Antibacterial Activity of Guttation Droplets from Penicillium pimiteouiense and Penicillium menonorum Against Clinically Relevant Bacterial Pathogens

“In this manuscript, the authors focus on evaluation of the antibacterial activity of the guttation droplets using agar well diffusion and microbroth dilution assays. Antibacterial activity against clinically relevant bacterial pathogens is noteworthy, as it may enhance potential discovery of antibacterial natural products and the resulting compounds could be of broad interest.

To strengthen the manuscript, it would be highly beneficial to include antibacterial experimental validation of the guttation droplets. Currently, the conclusions appear to be based on a single experimental result, which may not be sufficient to support the claims made. While the antibacterial activity of the guttation droplets is promising, the complex nature of these droplets warrants a more thorough discussion regarding the repeatability and reproducibility of the results. Without such a discussion, the significance of the antibacterial findings may be limited. Additionally, as highlighted in the manuscript, the chemical characterization of the bioactive compounds in the guttation droplets shows great potential and should be further explored if possible.”

Response

We appreciate the reviewer’s major concern, would like to clarify that the antibacterial activity reported in this study is not based on a single experimental observation. All agar well diffusion assays were performed in triplicate and reported as mean ± standard deviation. Moreover, the microbroth dilution assays for determining MIV and MBV were also performed in duplicate across two independent experimental setups. In order to improve clarity, we have revised the Methods and Results sections to emphasize the number of biological and technical replicates performed [Lines 189, 215, 317]..

We agree with the reviewer that the complex nature of guttation droplets raises concerns about reproducibility. We have expanded the Discussion section to address this aspect explicitly. We also acknowledge that guttation composition may vary with fungal growth phase/stage, representing both a limitation and an opportunity for future studies.

Finally, we agree that chemical characterization represents a critical next step. As the aim of the present study was to establish the antibacterial potential of fungal guttation droplets as a proof-of-concept, a comprehensive metabolomic analysis was beyond the scope of this work. Nevertheless, we have strengthened the Discussion to clearly state that this study provides a bioprospecting framework [Lines 411-420]..

1. The ABSTRACT for the Journal of Fungi should be a total of about 200 words maximum as indicated by the journal. Please revised the abstract in a more concise manner.

Response: We agree with the reviewer that the abstract exceeded the recommended length. It has been revised and condensed to 198 words, keeping in mind the conciseness while preserving the key findings. [Lines 19-36].

2. The manuscript necessities Graphical Abstract as indicated by the journal. Please provide it.

Response: We appreciate this suggestion. The graphical abstract was constructed but could not be included as a separate figure. The graphical abstract has been incorporated into the revised version of the manuscript.

3. Although the reference list is well-organized. However, it does not strictly adhere to the formatting guidelines of the prestigious journal Journal of Fungi. Please update the reference style to comply with the journal’s requirements.

Response: We thank the reviewer for the precise revision. The reference list has been carefully revised to comply with the JoF guidelines. [Lines 473-589].

Reviewer 2

Lines 65-66. It needs to be reformulated. Currently, there is insufficient experimental data to assess the qualitative and quantitative composition of exudates, but it is not only water and secondary metabolites, these are enzymes, ammonia and oxalic acid..

Response

We agree with the reviewer's comment. Lines 65-66 have been reformulated to provide a more accurate description of the exudate composition, explicitly including enzymes, ammonia, and oxalic acid, water, alongside secondary metabolites.

Check the spelling of the types in italics throughout the text. In table 1, S. typhimurium

Response: We appreciate the reviewer's attention to detail. We have thoroughly reviewed the manuscript to ensure that all scientific names are correctly italicized. Specifically, in Table 1 and throughout the text, the nomenclature for Salmonella has been corrected to conform to international standards (e.g., Salmonella enterica serovar Typhimurium or S. Typhimurium).[Tables 1 and 2]...

Lines 324-325 "secondary metabolites, possibly anthraquinones, naphthopyrones, or other polyketide groups." are written incorrectly. Anthraquinones is not polyketide groups. And it doesn't seem right to me to be tied to specific classes of antibiotics in your case. You can't prove that you have polyketides or anthraquinones, can you?

Response: We agree with the reviewer that the previous phrasing was imprecise and speculative. Since we did not perform a definitive structural characterization of the metabolites, we have reformulated the sentence to avoid overstating our findings. We have replaced the specific mentions of anthraquinones and naphthopyrones with more general terms supported by the existing literature for this species/genus. [Line 324-325].

Table 2 K, pneumoniae. You have a comma after the K.

Response: Thank you for your observation. Table 2 was modified.

Wouldn't it be more correct to call section 3 Results and discussions?

Response: Thank you for your observation. The section was modified.

This article is undoubtedly a timely and relevant work. The above remarks are editorial in nature, and their presence does not reduce the overall positive impression of the presented research. The work corresponds to the profile of the journal and can be recommended for publication.

Response: We sincerely thank the reviewer for their positive assessment of our work and for recognizing its relevance and timeliness. We are grateful for the constructive editorial remarks provided, as addressing them has significantly improved the precision and clarity of the manuscript. We have carefully incorporated all the suggested corrections, and we are pleased that the research is considered suitable for publication in the Journal of Fungi.

Reviewer 2

The study explores the antibacterial potential of fungal guttation against clinically significant pathogens. The researchers successfully isolated and identified two Penicillium species and demonstrated that their exudates possess bactericidal properties. Several methodological issues regarding taxonomic identification, sample standardization, and statistical rigor require attention.

Response

We thank the reviewer for the careful evaluation of our manuscript and for highlighting important methodological aspects. We have addressed each point in detail and revised the manuscript accordingly to improve the aforementioned concerns.

Line 160 - please clarify/explain is the componence of the exudates is stable in time (3 weeks of reseeding - this could influence the quality of the exudate) - MS or HPLC would be needed to present the final content and the bioactive compounds. This is a limitation that should be presented, and the bioactive main compounds to be discussed.

Response: Thank you. We agree with the reviewer that the chemical composition of guttation droplets may vary over time, which represents an important limitation. In this study, the guttation droplets were collected at a consistent time point under controlled growth conditions to minimize variability. However, we acknowledge that the metabolite composition of these exudates is dynamic and may depend on different factors, such as growth phase, nutrients availability, and other environmental conditions [Lines 413-420; 444-450]...

Line 265 - fungus 4 is not previously mentioned (is it fungus 2 most probably)

Response: We thank the reviewer for addressing this concern [Line 266]..

Chapter 3.3.1 - the use of clinical isolates is not ideal, as their behavior is unpredictable and non-reproductible. In this idea, the discussion from rows 382-398 may be related only to this particular clinical isolate. Other strains may behave differently. For these reasons, please strongly argument the use of these clinical strains, or remove them from the analysis. Indeed, a comprehensive similar study on clinical strains would be interesting in the future. Moreover, is there an ethical approval for using the clinical strains from the laboratory?

Response: Thank you for your observation. The inclusion of these strains was totally intentional, since it provides a more translational perspective by evaluating antibacterial activity against pathogens with potential clinical relevance. Moreover, all clinical isolates used in this study were anonymized and obtained from routine diagnostic procedures, without any patient-identifiable information. Therefore, in accordance with institutional and national regulations, formal ethical approval was not required. This clarification has been added to the Materials and Methods section. [Lines 98-101].