Investigating the impact of temperature on growth rate of the root rot fungus, Gymnopus fusipes
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Gymnopus fusipes is an understudied root rot pathogen associated with multiple tree species and is linked to episodes of oak decline across the United Kingdom and Europe. Although the reported distribution of G. fusipes is broad, many observations rely solely on visual identification of fruiting bodies, which can be unreliable, and lack confirmation by molecular and/or isolation data to verify this broad ecological range. Given the paucity of information regarding the true ecological distribution of G. fusipes , it is difficult to predict and model the potential distribution of the species under both current and future climate scenarios. In this study, to determine the growth capabilities of G. fusipes across a range of ecologically relevant temperatures , five geographically diverse isolates of G. fusipes were grown at five different temperatures ranging from 4–37°C, to determine the optimal temperature for G. fusipes growth, and to establish whether geographically diverse isolates exhibit local adaptation to temperature tolerance. Incubation temperature had a significant effect on G. fusipes growth rate, with 25°C representing the optimum ( P <0.001). Isolates had differing growth rates at each of the temperatures, with an isolate from the UK having the highest overall growth rate across all five temperatures tested ( P <0.001), and at the optimum, increased by a mean value of over 4915 mm 2 . Local adaptation to temperature tolerance was not found in the isolates tested. These data demonstrate the optimal incubation temperature for future laboratory studies on G. fusipes and provide the first data on the growth rate of this pathogen across ecologically relevant climate ranges that may inform land managers, modellers, and policy makers in predicting the current and potentially future geographical limits of this widespread root rot pathogen.
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This is a study that would be of interest to the field and community.
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Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
The topic of temperature impact on fungal growth is interesting in it's present context. However, there is evidence of inappropriate self citations with no justification from other literature. This is poor practice and must be corrected. The data also cannot support the conclusions or discussion in it's present state. Saying that a 'statistical analysis' was done is not sufficient justification. The analysis of data is a major flaw that must be corrected. Line 53 - It is poor practice to reference just yourself in such a context. You need to have at least 2 other reference to support this statement. Same for line 346 Line 69 - In what way? Line 70 - You say it's a significant influence. How significant? Where are you tests? If there are no statistical tests carried out, you cannot use the term …
Comments to Author
The topic of temperature impact on fungal growth is interesting in it's present context. However, there is evidence of inappropriate self citations with no justification from other literature. This is poor practice and must be corrected. The data also cannot support the conclusions or discussion in it's present state. Saying that a 'statistical analysis' was done is not sufficient justification. The analysis of data is a major flaw that must be corrected. Line 53 - It is poor practice to reference just yourself in such a context. You need to have at least 2 other reference to support this statement. Same for line 346 Line 69 - In what way? Line 70 - You say it's a significant influence. How significant? Where are you tests? If there are no statistical tests carried out, you cannot use the term 'significant'. Line 138 - The DNA methods should be in their own section Line 157 and 158 - This is fundamentally wrong. ITS sequencing cannot get to species level without further primers. Line 177 - You haven't said they are 5 strains. You've said they come from 5 sites. Line 181 - What are the parallel tests? Which sections of the methods is this linking to? Line 185 - Why are you telling us how to pour agar? Section 2.2 - If you're giving the diameter of the petri dish (which seems a little strange), what's the diameter of the plug you're using? Section 2.2 - This can be combined with the first paragraph of the methods section. You're repeating your media conditions. Line 225 - 'Statistical analysis was carried out', and? What tests? Section 2.4 - This needs a rewrite with comprehensive details and a change in tone. Line 284 - You say significantly again without any stats support. How 'significant'? What's the p value? What test have you done to know it's 'significant'. Section 3.2 - This section is a major problem. You cannot just say an analysis was done. You have no stats tests shown but use 'significantly' a lot. You have no support or justification to say anything is significant in your current manuscript.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
The manuscript titled "Investigating the impact of temperature on growth rate of the root rot fungus, Gymnopus fusipes" represents investigations regarding an ecological requirement of a pathogen that could affect the proper growth of trees. This could complicate afforestation contributing to climate change and associated effects. The study has strengths such as being conducted from two sites which can reduce random errors, proper verification of the materials (Isolate strains) using molecular means prior to experimentation, and statistical analysis. However, I seriously believe that the manuscript also has areas that require improvement before it can be published. This includes; a relatively poor structure involving mixture of some content in different sections (such as methods in results). Inadequate …
Comments to Author
The manuscript titled "Investigating the impact of temperature on growth rate of the root rot fungus, Gymnopus fusipes" represents investigations regarding an ecological requirement of a pathogen that could affect the proper growth of trees. This could complicate afforestation contributing to climate change and associated effects. The study has strengths such as being conducted from two sites which can reduce random errors, proper verification of the materials (Isolate strains) using molecular means prior to experimentation, and statistical analysis. However, I seriously believe that the manuscript also has areas that require improvement before it can be published. This includes; a relatively poor structure involving mixture of some content in different sections (such as methods in results). Inadequate justification of the problem being addressed by the research, undisclosed criteria for choice of study materials. Insufficient information is provided regarding quality control during the experimentation. There is no ethical clearance for the study. Insufficient literature review especially during results discussion, among others as described below. Title and Keywords Consider adding the study location such as "Northern Europe" if appropriate for specificity about the distribution of investigated strains. Another possible keyword could be the "oak" tree that is most affected by the studied agent to enable easier access to the article. Abstract The abstract is quite long with some unnecessary statements of repetition such as "… by having different growth rates at different temperatures", "(originating from the south of England)", "(at the optimal growth temperature of 25˚C).", "…, as there was no significant interaction between temperature and isolate." What does "UK" stand for? Also, there is mixture of results and methods statements such as ". Statistical analysis, and a generalised linear mixed-effects model fitted to the growth data, illustrated that …" Line 25-27: The statement "G. fusipes …... at 37˚C" can be omitted since the optimum temperature is the main result to be captured. Line 33-34: This last bit of the sentence "….as there was no significant interaction between temperature and isolate." is unnecessary since it is more of an explanation for the already mentioned point and should be kept for the discussion section. Despite being an unstructured abstract (No subtitles) which is okay, the author should re-organize the sentences to flow appropriately from the introduction, through the problem, goals, and most essential methodology, up to the main results and conclusion. Should define all abbreviations at first mention, then use them later in the text. Consider summarizing the abstract into 250-300 words for ease of the reader. Introduction In addition to distribution, consider the addition of some biological background of G. fusipes such as taxonomy, and genetic makeup that can aid molecular characterization, virulence factors, and others as appropriate. Line 87: Specifying the temperature range at this point could be quite valuable and clarify the limits in the statement "……require a much narrower margin …..." The author is encouraged to include some literature about G. fusipes regarding local adaptation just like done for Phytophthora infestans and Puccinia striiformis f. sp. tritici. Would give more attention to the subject organism for the study and only a mild reaction in relation to the organisms in comparison. Line 114: Further clarity is needed regarding the significance of the problem to be solved by learning the effect of temperature in this case within the problem statement. Since the infection by G. fusipes can take up to 30 years, how is it much of a problem in relation to humans? Are there any economic effects reported or anticipated? If so, how much? Or disease events due to habitat destruction? The author is encouraged to further write about the research gap with justification for it, and linkage to the contribution of the study to current literature. Line118: Would prefer if the statement "This work employed traditional culture-based methods" is removed from this sentence and transferred to the methods section. The same applies to any other one of a similar kind. Consider the addition of some background however little regarding the growth rates of the different G. fusipes strains and how it is affected by the temperature. Materials and Methods The author should think about adding a little description of the general study design, the study duration, and information about the study sites including the justification for their use during the study. Such as "This was a cross-sectional study conducted from XXXX to XXXX at XXXX laboratory…..." Line 129: What was the reason behind the choice of the strains used in this study and the choice of their origin? Are they the only ones found in Europe? If not, why were others not considered to provide better knowledge about the distribution of G. fusipes. Further information is needed from the author regarding the sampling, inclusion, and exclusion criteria for the studied strains of G. fusipes. Figure 1: This being a map, I was of the view that extra elements are added such as the compass direction, ground scale Line 138: The author should add subheadings before the paragraphs discussing new subjects such as "DNA extraction" in this case. Abbreviations should be written in full at first appearance. Line 157: Same as above. Line 184: Was Quality control (QC) done on the media such as using standard organisms to confirm the ability of the media to effectively support growth under routine conditions before the experiment? If so, kindly include how it was done. Otherwise, describe how it was observed in order to trust the results of growth from the media throughout the experiment. Line 194: There is a typing error "…optima..." that requires correction. Line 195: Kindly clarify to the reader how this replication number was arrived at and why it is sufficient for the reliability of the experiment outcomes. Consider the addition of a pictorial view of the workflow during the experimentation for easier following. Line 220: The spacing error "…timepoint,…" should be rectified. Line 221: Same here "..area was…" Line 239: Specify the threshold level (such as 95% confidence level) considered for statistical inference of significance in the study. The author should include an ethics statement specifying the ethical body that approved the study to be conducted and include the approval number if the results are to be trusted widely. If the ethical approval was not required, clearly explain why it is the case. Results Should add subheadings to clarify points revealing the results of the main study objectives. Consider the inclusion of some pictures of the growth plates from the experiment for easier comprehension by the reader. Line 243- 248: This paragraph consists of information that belongs to the methods section and much of it is already mentioned. Avoid unnecessary repetitions by merging this with the existing content in the methods. At this point, specify the outcomes from the experiment answering the research questions. Figure 2: Individual subfigures for the five studied temperatures can easily be misinterpreted. Consider displaying the different growth changes for all temperatures on a single row. Eliminate the shadows to enable clear differentiation of the growth patterns exhibited by the isolate strains. Line 267-271: Remove the statement "…...significant effect on the growth rate of G. fusipes." describing how statistical analysis was done and instead state what was found from the analysis. Line 275: The statement "Analysis was also conducted to determine whether the isolate of G. fusipes influenced the growth rate observed." Should be treated as mentioned above. Line 288-290: Transfer the statement "The final statistical analysis was conducted to determine if there was a relationship between incubation temperature and isolate on the growth of G. fusipes, which may indicate localized adaptation to temperature between the different isolates." to the methods section and specify the method used to test for the association. Line 293-295: Transfer the statement "In this analysis, the relationship between isolate and temperature was used in the generalised linear mixed-effects model (Figure 4), with temperature as a polynomial number." Line 297-300: The last two statements "This illustrates that……" would best fit in the discussion section. Consider transferring them there. Discussion Line 316-318: For consistency, consider putting the P values in brackets such as "A significant effect (P value
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
Reviewer Comments: Reviewer 1 In the manuscript entitled "Investigating the impact of temperature on growth rate of the root rot fungus, Gymnopus fusipes." Abstract: * Clear and concise writing: The text is easy to understand and follows a logical flow. * Well-organized structure: The introduction, methods, results, and discussion sections are clearly delineated. Suggestions for improvement: * Specific data: Consider adding specific data points, such as average growth rates at different temperatures, to strengthen your results. * Lines 15-18: Consider restructuring this sentence for clarity. Perhaps: "However, many observations rely solely on visual identification of fruiting bodies, which can be unreliable, and lack confirmatory molecular and/or isolation data to verify this broad ecological range."
Comments to Author
Reviewer Comments: Reviewer 1 In the manuscript entitled "Investigating the impact of temperature on growth rate of the root rot fungus, Gymnopus fusipes." Abstract: * Clear and concise writing: The text is easy to understand and follows a logical flow. * Well-organized structure: The introduction, methods, results, and discussion sections are clearly delineated. Suggestions for improvement: * Specific data: Consider adding specific data points, such as average growth rates at different temperatures, to strengthen your results. * Lines 15-18: Consider restructuring this sentence for clarity. Perhaps: "However, many observations rely solely on visual identification of fruiting bodies, which can be unreliable, and lack confirmatory molecular and/or isolation data to verify this broad ecological range." * Line 25: "Temperature tolerance" might be more accurate than "temperature preference" if different isolates can survive but not necessarily thrive at different temperatures. * Line 47: You could clarify whether the 4915 mm2 increase refers to individual growth or the difference between the UK isolate and other isolates. Introduction: Suggestions for improvement: * Sentence 49 could be rephrased as "Since its identification as a primary pathogen in the mid-1980s, Gymnopus fusipes has been linked to numerous episodes of oak decline in the UK and Europe." * Sentence 54 could be shortened by combining it with sentence 53: "Though confirmed distribution based on molecular analysis or fungal isolation is limited to Europe and the UK, less conclusive methods suggest a wider range, potentially encompassing Europe, America, Asia, and northern Africa." * Citations: Ensure all citations follow the journal's specific style guide. For example, you might need to add full page numbers for some references. * Sentence 84 could be rewritten for clarity. Consider "Much uncertainty surrounds how pathogens and tree hosts will respond to a changing climate, partly due to a lack of empirical data on their tolerances and limits to different climatic elements." * Sentence 89 could be rephrased for conciseness. "Local adaptation to specific climates can cause geographic variation in virulence, evident in forest pathogens where isolates from different locations react differently to temperature." Material and Methods: Suggestions for improvement: * Some sentences can be shortened or restructured for improved clarity and flow. (e.g., lines 122-125 & 149-152) * Consider removing repeating information (e.g., "14,000 xg for 2 minutes" is mentioned multiple times). * Use simpler terms where possible ("RPM 150" could be replaced with "moderate speed") * Abbreviations: Define any abbreviations used, like T0, T7, etc., on first mention. * Statistical analysis: Mention the intended statistical analysis for the data collected. Discussion: Suggestions for improvement: * Consider rearranging the discussion: The section currently starts with conclusions and then moves to limitations and future research. It might be more impactful to start with the main findings and their significance, then discuss limitations and future directions to address them. * Strengthen the connection to local adaptation: Although you state the hypothesis for local adaptation was not supported, the discussion could delve deeper into possible explanations for this result. Was the sample size sufficient? Were the geographical locations diverse enough? * Expand on predictive modeling potential: The potential use of your data for predictive modeling is interesting. Consider providing more details about specific modeling tools and how they could be used with G. fusipes data.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Very good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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