.

 

2. Line 195-196:'the phytopathology

laboratory of CEPROBI.":

The authors must write full affiliation for this lab

 

3. The section:"3.3. Molecular identification":

The authors must add similarity with other isolates in NCBI

 

4. Line 504-506:

The discussion for multilocus is unacceptable. It is necessary used some primers, one as universal primer and others specific depending on researching. Therefore, you must re-discuss again

 

5. Line 557-560: from "These" to end:

Delete it

 

6. Line 562:"morphological characterization":

Mention to the main characterizes that you are used and useful in identify the plant fungal pathogens or fungi isolates  in this study

Author Response

Dear reviewer, we appreciate the time you dedicated to reviewing the document; the answers to your comments can be found in the attached file.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have substantially improved the manuscript with clear methodology, well-structured results, and comprehensive figures. The revisions enhance readability and strengthen scientific rigor. Minor points remain for consideration: 
Line 54–57 in introduction section need to clarify that charcoal rot caused by Macrophomina phaseolina is the primary yield-limiting disease in sesame. 
The figure 1 captions should mention the scale of symptoms or sampling size to improve reproducibility in the manuscript from line 126–128. 
Line 193–197 (Antagonism tests)
I suggest the author to mention the molecular confirmation of Trichoderma sequences and may state that the Trichoderma sequences were characterized by morphology and by amplification of the ITS region and cite https://doi.org/10.1128/aem.00218-25 describing biofilm and microbial interaction studies.
Line 235–245 regarding the Statistical analysis, the author should indicate software version for reproducibility.
Line 411–415 (Fungicide sensitivity results)
The results are well written but author can add context on resistance mechanisms.
Line 499–525 (Discussion)
The discussion section is well developed and author can add references to recent molecular studies linking fungal pathogenicity to host response. Cite (https://doi.org/10.1002/ps.70649) to illustrate recent mechanistic studies on pathogen inhibition by small molecules.
Check consistency of isolate codes like CCLF674 vs. CCLF74 on line 359.
The manuscript is suitable for publication after these minor suggestions.

The authors have substantially improved the manuscript with clear methodology, well-structured results, and comprehensive figures. The revisions enhance readability and strengthen scientific rigor. Minor points remain for consideration: 
Line 54–57 in introduction section need to clarify that charcoal rot caused by Macrophomina phaseolina is the primary yield-limiting disease in sesame. 
The figure 1 captions should mention the scale of symptoms or sampling size to improve reproducibility in the manuscript from line 126–128. 
Line 193–197 (Antagonism tests)
I suggest the author to mention the molecular confirmation of Trichoderma sequences and may state that the Trichoderma sequences were characterized by morphology and by amplification of the ITS region and cite https://doi.org/10.1128/aem.00218-25 describing biofilm and microbial interaction studies.
Line 235–245 regarding the Statistical analysis, the author should indicate software version for reproducibility.
Line 411–415 (Fungicide sensitivity results)
The results are well written but author can add context on resistance mechanisms.
Line 499–525 (Discussion)
The discussion section is well developed and author can add references to recent molecular studies linking fungal pathogenicity to host response. Cite (https://doi.org/10.1002/ps.70649) to illustrate recent mechanistic studies on pathogen inhibition by small molecules.
Check consistency of isolate codes like CCLF674 vs. CCLF74 on line 359.
The manuscript is suitable for publication after these minor suggestions.

Author Response

Dear reviewer, we appreciate the time you dedicated to reviewing the document; the answers to your comments can be found in the attached file.

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report

Just need to some improvements  

I have some comments, as follows:

 

1. Trichoderma sp.:

Rewrite in all manuscript from "Trichoderma sp." to "Trichoderma spp."

 

2. Line 193-205: From "Tow isolates of" to end:

Remove to separate section, and a title as example: " Isolate and identification of Antagonistic fungi". In this section, you can talk on prepare your collecting of three Trichoderma isolates, and isolate of two Trichoderma spp.,  then mention to identify depending on morphological characteristics and molecular method "Need to write some details and sequences for Two primers"

 

3. Line 202-203:"the phytopathology laboratory of Center for the Development of Biotic Products (CEPROBI).":

The affiliation is incomplete and needs to add, what is the collage, what is university, what is status, and what is the country?

 

4. Line 322-323: "98% similarity":

The similarity is unacceptable

 

5. Line 391-399:

Write this paragraph in separate section under a title "Identification of antagonistic fungi"

 

6. Figure 6:

I found some numbers as "431 - 441"  on isolates of Trichoderma  spp. what   is this meant?

 

7. Three papers must use because they related with the idea of this manuscript, as follows::                                    A. Mitrović, I., Vučurović, D., Al-Ani, L.K.T., Mitrović, B., Bajić, B., Dodić, S. and Živanov, S.T. (2023). Production of Trichoderma harzianum K179 bioagent for maize diseases control: complete laboratory stage bioprocess development. Journal of applied microbiology, 134(6), lxad115. DOI: https://doi.org/10.1093/jambio/lxad115.  B.  Hajji-Hedfi, L., Al-Ani, L. K. T., Wannassi, T., Khlif, A., L’taief, B., & Acheampong, M. A. (2026 a). Estimating Efficacy of Indigenous Isolates of Three Trichoderma Species as Biocontrol Agents Against Alternaria alternata and Curvularia spicifera. Journal of Fungi, 12(6), 421. DOI: https://doi.org/10.3390/jof12060421.                         C. Hajji-Hedfi, L., Bargougui, O., Al-Ani, L.K.T., Oueslati, S., Sukmawati, D., Wannassi, T., & Triki, M. A. (2026 b). Sustainable Integrated Management of Olive Verticillium Wilt (Verticillium dahliae) Using a Microbial Consortium of Pseudomonas yamanarum - Trichoderma longibrachiatum, and Compost Mill Waste. Journal of Soil Science and Plant Nutrition, 1-17. DOI: https://doi.org/10.1007/s42729-026-03297-3.

 

Author Response

Dear reviewer, thank you for your comments. You will find the answer in the attached document.

Author Response File: Author Response.docx