Isolation of Bioactive Metabolites from Fusarium fujikuroi: GC-MS Profiling and Bioactivity Assessment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study presents interesting findings, however, there are aspects that need improvement before it can be considered for publication. My specific comments are as follows:

Formatting corrections

1.       Line 36: “medicins” should be corrected to medicines.

2.       Line 47: “[7],8]” should be corrected to [7,8].

3.       Line 68: a space is required between “application” and [19].

4.       Line 189: “n-heax” should be corrected to n-hexane.

5.       Line 200: “have” should be corrected to has.

6.       Line 210: “again” should be corrected to against.

7.       Line 263: “show” should be corrected to shows.

8.       Line 273: “[34],35]” should be corrected to [34,35].

9.       Line 276: “comound” should be corrected to compound.

10.   Line 295: “F.” should be corrected to F. fujikuroi.

11.   Lines 302, 303, 305, 306: “activites” should be corrected to activities.

Fundamental corrections

1.       In section 2.6, the authors indicate that ethyl acetate was used as a negative control. Since the antibacterial assays included fractions obtained from different solvents, each solvent used in the fractionation process should have been incorporated as a separate negative control.

2.       The manuscript does not specify the selection criteria for the bacterial strains nor their origin (clinical or from a strain collection). This information is essential for assessing relevance and reproducibility.

3.       The antibacterial activity evaluation lacks a positive control, such as a commercially available antibiotic. Without a known standard for comparison, it is not possible to determine whether the reported inhibition zones represent meaningful antibacterial activity.

4.       In Table 1, the standard deviations should be reported with the same number of significant figures as the corresponding mean values.

5.       Figure 3 lacks error bars (standard deviation) and statistical analysis. Furthermore, there is no discussion of whether significant differences exist among fractions or bacterial species.

6.       The manuscript compares mean ZOI values with results from studies on other fungi; however, no information is provided on the compounds responsible for the activity in those studies. Without such context, the comparison is weak.

7.       Table 2 lists compounds as “not reported”, yet the terms shown in column 4 (“alkane”, “phenol derivative”, “high molecular weight”, “cyclic alcohol”) correspond to broad chemical classes, not specific compounds. This should be corrected.

Additionally, there is extensive literature on secondary metabolites of Fusarium fujikuroi (e.g., Janevska et al., 2017; Avalos et al., 2007; Amuzu et al., 2024), but these references are not mentioned in the introduction or discussion. Including them would strengthen the manuscript and better contextualize the findings.

Comments on the Quality of English Language

Given that English is probably not the authors' primary language, it is necessary to pay
closer attention to the writing and thoroughly proofread before submitting a manuscript
for publication consideration.

The English corrections can be found in the previous comments.

Author Response

The study presents interesting findings, however, there are aspects that need improvement before it can be considered for publication. 

Response: We are thankful to the reviewers for their valuable comments and carefully reviewing the manuscript. A point-by-point response to the reviewers’ comments are appended below and the corresponding changes have been carried out in the revised manuscript. The changes carried out in the revised manuscript are highlighted in red color.

My specific comments are as follows:

Formatting corrections

Comment 1. Line 36: “medicins” should be corrected to medicines.

Response: Corrected

Comment 2.  Line 47: “[7],8]” should be corrected to [7,8].

Response: Corrected

Comment 3. Line 68: a space is required between “application” and [19].

Response: Correction done.

Comment 4. Line 189: “n-heax” should be corrected to n-hexane.

Response: Corrected

Comment 5. Line 200: “have” should be corrected to has.

Response: Corrected

Comment 6. Line 210: “again” should be corrected to against.

Response: Corrected

Comment 7. Line 263: “show” should be corrected to shows.

Response: Corrected

Comment 8. Line 273: “[34],35]” should be corrected to [34,35].

Response: Corrected

Comment 9. Line 276: “comound” should be corrected to compound.

Response: Corrected

Comment 10. Line 295: “F.” should be corrected to F. fujikuroi.

Response: Corrected

Comment 11. Lines 302, 303, 305, 306: “activites” should be corrected to activities.

Response: Corrected

Fundamental corrections

Comment 12. In section 2.6, the authors indicate that ethyl acetate was used as a negative control. Since the antibacterial assays included fractions obtained from different solvents, each solvent used in the fractionation process should have been incorporated as a separate negative control.

Response: We agree. In the revised manuscript, we have clarified that each solvent employed in the fractionation process (n-hexane, chloroform, ethyl acetate, and methanol) was tested separately as a solvent control in antibacterial assays. None of the solvents alone produced measurable inhibition zones.

Comment 13. The manuscript does not specify the selection criteria for the bacterial strains nor their origin (clinical or from a strain collection). This information is essential for assessing relevance and reproducibility.

Response: We have added this essential information in Section 2.6 to clarify that the bacterial strains were obtained from the departmental culture collection, originally isolated from clinical samples, and selected based on their relevance as common human pathogens.

Comment 14. The antibacterial activity evaluation lacks a positive control, such as a commercially available antibiotic. Without a known standard for comparison, it is not possible to determine whether the reported inhibition zones represent meaningful antibacterial activity.

Response: We acknowledge this omission. We have repeated assays including a positive control (ciprofloxacin 10 µg/disc equivalent) and now report the comparative inhibition zones. The inclusion allows better interpretation of the biological significance.

Comment 15. In Table 1, the standard deviations should be reported with the same number of significant figures as the corresponding mean values.

Response:  Corrected in Table 1, the SD values are now reported consistently (e.g., 13.7 ± 1.5 mm instead of 13.7 ± 1.52 mm).

Comment 16. Figure 3 lacks error bars (standard deviation) and statistical analysis. Furthermore, there is no discussion of whether significant differences exist among fractions or bacterial species.

Response: Figure 3 has been revised to include mean ± SD error bars, and significance among fractions and bacterial species was assessed by one-way ANOVA followed by Duncan’s multiple range test (p < 0.05). The following text has been added to results and discussion section 3.4. Error bars represent mean ± SD (n = 3). Statistical analysis (ANOVA, p < 0.05) indicated significant differences among solvent fractions, with the n-hexane/chloroform (1:1, v/v) fraction showing the highest inhibition against B. subtilis and E. coli.

Comment 17. The manuscript compares mean ZOI values with results from studies on other fungi; however, no information is provided on the compounds responsible for the activity in those studies. Without such context, the comparison is weak.

Response: Most of the studies do not reported the purified compounds, rather they have reported class of compounds such as phenolic, or other metabolites. We have expanded the discussion to include specific compounds responsible for activity in referenced studies. Text is added to the revised manuscript, section 3.5. They reported that hentriacontane, dodecane, phenols, and benzene derivatives identified in GC-MS displayed significant activity against clinical pathogens and multi-drug-resistant organisms. Similarly, Emericella quadrilineata produced 2,4-di-tert-butylphenol and long-chain alkanes responsible for antibacterial activity 33. The presence of similar phenolic and hydrocarbon compounds in F. fujikuroi fractions suggests comparable bioactivity mechanisms. Moreover, 2,4-di-tert-butylphenol (2,4-DTBP) has been isolated from multiple endophytes 34. This compound exerts antibacterial and an-ti-virulence activity against Pseudomonas aeruginosa, Staphylococcus aureus and other patho-gens. Similarly, Fatty-acid esters and phthalate esters such as bis-(2-ethylhexyl) phthalate/di-(2-ethylhexyl) phthalate have been isolated from fungi which have shown antibacterial activity 35.

Comment 18. Table 2 lists compounds as “not reported”, yet the terms shown in column 4 (“alkane”, “phenol derivative”, “high molecular weight”, “cyclic alcohol”) correspond to broad chemical classes, not specific compounds. This should be corrected.

Response: We have revised Table 2 by removing the column “Compound name” and replacing generic terms with the specific compound names identified via NIST matching (e.g., hentriacontane, 2,4-di-tert-butylphenol). We clarified that “not reported” refers to “not previously reported from F. fujikuroi.”

Additionally, there is extensive literature on secondary metabolites of Fusarium fujikuroi (e.g., Janevska et al., 2017; Avalos et al., 2007; Amuzu et al., 2024), but these references are not mentioned in the introduction or discussion. Including them would strengthen the manuscript and better contextualize the findings.

Response: These references are incorporated in both Introduction and Discussion section 3.5.

Comments on the Quality of English Language

Given that English is probably not the authors' primary language, it is necessary to pay

closer attention to the writing and thoroughly proofread before submitting a manuscript

for publication consideration.

Response: All grammatical and typographical errors have been corrected throughout the manuscript. The entire text was proofread by a native English speaker.

The English corrections can be found in the previous comments.

 

Reviewer 2 Report

Comments and Suggestions for Authors

    This manuscript presents interesting findings on bioactive metabolites from an endophytic fungus with antimicrobial potential. The paper is generally well structured, but several aspects require attention before publication. The experimental approach is sound, although some methodological details require clarification, and the presentation could be improved in places.

Comments for author File: Comments.pdf

Author Response

Comment 1. Page 1, lines 2-24: My recommendation is that the Abstract includes quantitative details about the experimental setup, such as exact solvent ratios and number of replicates. It is also essential to include statistical values (p-values) to support claims regarding antimicrobial activity. It is also important to state whether a positive control (standard antibiotic) was used and, if so, to specify it.

Response: Thank you for your valuable suggestion. The Abstract now specifies solvent ratios (n-hexane/chloroform 1:1 v/v), triplicate assays, and statistical validation (p < 0.05). Ciprofloxacin was used as a positive control. The information is added to abstract and results & discussion sections.

Comment 2 Page 1, lines 28-29: Page 1, lines 28-29: The introduction provides adequate context, but is too general and contains typographical errors (e.g. "medicins"[3] should be "medicines"). Recommendations: Condensate the first two paragraphs and emphasize the biosynthetic potential of Fusarium fujikuroi. State what is new about this study and what gap it fills in relation to previous research. What is the main innovation, beyond the confirmation of antibacterial activity? Is a new metabolite identified or a proposed mechanism of action?

Response: We condensed the first two paragraphs and emphasized novelty in the third paragraph in the revised manuscript. The changes are highlighted in red color.  

Comment 3. Page 2, line 47: Citation format “[7], 8]” is incorrect. Should be “[7,8]”.

Response:  The citations are corrected throughout the manuscript.

Comment 4. Page 3-5: Sections 2.1–2.6 – The methodology follows standard protocols, but lacks critical details regarding reproducibility, namely: sample size, number of replicates, and precise solvent ratios for all chromatographic steps.

Response: We thank the reviewer for this valuable observation. We have carefully revised Sections 2.1–2.6 to ensure full methodological transparency and reproducibility. Specifically, we have now included the number of biological and technical replicates for all experiments, the sample sizes used for each assay, and the exact solvent ratios and volumes employed during chromatographic fractionation. Each experiment (antibacterial assay, extraction, fractionation, and TLC) was conducted in triplicate (n = 3) to ensure reproducibility. Each chromatographic step used precisely measured solvent mixtures, including n-hexane/chloroform (1:1, v/v), chloroform/ethyl acetate (1:1, v/v), and ethyl acetate/methanol (1:1, v/v). The sample size for antibacterial assays was standardized to three biological replicates per bacterial strain, with three technical repeats per replicate. Additional details on the volume of solvent applied per gradient (200 mL per solvent phase) and the column dimensions (700 mm × 18 mm) are now clearly reported

Comment 5. Page 4, lines 154–158: Statistical analysis is briefly mentioned – please describe the type of data transformation or normalization applied prior to ANOVA. Were all assays performed in triplicate or more? Please specify the sample size for each antibacterial assay.

Response: We appreciate this constructive comment and have revised Section 2.9 (Statistical analysis) to clearly describe the statistical procedures, data transformation, and replication scheme. All antibacterial assays were conducted in triplicate (n = 3) for each bacterial strain and solvent fraction. Each experiment was repeated three times independently on different days using freshly prepared cultures (three biological replicates), and each measurement was based on three technical replicates per treatment. Prior to analysis of variance (ANOVA), the raw data (zone of inhibition diameters) were checked for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene’s test. As data met the assumptions of ANOVA, no further transformation was required. When minor deviations occurred, data were log₁₀-transformed to stabilize variance. The overall effects of solvent fraction and bacterial species, as well as their interaction, were then analyzed using a two-factor factorial ANOVA within a completely randomized design (CRD). Mean separations were conducted by Duncan’s multiple range (DMR) test at a significance level of p < 0.05. Results are reported as mean ± standard deviation (SD).

Comment 6. Page 5: The GC-MS method uses the NIST library for identification. Are the authors referring to compounds not confidently identified by NIST or compounds with retention times that were not listed in Table 2? Please specify the nature of these "unidentified compounds."

Response: We thank the reviewer for requesting clarification. In the original manuscript, the term “unidentified compounds” referred to peaks detected by GC–MS that did not produce reliable spectral matches (similarity index < 80%) in the NIST (National Institute of Standards and Technology) library, or for which the mass spectra corresponded to broad chemical classes (e.g., “phenol derivative,” “cyclic alcohol”) rather than specific molecular structures. These peaks were therefore not assigned a definitive compound name. To enhance transparency, we have revised Section 2.8 (GC–MS analysis) to explicitly define the identification criteria used, the confidence thresholds applied, and the reason certain peaks were not reported in Table 2. Furthermore, we now state that all retention times listed in Table 2 correspond only to peaks that met the NIST match confidence level of ≥80%. This clarification distinguishes between “unidentified compounds” (spectral match below threshold) and “identified but previously unreported compounds”.

Comment 7. Page 5, line 176: Grammatical error: “aquaous” should be “aqueous”; Page 8 , line 249: Grammatical error:’’ethyl benzene” should be “ethylbenzene”

Response: Thanks for pointing out these grammatical mistakes which are corrected in the revised manuscript.

Comment 8. Page 6, Table 1: The statistical methodology mentions Duncan multiple-range test, but Table 1 presents data as mean ± SD without any statistical grouping letters or significance indicators. This makes it difficult to assess which differences are statistically meaningful. Were post-hoc comparisons actually performed? If so, why aren't the results shown? The table would benefit from superscript letters (a, b, c, etc.) to indicate significant differences between treatments.

Response: We thank the reviewer for highlighting this important point. Indeed, Duncan’s Multiple Range (DMR) post-hoc test was performed after ANOVA to determine statistically significant differences among mean inhibition zone diameters for different solvent fractions and bacterial strains. In the original submission, we inadvertently omitted the superscript letters denoting these differences. To address this, Table 1 has been revised to include superscript lowercase letters (a, b, c, d, etc.) next to each mean value. These letters indicate statistically distinct groups at p < 0.05 according to Duncan’s test. Means sharing the same letter are not significantly different, whereas those with different letters represent significant differences among treatments. We have also clarified this in the table legend and in Section 2.9 (Statistical analysis). The revised Table 1 now clearly conveys the statistical significance of variations in antibacterial activity among the tested fractions and bacterial strains.

Comment 9. Page 6,line 189: Grammatical error: “n- heaxe” should be “n-hexane.”

Response: Corrected.

Comment 10. There's confusion in how fractions are named. For instance, line 199 mentions "hexane/chloroform fraction" while line 228 refers to "n-hexane/chloroform (1:1, v/v) fraction." Are these the same? Table 1 uses "Hexane/chloroform" without specifying ratios.Recommendation: Standardize the nomenclature throughout. Either always include the ratio or establish abbreviations clearly in the methods section (e.g., "HCF" for hexane/chloroform 1:1 v/v).

Response: We thank the reviewer for pointing out this inconsistency. We agree that the nomenclature for the hexane/chloroform fractions should be standardized for clarity. In the revised manuscript, we have: Updated all mentions of “hexane/chloroform fraction” to “n-hexane/chloroform (1:1, v/v) fraction” to specify the solvent ratio consistently. Added a note in the Methods section defining the abbreviation “HCF” for n-hexane/chloroform (1:1, v/v) fraction, which is now used consistently throughout the text and tables.

Comment 11. Page 8: I have a question about the comparison in lines 236-243. To fully understand it, we need to know the concentration in mg/mL of the fractions you tested. The 80 μL volume tells us "how much" was added, but not "how strong" that extract was. Without this information, it is difficult to assess whether the result is very promising or only moderate compared to other studies.

Response: The concentration is clarified in the revised manuscript.  For each assay, 100 µL of bacterial inoculum (~1×10⁸ CFU/mL) was spread uniformly on Mueller–Hinton agar plates. Wells of 6 mm diameter were filled with 80 µL of each fraction (dissolved at 50 mg/mL in the corresponding solvent).

Comment 12. Page 10, line 277: Grammatical error:”comound” should be “compound”.

Response: Corrected.

Comment 13. Page 11, Table 2: The header says "not reported so far" but the Status column repeats "not reported" for each compound. What does "not reported" mean exactly? Not reported in F.

Response: The term not reported is corrected in the Table 2.

Comment 13. fujikuroi specifically, or never reported anywhere? Some retention times don't match those

mentioned in the text (e.g., 2,4-di-tert-butylphenol is listed at 21.21 min in line 250 but this doesn't appear in Table 2). Are these compounds truly novel to science, or just not previously reported from this fungus? This distinction is crucial and should be clarified.

Response: Table 2 has been revised and the term not reported is classified, the retention times of eluted compounds are mentioned in Table 2.

Comment 14. Page 11, lines 311-331: You mention "novel constituents not yet documented in the literature" but the manuscript doesn't provide structural elucidation or confirmation beyond GC-MS library matching. Isn't this overclaiming?

Response: The sentence has been modified in the revised manuscript as “The GC-MS results revealed that F. fujikuroi produces a diverse array of bioactive metabolites, including both previously reported compounds and several new constituents not yet documented in the literature which need to be further purified and characterize to find out their chemical structure and composition.

Comment 15. Page 12, References list does not include DOIs for any of the cited articles, even though most are recent and may have DOIs. According to the MDPI author guidelines, all references must include a full DOI when available. Please review the entire references section to add the appropriate DOIs.

Response: The references style was changed to MDPI, each reference has a DOIs.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript clearly describes a study on the isolation and characterization of bioactive metabolites from the endophytic fungus Fusarium fujikuroi. It is well-organized, moving logically from isolation and extraction to antimicrobial testing and compound identification. The findings—especially the discovery of potentially novel compounds—are clearly highlighted and give the text strong scientific value.

Row 1 The word Article is misspelled: Artile. Please correct.

Row 36 "medicins" instead of "medicines" – please correct.

Row 47, 57 … Citation formatting is inconsistent; some references include brackets with commas or spaces misplaced. Please revise all.

Table 2. I don’t see reason for the column “Compound name” as the authors do not report the name of the compound. Please remove. If you have identified the structures (using for example MS fragmentation) please provide the IUPAC name.

Toxicity or safety evaluation of bioactive compounds is mentioned as future work but no preliminary toxicity assessments are provided. The authors needs to provide at least In silico toxicity predictions based on the main components to strengthen the manuscript.

Based on the above I think that the manuscript is interesting, but needs a minor revision.

Author Response

The manuscript clearly describes a study on the isolation and characterization of bioactive metabolites from the endophytic fungus Fusarium fujikuroi. It is well-organized, moving logically from isolation and extraction to antimicrobial testing and compound identification. The findings—especially the discovery of potentially novel compounds—are clearly highlighted and give the text strong scientific value.

Comment 1. Row 1 The word Article is misspelled: Artile. Please correct.

Response: We are thankful to the reviewer for his/her constructive comments. The spelling has been corrected.

Comment 2. Row 36 "medicins" instead of "medicines" – please correct.

Response: Corrected.

Comment 3. Row 47, 57 … Citation formatting is inconsistent; some references include brackets with commas or spaces misplaced. Please revise all.

Response: The citations were carefully checked and corrected where necessary.

Comment 4. Table 2. I don’t see reason for the column “Compound name” as the authors do not report the name of the compound. Please remove. If you have identified the structures (using for example MS fragmentation) please provide the IUPAC name.

Response: Yes, we agree with the reviewer, we have identified their structures using MS fragmentation. The IUPAC names of some of these compounds are presented in the revised Table 2.

Comment 5. Toxicity or safety evaluation of bioactive compounds is mentioned as future work but no preliminary toxicity assessments are provided. The authors needs to provide at least In silico toxicity predictions based on the main components to strengthen the manuscript. Based on the above I think that the manuscript is interesting, but needs a minor revision.

Response: Thanks for your valuable insights, the manuscript has been revised according to the reviewers’ suggestions and we hope that the revised manuscript will be good enough for publication.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors I believe the corrections have been made satisfactorily.