Preliminary Report of Three Entomopathogenic Fungi as Potential Biocontrol Agents Against the Oak Wilt Vector, Platypus koryoensis
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe use of biological methods in forest protection is becoming a challenge in the coming years due to environmental, social, and economic aspects related to climate change and the mass occurrence of tree pests, as well as pathogens causing diseases. In this aspect, I consider the topic taken up by the authors to be very important and worth continuing.
I have some comments and questions related to the manuscript.
Title:
Should be changed. After reading the title, I expected results of field experiments connecting with the application of biological control agent formulations against insects in the natural environment. So I found the preliminary results of using fungi against insects in vitro. Well, it is a first step before the real application, but the way is long.
Maybe: Preliminary report of using three entomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae, and Purpureocillium lilacinum, as potential biological control agents against Platypus koryoensis, associated with oak wilt disease in South Korea.
Materials and methods
Isolates of entomopathogenic fungi:
Why did you explore only one soil to look for fungi? Perhaps if you use more soil from not only oak stands, you would find more isolates and different entomopathogenic species to check in the experiment.
Line 139: The four entomopathogenic fungal strains were cultured on SDA and PDA media at 25°C for 10-14 days to induce sporulation
Where are the results of the fourth isolate – B. bassiana. In all parts of the results, you showed data for three isolates, and in the Materials section, you mentioned four.
Line 145: A total of 25 mL glass vials were sterilized using a high-pressure autoclave. A conidial suspension (100 μL, 1 × 10⁸ conidia/mL)
How many mL did you pour into the glass vials? And how many spores did you put inside the small vial to infect five Platypus? What about the control – you introduce bark beetles to empty and sterilized vials or vials filled with sterile water, that dried, similar to introducing the spores of fungi.
Line 148: vial was inoculated with the 148 vector beetle P. koryoensis
Avoid using the word inoculation in relation to insect; inoculation occurs thanks to disease agents
Line 186: After conidia were collected, a conidial suspension (1 × 10⁷ conidia/mL with 0.02% 186 Tween-20) was prepared.
Why did you use a different conidial suspension than in other experiments?
Results
Line 223: The pesticidal activity of the selected three strains was tested against the control treatment, showing significantly higher insecticidal effects ….
I doubt it, because you obtained a high death rate of control insects. How do you explain this fact? All control insects died 2-3 days after the insects from infected vials. Maybe on the third day, the effect of entomopathogenic fungi treatment was visible, but after that, the sudden decline of the control population showed the same trend a in the infection experiment? In a situation where the control population dies so quickly, there is no need to control it.
Line 304: These results indicate that the fungal strains respond sensitively to low temperatures and exhibit optimal growth under temperature conditions typical for fungal development.
Yes, it's true for 4oC. Could you comment on the range between 15oC and 25oC. In lower temperatures, the germination is also good, maybe takes a little more time, but in the experiment, all isolates rich 100% germination of spores. Another question is the threshold of temperature at which the spores germinate…
I wish you to rich a successful future application of the biological method in the forest against the bark beetle, probably the most difficult group of insects to fight with the aid of fungi. There are some failures of this method against bark beetles e.g. Grodzki & Kosibowicz 2015. DOI: 10.1515/frp-2015-0001
Author Response
Comments: Should be changed. After reading the title, I expected results of field experiments connecting with the application of biological control agent formulations against insects in the natural environment. So I found the preliminary results of using fungi against insects in vitro. Well, it is a first step before the real application, but the way is long.
Maybe: Preliminary report of using three entomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae, and Purpureocillium lilacinum, as potential biological control agents against Platypus koryoensis, associated with oak wilt disease in South Korea.
Response: We appreciate the reviewer’s insightful comment; to take considerations raised by three reviewers into account, we have revised the title as followed.
Revised Title: Preliminary report of three entomopathogenic fungi as potential biocontrol agents against the oak wilt vector, Platypus koryoensis
Comments: Why did you explore only one soil to look for fungi? Perhaps if you use more soil from not only oak stands, you would find more isolates and different entomopathogenic species to check in the experiment.
Response: Thank you for your comment. In this initial investigation, we focused on soil from a Quercus dentata stand due to its ecological relevance and to ensure a controlled approach to isolate fungi specifically associated with oak environments. We agree that broader sampling across diverse forest types could uncover additional entomopathogenic species. We plan to pursue such expanded surveys in future studies and have added a sentence in the discussion (Lines 344–352) acknowledging this limitation and future direction.
Comments: Line 139: The four entomopathogenic fungal strains were cultured... Where are the results of the fourth isolate – B. bassiana. In all parts of the results, you showed data for three isolates, and in the Materials section, you mentioned four.
Response: We appreciate this observation. While four strains were initially included (two B. bassiana, one M. anisopliae, and one P. lilacinum), only three strains were ultimately used in the insecticidal efficacy experiments. The strain B. bassiana KACC43988 was excluded due to insufficient sporulation and viability. This clarification has been added to the Materials and Methods section (Lines 124-129).
Comments: Line 145: How many mL did you pour into the glass vials? And how many spores did you put inside the small vial to infect five Platypus? What about the control—did you introduce beetles to empty vials or to ones with sterile water dried similarly to the treatment vials?
Response: Thank you for raising this important detail. We applied 100 µL of a conidial suspension (1 × 10⁸ conidia/mL), amounting to 1 × 10⁷ conidia per 25 mL vial. Five beetles were introduced into each treated vial. For the control group, vials were treated with 100 µL of 0.02% Tween-20 solution (without fungal spores) and dried under the same conditions. This protocol ensured that the control group underwent the same treatment process, minus the fungal exposure. Clarifications have been made in the revised manuscript (Lines 159-165).
Comments: Line 148: Avoid using the word inoculation in relation to insects; inoculation occurs thanks to disease agents.
Response: We agree with the reviewer and have replaced “inoculated” with “introduced” when referring to the placement of beetles into vials (Line 168).
Comments: Line 186: Why did you use a different conidial suspension than in other experiments?
Response: We appreciate this point. The conidial suspension with a concentration of 1 × 10⁷ conidia/mL was used specifically for cold germination tests, where lower concentrations helped avoid overlapping spores and allowed for clearer microscopic observations. In contrast, the insect bioassays and thermal/UV tolerance tests required a higher dose (1 × 10⁸ conidia/mL) to simulate effective biological control conditions. This rationale has been clarified in the Methods section (Lines 159-165).
Comments: Line 223: I doubt the statement about significantly higher insecticidal effects, because the control group also died quickly. How do you explain this? In such a case, it may not be necessary to apply fungal control.
Response: Thank you for this critical insight. The control group did experience mortality (LT₅₀ ≈ 137.76 hours), which we attribute to handling stress, age, and confinement. However, treated beetles died significantly earlier (e.g., 58.7 hours for M. anisopliae), and fungal infection was microscopically confirmed only in the treatment groups. Thus, although the control mortality was notable, the rapid and statistically distinct mortality curves in the fungal groups validate the treatment effect. We have added this discussion and clarified limitations in the manuscript (Lines 251–257).
Comments: Line 304: Could you clarify your statement on low temperature response? Between 15°C and 25°C, all isolates reached 100% germination, though more slowly at 15°C. Also, what is the actual threshold temperature for germination?
Response: We thank the reviewer for pointing this out. We agree that the original statement was imprecise. We now clarify that while 4°C completely inhibited germination, all strains showed strong viability at 15°C, reaching 100% germination by Day 10. Therefore, the spores are capable of germinating efficiently at moderate temperatures relevant to field conditions, albeit at slower rates. Our data suggest that the threshold temperature for germination lies between 4°C and 15°C. We have revised the text to reflect this interpretation more accurately (Lines 326-332).
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsManuscript Title: Application of three entomopathogenic fungi, Beauveria bassi-ana, Metarhizium anisopliae and Purpureocillium lilacinum, as potential biological control agents against Platypus koryoensis, associated with oak wilt disease in South Korea
Manuscript ID: (forests-3669321). Although the manuscript addresses an important topic, namely biological control of oak wilt disease, it needs major revisions.
- The experiments were conducted entirely under laboratory conditions. While this is acknowledged, the manuscript would be improved by providing a more detailed proposal on how these results could be applied in the field. What delivery mechanisms are being considered for spore deployment in forest ecosystems?.
- The title is too long... I suggest changing it to: Biological Control of the Oak Wilt Vector Platypus koryoensis Using Three Entomopathogenic Fungi
- Abstract: Add a short sentence highlighting the broader impact of IPM in forests.
- Explain the meaning of the abbreviation LT50 when it is first mentioned in the Abstract.
- Line 39: Correct the spelling of Raffaelea qeurcus-mongolicae to "Raffaelea quercus-mongolicae"
- Line 109 and 110, what is the difference between potato dextrose agar (PDA) and Sabouraud dextrose agar (SDA).
- What is the basis on which the media used for fungal growth is chosen? Sometimes SDA is mentioned, and other times PDA is mentioned.
- Add a photo of Purpureocillium sp growing on SAD medium.
- Identification of P. lilacinum: The molecular identification section should include GenBank accession numbers for the ITS and tef1 sequences used for phylogenetic confirmation.
- Improve the resolution of Figures 1 and 2.
- Figures 1 and 2 (phylogenetic trees) should be improved by including branch lengths, clearer labels, and more readable layouts.
- All figures require improved formatting, including complete axis labels, and clearly distinguish statistical groups.
- The discussion needs improvement.
- Language: A few sentences are awkward or unclear and would benefit from editing
Author Response
Reviewer 2
Comments: The experiments were conducted entirely under laboratory conditions. While this is acknowledged, the manuscript would be improved by providing a more detailed proposal on how these results could be applied in the field. What delivery mechanisms are being considered for spore deployment in forest ecosystems?
Response: We appreciate this valuable suggestion. We have added a new paragraph in the Discussion section outlining potential delivery mechanisms for applying entomopathogenic fungi in forest settings. These include bark spray applications, fungal-coated traps, and sawdust inoculum near entry holes, which are compatible with the beetle’s biology. Additionally, we propose future exploration of solid formulations or slow-release carriers to improve field stability. The corresponding text has been inserted in the Discussion (Lines 344–352).
Comments: The title is too long. I suggest changing it to: Biological Control of the Oak Wilt Vector Platypus koryoensis Using Three Entomopathogenic Fungi.
Response: Thank you for the suggestion. We agree with the reviewer and have shortened the title as follows:
Revised Title: Preliminary report of three entomopathogenic fungi as potential biocontrol agents against the oak wilt vector, Platypus koryoensis
Comments: Abstract: Add a short sentence highlighting the broader impact of IPM in forests.
Response: We have revised the Abstract to include a sentence emphasizing the broader role of entomopathogenic fungi as part of integrated pest management (IPM) strategies for sustainable forest health management (Lines 30-31).
Comments: Explain the meaning of the abbreviation LT₅₀ when it is first mentioned in the Abstract.
Response: We have revised the Abstract to define LT₅₀ at its first appearance as “lethal time to 50% mortality (LT₅₀)” (Lines 38-39).
Comment: Line 39: Correct the spelling of Raffaelea qeurcus-mongolicae to "Raffaelea quercus-mongolicae".
Response: Thank you for pointing out this error. The spelling has been corrected in the manuscript (Line 42).
Comment: Line 109 and 110: What is the difference between potato dextrose agar (PDA) and Sabouraud dextrose agar (SDA)?
Response: We thank the reviewer. A sentence explaining the differences has been added: PDA is a general-purpose medium rich in carbohydrates used for promoting overall fungal growth, while SDA is optimized for the growth of pathogenic fungi due to its lower pH and high dextrose content. This has been added to the Materials and Methods section (Lines 117–119).
- Comment: Add a photo of Purpureocillium sp growing on SDA medium.
Response: Thank you for your suggestion; Due to the deadline for the revised manuscript submission, the suggestions could not be reflected in the current version. However, the strain is currently being cultured, and once the cultivation is complete, colony images will be included in the manuscript as previously mentioned.
- Comment: Identification of P. lilacinum: The molecular identification section should include GenBank accession numbers for the ITS and tef1 sequences used for phylogenetic confirmation.
Response: The sequence submissions to GenBank have been completed, and we are currently awaiting the assignment of the accession number. Although the sequence numbers have not been included in the current version of the manuscript, it will be provided. For reference, the submission numbers are SUB15373188 for ITS and Submission2967784 (tef1).
SUB15373188 for ITS
Submission2967784 for tef1
- Comment: Improve the resolution of Figures 1 and 2.
- Comment: Figures 1 and 2 (phylogenetic trees) should be improved by including branch lengths, clearer labels, and more readable layouts.
- Comment: All figures require improved formatting, including complete axis labels, and clearly distinguish statistical groups.
Response: Thank you for your suggestion, and we have reformatted to improve clarity and print quality for all the figures that you mentioned as accordingly.
Comment: The discussion needs improvement.
Response: The Discussion section has been revised for clarity and depth. We expanded the interpretation of results, emphasized ecological implications, acknowledged limitations (e.g., high control mortality), and added practical suggestions for field application of fungal biocontrol. These enhancements appear in Section 4.
Comment: Language: A few sentences are awkward or unclear and would benefit from editing.
Response: Thank you for your careful revision on is. We have carefully reviewed the manuscript for awkward phrasing and have revised multiple sentences for improved clarity, grammar, and academic tone throughout the text.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript “Application of three entomopathogenic fungi, Beauveria bassiana, Metarhizium anisopliae and Purpureocillium lilacinum, as potential biological control agents against Platypus koryoensis, associated with oak wilt disease in South Korea” is worthy study and important biocontrol potential. There are few points which need to be addressed before further consideration.
Please revised the title and short in meaningful words. It has two points one is entomopathogenic fungi and other is oak wilt disease. However, there is no correlation or is not clearly articulated. The transition from general pest management to oak wilt control with fungal agents should be better structured to help the reader.
While the study focuses on Beauveria bassiana, Metarhizium anisopliae, and Purpureocillium lilacinum, no rationale is provided for selecting P. lilacinum alongside the other two, which are more commonly associated with insect control. A brief explanation of its relevance or prior use would strengthen the justification.
The phrase “rapid insecticidal effects” is vague. Quantitative descriptors or statistical comparisons should be included to provide a more accurate scientific assessment of efficacy across treatments.
The manuscript refers to Platypus koryoensis as the insect vector for Korean oak wilt disease but does not briefly describe its role in disease epidemiology. A concise explanation would help contextualize the vector’s importance.
While it is noted that M. anisopliae spores are temperature-sensitive but UV-tolerant, no further interpretation or implication of this finding is provided. How do these findings affect the field applicability of the fungus?
Several sentences are lengthy and may benefit from restructuring for readability. For instance:
Original: “Entomopathogenic fungi ecologically refer to fungi that utilize insects as hosts to obtain nutrients...”
Suggestion: “Ecologically, entomopathogenic fungi are those that parasitize insects to derive nutrients...” in the abstract
The results, particularly the LT₅₀ of M. anisopliae, are presented without statistical comparison to the other fungi. Including comparative metrics would provide a clearer picture of relative effectiveness.
The statement about "environmental side effects of insecticides" could be better supported with a reference or more specific examples (e.g., non-target effects, soil toxicity, resistance).
Authors are requested to revise the entire manuscript, as there are many mistakes in both grammar and scientific style. Please relook at the references, especially the material methods and results.
Author Response
Reviewer 3
Comment: Please revised the title and short in meaningful words. It has two points one is entomopathogenic fungi and other is oak wilt disease. However, there is no correlation or is not clearly articulated. The transition from general pest management to oak wilt control with fungal agents should be better structured to help the reader.
Response: Thank you for the suggestion. We agree with the reviewer and have shortened the title as follows:
Revised Title: Preliminary report of three entomopathogenic fungi as potential biocontrol agents against the oak wilt vector, Platypus koryoensis
Comment: While the study focuses on Beauveria bassiana, Metarhizium anisopliae, and Purpureocillium lilacinum, no rationale is provided for selecting P. lilacinum, which is less commonly associated with insect control. A brief explanation of its relevance or prior use would strengthen the justification.
Response: Thank you for this important comment. We have now added a brief rationale in the Introduction (Lines 73-77) [as well as the relevant reference] explaining that P. lilacinum was selected due to its increasing recognition as a biocontrol agent against insect pests in recent studies and its successful recovery from native forest soil using baiting techniques. Although traditionally associated with nematode and fungal pathogens, recent literature supports its potential as an insect-pathogenic fungus. This addition provides context for its inclusion alongside more conventional species like B. bassiana and M. anisopliae.
Comment: The phrase “rapid insecticidal effects” is vague. Quantitative descriptors or statistical comparisons should be included to provide a more accurate scientific assessment of efficacy across treatments.
Response: We fully agree. We have revised the phrasing throughout the Results and Abstract sections to specify LT₅₀ values for each fungus and provide comparative interpretations (e.g., “M. anisopliae KACC40969 exhibited the fastest lethal effect, with an LT₅₀ of 58.7 hours, significantly shorter than B. bassiana GHA (90.17 hours, p < 0.05)”). These changes clarify the differences in efficacy (Lines 37–39).
Comment: The manuscript refers to Platypus koryoensis as the insect vector for Korean oak wilt disease but does not describe its role in disease epidemiology.
Response: Thank you for pointing this out. To make it clarified, we have revised it concisely in the Introduction (Lines 59-61) stating that P. koryoensis introduces the pathogenic fungus Raffaelea quercus-mongolicae into oak trees during its boring activity, facilitating systemic infection and wilting.
Comment: Several sentences are lengthy and may benefit from restructuring for readability. For instance, rephrasing the sentence on entomopathogenic fungi in the Abstract.
Response: Thank you for your suggestion, we have revised and rephrased the sentence on entomopathogenic fungi in the Abstract as suggested (Lines 19-22).
Comment: The results, particularly the LT₅₀ of M. anisopliae, are presented without statistical comparison to the other fungi. Including comparative metrics would provide a clearer picture of relative effectiveness.
Response: We agree and have included statistical comparisons using 95% confidence intervals for LT₅₀ values and p-values where applicable. Table 1 now clearly presents these comparisons (Line 253).
Comment: The statement about "environmental side effects of insecticides" could be better supported with a reference or more specific examples (e.g., non-target effects, soil toxicity, resistance).
Response: We agree and have added a more and detailed explanation in the section 4 to take your suggestion into account as well as relevant references (Lines 338-343).
Comment: Authors are requested to revise the entire manuscript, as there are many mistakes in both grammar and scientific style. Please relook at the references, especially the Materials and Methods and Results.
Response: We thank the reviewer for this overarching recommendation. In response, we have completed a full revision of the manuscript for grammar, punctuation, sentence structure, and scientific clarity. Specific attention was given to the Materials and Methods and Results sections to ensure consistency in terminology, tense, unit usage, and formatting.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Sirs,
The authors' answers to my questions and the corrections made to the manuscript text are satisfactory. Therefore, I recommend the article for printing.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors conducted all reviews satisfactorily.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have revised the manuscript meticulously, and now it can be accepted in its current form.
