Identification and Characterization of Pathogenic Fusarium Species Causing White Mold Disease in Cultivated Morels (Morchella spp.) in China

Round 1

Reviewer 1 Report

 GOOD PAPER. The corrections, the form. not the substance, are indicated in the same manuscript which I am attaching.

None

Comments for author File: Comments.pdf

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted (red) in the re-submitted files.

 

Abstract

Line 24: We added a space before “%” and applied this formatting change throughout the entire text.

 

Introduction

Line 34: We replaced “family Morchellaceae (order Pezizales)” with “order Pezizales, family Morchellaceae”.

Line 35: We replaced “ascomycetes” with “Ascomycetes”.

Line 69: We added "(Fig. 1A)" after "M. sextelata".

 

Materials and Methods

Lines 92-94: We provided additional explanations for "serial dilution".

Line 103: We replaced “½-strength” with “half-strength”.

 

Results

Lines 241-242: We replaced ”FTSC、FOCS、FFSC、FSSC、FIESC“ with ”FtSC、FoCS、FfSC、FsSC、FieSC“ respectively, and corresponding revisions were also made to Figures 3 and 4.

Line 267: We replaced “six out of twelve Fusarium species identified” with “Fusarium species”, and the same revision was made at line 274.

Line 312: We replaced “Fusarium spp.” with “the twelve Fusarium species identified”, and the same revision was made at lines 317–318.

 

In addition to the above changes, we also: (1) removed the redundant sentence: "The frequency of isolation for each Fusarium species was calculated as a proportion of the total recovered isolates" (2.7 Data Analysis section); (2) replaced ”To evaluate the pathogenicity of Fusarium isolates associated with white mold disease (WMD) in cultivated morels, representative isolates were selected based on geographic origin and colony morphology“ with ” To determine whether the 12 Fusarium species isolated from diseased Morchella ascocarps were all pathogenic, pathogenicity verification was carried out according to Koch’s postulates. One strain from each of the 12 Fusarium species was selected as a test isolate (representative isolates were selected based on geographic origin and colony morphology, Supplementary Table 2)“(2.6. Pathogenicity testing section, lines 168–172); (3) updated the corresponding author's email address from “458560969@qq.com” to zhangcl88@ccu.edu.cn; (4) added “Supplementary Table 1” after "features" at line 109.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript provides new and interesting information on the occurrence of Fusarium as pathogens in morel cultivations in China. This provides novel information on the occurrence of this genus in mushroom cultivation worldwide, yet the authors have not introduced the knowledge of this genus or related fungi in Nectriaceae as fungal pathogens.

The wide sampling was accompanied by rigorous work on isolation and pathogenicity tests using established pure cultures that were, at least partially, identified using several genetic markers.  However, no information is provided as how the rest of the isolates were identified and what was the selection of 27 allocated for sequencing based on. Were the isolates grouped based on colony morphology or micromorphology and then a certain amount of isolates from each chosen for sequencing? On line 207 it is written that ITS rDNA was sequenced for all 120 isolates but no info on extended set of ITS sequences is given. This would provide a way for selecting isolates for sequencing additional genes, yet still some sibling species could be missed (not selected for protein-coding gene sequencing) due to the low variation of ITS in Fusarium. As the main results compare the distribution of Fusarium species among collection sites and describe their pathogenicity the species identification is key to verify the correctness of the results that cannot be done at the moment.

The same question applies to the selection of strains derived from GenBank to include these in the phylogenetic analyses. On line 147: “Reference sequences from type or ex-type strains of Fusarium species were retrieved from GenBank and previous taxonomic studies.” Omit the second part of the sentence as you didn’t retrieve sequences from studies but only GenBank and mark ex-type sequences in Table 3 and provide studies on which the selection of sequences from other voucher strains was based on.

In addition to listing pathogens in morel cultivations, information on Fusarium and/or related fungi as pathogens of mushroom farms or fungi in general (including plant pathogens) should be added to the Introduction and/or Discussion. Several members of Nectriaceae, e.g. in the former section/genus Dialonectria occur on fungal sporomata. The scope of Fusarium as pathogens has recently also been broadened by realizing these as entomopathogens (https://doi.org/10.1016/j.fbr.2019.12.002).

The term ‘ascocarp’ used on line 87 could be applied throughout the text to replace the ‘fruiting body’ which scientifically is less sound taken that fungi do not fruit.

Abbreviate Morhcella after its first mention.   

In Materials and methods define how virulence was evaluated.

Include info on cultivations sampled but where no Fusaria were detected as depicted on Fig. 1.

Line 185: “Correlations between isolation frequency and virulence were assessed descriptively.” – What does this mean?

The list of isolates selected for pathogenicity tests should be deleted from lines 265-269 and presented with compared measurements in a table in supplementum. One isolate per species does not allow to compare pathogenicity (and virulence) among species, if that is aimed then more isolates should be included in the comparison.

Table 2 columns for host and CaM should be omitted indicating M. importuna for the 2 isolates by asterisk and providing the only CaM sequence number in the text. The need to obtain that sequence should be explained and the two sets of genes described in Materials and methods.

Results

Figures should appear in the order of their mentioning in the text, currently 1B before 1A and 2 introduced after 3 and 4.

Delete ‘symptom desctriptions’ from here (or from Materials and methods), no need to duplicate, the same applies to the first sentences of both paragraphs in 3.1 that has been said earlier.

What is isolation frequency (line 199), how measured?

Did you isolate also fungi from other genera not included in this study and how you distinguished Fusaria from these?

Fig. 2 – pie charts have to be larger to follow the colors. In legend Each colored circle represents one identified species. -> Colors are used to distinguish Fusarium species.

Line 209 delete ‘boundaries’

Fig. 3. What is this tree – representing the whole genus Fusarium or selected lineages/complees/species? If there are just species that occurred in morel farms – these cannot be all analysed together while omitting the rest of the diversity in Fusarium which also need to be included or alternatively, (related) complexes analysed separately, including also species closely related to the target ones. If, however, the phylogeny includes representative of main lineages then rename the legend properly – in any case it is not based on 26 isolates as now on line 221. Outgroup can be removed from the legend and species-complexes marked.

For example, blasting ITS sequences of strains identified as F. acuminatum resulted three sequences identified as F. citricola as the best matches (100% similarity). Although, the similarity of the ex-type sequence of F. citricola (LT746245) is 99.65, the species should still be included in comparison and the phylogenetic trees if these will be retained.

The colors in the tree do not all correspond to those explained on the left-hand side, e.g. what is yellow? Hard to distinguish the different violet-puprlpe.

Fig. 4 to rename it in the legend- what a tree is this? Why such species were included?

Line 231 specify here and in Suppl table that the characteristics are derived from culture, grown on PDA?

Fig. 5 – you illustrate aerial and sporodochial conidiophores – aren’t the latter also aerial, i.e. not submerged?

249 consistent with descriptions of respective species …

260 absent -> lacking

262-3 supported delimitation of Fusarium species.

Discussion

Avoid just retelling the results here, omit list of 12 species names and their descriptions. Rather evaluate your results with respect to previous knowledge, e.g. do you agree or argue the identification of the only previous records of Fusarium on morels in the two species (line not identified in your study? Where there any characteristics of species found to be different fromprevious reports? From which complexes these are – enough carefully identified? What would be the use of your results for WMD management, how would it depend on identifying the pathogen to different species or genera? What could be the inoculum for Fusaria in morel cultivations – soil, substrate, plants.

line 302 delete ‘meanwhile’

line 307 add ‘tested’ after ‘all’

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted (red) in the re-submitted files.

 

Comments 1: However, no information is provided as how the rest of the isolates were identified and what was the selection of 27 allocated for sequencing based on. Were the isolates grouped based on colony morphology or micromorphology and then a certain amount of isolates from each chosen for sequencing? On line 207 it is written that ITS rDNA was sequenced for all 120 isolates but no info on extended set of ITS sequences is given. This would provide a way for selecting isolates for sequencing additional genes, yet still some sibling species could be missed (not selected for protein-coding gene sequencing) due to the low variation of ITS in Fusarium. As the main results compare the distribution of Fusarium species among collection sites and describe their pathogenicity the species identification is key to verify the correctness of the results that cannot be done at the moment.

Response 1: We thank the reviewer for this important comment and for highlighting the need to clarify the identification workflow and isolate selection strategy. In response, we have revised the manuscript to explicitly describe how the 120 isolates were processed and how the 27 representative isolates were selected for multilocus phylogenetic analysis.

Briefly, all 120 isolates recovered from diseased morel ascocarps were initially subjected to ITS rDNA sequencing for preliminary genus-level identification, which confirmed that all isolates belonged to Fusarium. We acknowledge that ITS has limited resolution for species-level identification within Fusarium; therefore, ITS sequences were not used alone for species delimitation.

To avoid misidentification and the omission of closely related (sibling) species, isolates were subsequently grouped based on a combination of colony morphology (growth rate, pigmentation, colony texture) and micromorphological characteristics (macroconidia, microconidia, chlamydospores, and conidiophore structures). Within each morphotype group, representative isolates were selected proportionally according to their geographic distribution and isolation frequency, ensuring that dominant and less frequent morphotypes from different sampling sites were included.

Based on this integrative screening approach, 27 representative isolates were selected for multilocus phylogenetic analyses using ITS, tef1, CaM, and rpb2 gene regions, which provide higher resolution for species-level identification in Fusarium. This strategy minimized the risk of overlooking cryptic or closely related species that might not be distinguishable using ITS alone.

These methodological details and the rationale for isolate selection have now been clearly added to the manuscript (Page 9, Section 3.2 “Molecular identification”, Lines 227–235). We believe that this clarification strengthens the robustness and transparency of species identification underlying the distribution and pathogenicity analyses presented in this study.

 

Comments 2: The same question applies to the selection of strains derived from GenBank to include these in the phylogenetic analyses. On line 147: “Reference sequences from type or ex-type strains of Fusarium species were retrieved from GenBank and previous taxonomic studies.” Omit the second part of the sentence as you didn’t retrieve sequences from studies but only GenBank and mark ex-type sequences in Table 3 and provide studies on which the selection of sequences from other voucher strains was based on.

Response 2: We thank the reviewer for this helpful suggestion. As recommended, we have revised the sentence on Line 147 to remove the phrase “and previous taxonomic studies,” as all reference sequences were retrieved directly from GenBank.

In addition, ex-type strain sequences have now been clearly marked with an asterisk (*) in Table 3 to distinguish them from other reference strains. For non–type voucher strains included in the phylogenetic analyses, we have added citations to the corresponding taxonomic studies that informed their selection. These references are now explicitly provided in the revised manuscript (Line 147: “[29-31]”).

These revisions improve the clarity and traceability of reference sequence selection used in the phylogenetic analyses.

 

 

Comments 3: The term ‘ascocarp’ used on line 87 could be applied throughout the text to replace the ‘fruiting body’ which scientifically is less sound taken that fungi do not fruit.

Abbreviate Morhcella after its first mention.   

Response 3: We agree with the reviewer’s suggestion. The term “ascocarp” has now been used consistently throughout the manuscript to replace “fruiting body,” and the genus name Morchella has been abbreviated after its first occurrence. These changes have been applied throughout the revised text.

 

Comments 4: In Materials and methods define how virulence was evaluated.

Response 4: We thank the reviewer for this comment. The criteria used to evaluate virulence have now been explicitly defined in the Materials and Methods section. Specifically, virulence was assessed based on the development of brown necrotic lesions around inoculation sites on morel ascocarps following inoculation with Fusarium isolates, with lesion size used as a quantitative measure. This description has been added to Section 3.4 “Pathogenicity tests” (Page 12, Lines 298–300).

 

Comments 5: Include info on cultivations sampled but where no Fusaria were detected as depicted on Fig. 1.

Fig. 2 – pie charts have to be larger to follow the colors. In legend Each colored circle represents one identified species. -> Colors are used to distinguish Fusarium species.

Response 5: We thank the reviewer for this comment. Information on cultivation sites where no Fusarium was detected has now been explicitly included and is indicated in Figure 2 and its caption. Specifically, the names of sampling sites without Fusarium isolation are now described in the figure legend.

In addition, Figure 2 has been revised by enlarging the pie charts to improve color discrimination. The figure legend has also been corrected by revising the description from “Each colored circle represents one identified species” to “Colors are used to distinguish Fusarium species.”

These revisions have been incorporated in Section 3.1 “Symptom description and survey findings” (Page 9, Line 220) and in the updated Figure 2.

 

 

Comments 6:

Line 185: “Correlations between isolation frequency and virulence were assessed descriptively.” – What does this mean?

Response 6:

We thank the reviewer for pointing out this ambiguity. The statement “Correlations between isolation frequency and virulence were assessed descriptively” was unclear and could not be adequately supported by the data presented. As this analysis was not formally conducted in the present study, the sentence has been removed from the Data Analysis section.

The manuscript has been revised accordingly, and no inference regarding correlations between isolation frequency and virulence is now made (Section 2.7 “Data Analysis”).

 

Comments 7:

The list of isolates selected for pathogenicity tests should be deleted from lines 265-269 and presented with compared measurements in a table in supplementum. One isolate per species does not allow to compare pathogenicity (and virulence) among species, if that is aimed then more isolates should be included in the comparison.

Response 7:

We thank the reviewer for this important comment. In accordance with the suggestion, the list of isolates previously presented in Lines 265–269 has been removed from the main text. All pathogenicity assay data, together with strain information, are now presented in Supplementary Table 2.

We agree that using a single isolate per species does not allow for a robust comparison of virulence among species. The objective of the pathogenicity assays in this study was therefore not to compare relative virulence levels among Fusarium species, but rather to confirm whether the identified species are pathogenic to morel ascocarps. Given the large number of isolates collected from diverse geographic regions, we acknowledge that substantial intraspecific variation in pathogenicity is likely to exist, and comparative virulence analyses will be addressed in future studies.

To avoid overinterpretation of the data, we have revised the manuscript accordingly by removing statements implying differences in virulence among species. Specifically:

• Lines 24–25: “Pathogenicity assays showed that F. acuminatum had the highest virulence…” was revised to “Pathogenicity assays showed that all the identified twelve Fusarium species were virulent to morel ascocarps.”
• Lines 301–305: Comparative statements describing differences in lesion size among species were removed and replaced with a general description confirming virulence of all tested isolates.
• Lines 388–389: The phrase “with clear differences in their ability to cause disease” was revised to “and all these species exhibited virulence to morel ascocarps.”

These revisions ensure that conclusions drawn from the pathogenicity assays are fully supported by the experimental design.

 

 

Comments 8:

Table 2 columns for host and CaM should be omitted indicating M. importuna for the 2 isolates by asterisk and providing the only CaM sequence number in the text. The need to obtain that sequence should be explained and the two sets of genes described in Materials and methods.

Response 8:

We thank the reviewer for this helpful suggestion. As recommended, the columns “Host” and “Calmodulin gene (CaM)” have been removed from Table 2. Host information is now provided in the table footnote, where the two isolates derived from M. importuna are clearly indicated (Lines 136–137).

In addition, because only a single isolate (TJS-16-2) required CaM sequencing for accurate identification, the CaM accession number is now provided directly in the main text rather than in Table 2. Specifically, the following statement has been added (Lines 153–157):

“The CaM gene sequence of TJS-16-2 was deposited in GenBank under accession number PQ595068. The CaM gene was selected because it is a core barcode for the identification of Fusarium species and provides high resolution for distinguishing closely related taxa, including strains such as TJS-16-2 that are prone to misidentification.”

The use of the two multilocus datasets (ITS–tef1–rpb2 and CaM–tef1–rpb2) has already been described in detail in Sections 2.4 and 2.5 of the Materials and Methods.

 

Comments 9:

Figures should appear in the order of their mentioning in the text, currently 1B before 1A and 2 introduced after 3 and 4.

Response 9:

We have revised the manuscript to ensure that all figures are cited in the correct numerical order at their first mention in the text. Specifically, references to Fig. 1A and Fig. 1B have been reordered appropriately, and Fig. 2 is now introduced before Figs. 3 and 4. These changes ensure consistency between figure numbering and textual presentation.

 

Comments 10:

Delete ‘symptom desctriptions’ from here (or from Materials and methods), no need to duplicate, the same applies to the first sentences of both paragraphs in 3.1 that has been said earlier.

Response 10:

We agree with the reviewer that the symptom description was unnecessarily duplicated. Accordingly, all content related to “symptom description” has been removed from the Materials and Methods section.

In addition, to avoid repetition in the Results section, we deleted the sentence “Field surveys were conducted from 2023 to 2025 across 22 cultivation sites in 16 provinces of China to assess the incidence and distribution of white mold disease (WMD) in cultivated Morchella spp.” from Section 3.1.

The second paragraph of Section 3.1 “Symptom description and survey findings” has also been revised to eliminate overlap with earlier content.

 

Comments 11:

What is isolation frequency (line 199), how measured?

Did you isolate also fungi from other genera not included in this study and how you distinguished Fusaria from these?

Response 11:

We thank the reviewer for this comment. The term “isolation frequency” has now been clearly defined in the main text as the proportion of Fusarium isolates recovered from a given sampling site relative to the total number of Fusarium isolates obtained across all sites. The method of calculation is now explicitly stated.

In addition, we clarified that fungi belonging to other genera were also isolated during the surveys but were not included in this study. These non-Fusarium isolates were distinguished based on macroscopic colony characteristics, micromorphological features, and ITS-based phylogenetic analysis. These clarifications have been added to the revised manuscript (Lines 200–209).

 

Comments 12:

Line 209 delete ‘boundaries’

Response 12:

As suggested, the term “boundaries” has been deleted from the manuscript (Line 227).

 

Comments 13:

Fig. 3. What is this tree – representing the whole genus Fusarium or selected lineages/complees/species? If there are just species that occurred in morel farms – these cannot be all analysed together while omitting the rest of the diversity in Fusarium which also need to be included or alternatively, (related) complexes analysed separately, including also species closely related to the target ones. If, however, the phylogeny includes representative of main lineages then rename the legend properly – in any case it is not based on 26 isolates as now on line 221. Outgroup can be removed from the legend and species-complexes marked.

For example, blasting ITS sequences of strains identified as F. acuminatum resulted three sequences identified as F. citricola as the best matches (100% similarity). Although, the similarity of the ex-type sequence of F. citricola (LT746245) is 99.65, the species should still be included in comparison and the phylogenetic trees if these will be retained.

The colors in the tree do not all correspond to those explained on the left-hand side, e.g. what is yellow? Hard to distinguish the different violet-puprlpe.

Response 13:

We thank the reviewer for this detailed and constructive comment. The phylogenetic tree presented in Figure 3 does not represent the entire genus Fusarium. Instead, it is a targeted phylogeny designed to resolve the taxonomic placement of Fusarium isolates recovered from morel cultivation systems.

Reference sequences were selected from GenBank based on tef1 BLAST similarity to our isolates and represent closely related species within the corresponding species complexes. The phylogenetic analysis was conducted using a concatenated ITS, tef1, and rpb2 dataset, including 26 representative isolates obtained in this study together with relevant reference strains. This approach was adopted to ensure accurate species delimitation rather than to reconstruct a genus-wide phylogeny.

We acknowledge that ITS-based BLAST searches may yield high similarity among closely related species (e.g., F. acuminatum and F. citricola). For this reason, species identification in the present study was based on multilocus phylogenetic analyses rather than ITS alone, thereby minimizing the risk of misidentification due to limited ITS resolution. Closely related species were included in the phylogenetic analyses to allow reliable differentiation.

To improve clarity, the Figure 3 legend has been revised to explicitly state that the tree includes representative isolates and closely related species rather than the full diversity of Fusarium. Species complexes are now clearly indicated in the figure, and the outgroup information has been simplified.

In addition, Figure 3 has been revised to improve color contrast and legend accuracy. Ambiguous colors have been adjusted so that all clades and species complexes are clearly distinguishable.

 

 

Comments 14:

Fig. 4 to rename it in the legend- what a tree is this? Why such species were included?

Response 14:

We thank the reviewer for this comment. The title of Figure 4 has been revised to clarify the scope and purpose of the phylogenetic analysis. The revised title now reads:

“Phylogenetic tree based on combined CaM, tef1, and rpb2 gene regions of Fusarium pathogens causing WMD in Morchella (isolate TJS-16-2) and related species.”

In addition, we have clarified the rationale for species selection in the main text (Lines 232–235). The reference species included in Figure 4 belong to the same species complex and show high phylogenetic relatedness to isolate TJS-16-2 based on tef1 sequence comparisons. This targeted selection improves the robustness and accuracy of species identification for isolates that are prone to misidentification.

 

Comments 15:

Line 231 specify here and in Suppl table that the characteristics are derived from culture, grown on PDA?

Response 15:

We agree with the reviewer and have clarified that the morphological characteristics described were derived from cultures grown on PDA. This information has now been explicitly stated in the main text (Lines 264–265) and in Supplementary Table 1.

 

Comments 16:

Fig. 5 – you illustrate aerial and sporodochial conidiophores – aren’t the latter also aerial, i.e. not submerged?

Response 16:

We thank the reviewer for this clarification. Sporodochial conidiophores are indeed aerial structures and are not submerged. To avoid ambiguity, we have added the clarification “(not submerged)” after “sporodochial conidiophores” in the figure legend and text (Lines 271 and 278).

 

Comments 17:

249 consistent with descriptions of respective species …

260 absent -> lacking

262-3 supported delimitation of Fusarium species.

Response 17:

We have revised the wording as suggested to improve accuracy and consistency. Specifically, “consistent with…” was revised in Line 280, “absent” was replaced with “lacking” in Line 291, and the phrase was revised to “supported the delimitation of Fusarium species” in Line 295.

 

Comments 18:

In addition to listing pathogens in morel cultivations, information on Fusarium and/or related fungi as pathogens of mushroom farms or fungi in general (including plant pathogens) should be added to the Introduction and/or Discussion. Several members of Nectriaceae, e.g. in the former section/genus Dialonectria occur on fungal sporomata. The scope of Fusarium as pathogens has recently also been broadened by realizing these as entomopathogens (https://doi.org/10.1016/j.fbr.2019.12.002).

Avoid just retelling the results here, omit list of 12 species names and their descriptions. Rather evaluate your results with respect to previous knowledge, e.g. do you agree or argue the identification of the only previous records of Fusarium on morels in the two species (line not identified in your study? Where there any characteristics of species found to be different fromprevious reports? From which complexes these are – enough carefully identified? What would be the use of your results for WMD management, how would it depend on identifying the pathogen to different species or genera? What could be the inoculum for Fusaria in morel cultivations – soil, substrate, plants.

line 302 delete ‘meanwhile’

line 307 add ‘tested’ after ‘all’

Response 18:

We sincerely thank the reviewer for these constructive and insightful suggestions. In response, we substantially revised the Discussion section to broaden the contextual framework and better integrate our findings with existing knowledge on Fusarium and related fungi as pathogens of mushrooms, fungi in general, and plants.

Specifically, we expanded the Discussion to include:
(i) the occurrence of members of Nectriaceae (e.g., Dialonectria) on fungal sporomata;
(ii) the broader pathogenic scope of Fusarium, including plant and entomopathogenic lifestyles;
(iii) a critical evaluation of our findings in relation to previous reports of Fusarium on morels, including similarities and differences in species composition and morphological characteristics;
(iv) discussion of species complexes identified in this study and the robustness of their taxonomic delimitation; and
(v) implications of our results for WMD management, including the importance of accurate species identification and potential sources of inoculum (e.g., soil, cultivation substrates, and plant-derived materials).

To avoid repetition, we removed detailed re-listing of the twelve identified species and focused instead on interpretation and synthesis. These revisions are reflected in the revised Discussion section (Lines 320–396).

 

In addition to the above changes, we also: (1) removed the redundant sentence: "The frequency of isolation for each Fusarium species was calculated as a proportion of the total recovered isolates" (2.7 Data Analysis section); (2) replaced ”To evaluate the pathogenicity of Fusarium isolates associated with white mold disease (WMD) in cultivated morels, representative isolates were selected based on geographic origin and colony morphology“ with ” To determine whether the 12 Fusarium species isolated from diseased Morchella ascocarps were all pathogenic, pathogenicity verification was carried out according to Koch’s postulates. One strain from each of the 12 Fusarium species was selected as a test isolate (representative isolates were selected based on geographic origin and colony morphology, Supplementary Table 2)“(2.6. Pathogenicity testing section, lines 168–172); (3) updated the corresponding author's email address from “458560969@qq.com” to zhangcl88@ccu.edu.cn; (4) added “Supplementary Table 1” after "features" at line 109.

 

Author Response File: Author Response.docx

Reviewer 3 Report

Thank you for submitting this comprehensive study on Fusarium diversity associated with WMD in cultivated morels in China. The manuscript is strong and addresses an applied problem of clear economic importance.

Major strengths

• Excellent geographic and temporal coverage (2023–2025; 22 sites/16 provinces; 832 symptomatic ascocarps), which makes the dataset valuable for surveillance and future risk mapping.
• Robust species delimitation using multilocus phylogenetics (ITS, tef1, CaM, rpb2) with ML and BI frameworks and strong support values; the assignment to major Fusarium species complexes is particularly useful for readers.

Integration of morphology + molecular data. The supplementary morphology table is unusually detailed and will help diagnostic labs and growers’ extension services distinguish taxa (e.g., conidial dimensions, colony traits, growth rates).

• Biological relevance supported by pathogenicity assays (standardized inoculum, controlled humidity/temperature, lesion measurement, and re-isolation to satisfy Koch’s postulates).
• Clear take-home message: multiple Fusarium spp. can cause WMD, and management must consider this diversity rather than a single causal agent.

Points to reinforce (targeted, feasible revisions)

1. Statistics/reporting clarity: You state ANOVA + Duncan’s multiple range test; please add a short justification for Duncan vs. Tukey/other methods and clarify what constitutes an experimental unit (fruiting body, isolate, replicate) and whether assumptions were checked.
2. Virulence interpretation: Since you note differences in frequency and virulence (e.g., common vs. highly virulent taxa), consider adding one paragraph explicitly discussing how growers should interpret “high frequency” vs. “high virulence” for monitoring priorities (even if you keep recommendations conservative).
3. Practical management link: Your conclusions already point toward diagnostics and integrated management; you could strengthen impact by adding 3–5 sentences on actionable monitoring (e.g., when/where to sample, what loci are most informative for routine identification) without expanding experiments.
4. Figures/tables usability: Ensure that key figures (sampling map; phylogenies) have fully self-contained captions (what colors/symbols mean, what loci were concatenated in each tree), consistent with what you describe in Methods.

 English-language quality assessment (English)

The English is generally clear and publishable, but it would benefit from light professional polishing, mainly for readability and consistency. Specifically, please address:

• Sentence length and redundancy: Several passages are long and can be split for clarity, especially in the Introduction and Discussion.
• Consistency in terminology/abbreviations: Use one form consistently (e.g., “white mold disease (WMD)”) and avoid redefining. Keep species names italicized throughout.
• Minor grammar and style issues: Ensure consistent tense in Methods, consistent hyphenation (e.g., “north-central”), and uniform wording for symptoms (e.g., “villous mycelial overgrowth”).
• Precision in phrasing: A few statements would read more scientifically with hedging (e.g., “suggests/indicates”) unless directly supported by data.

These edits are straightforward and should not require substantive restructuring.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions highlighted (red) in the re-submitted files.

 

Comments 1:

Statistics/reporting clarity: You state ANOVA + Duncan’s multiple range test; please add a short justification for Duncan vs. Tukey/other methods and clarify what constitutes an experimental unit (fruiting body, isolate, replicate) and whether assumptions were checked.

Response 1:

We thank the reviewer for this comment. We have revised the Data Analysis section to improve clarity and transparency. Specifically, we added a brief justification for the use of Duncan’s multiple range test, noting its suitability for balanced designs with multiple treatments, and clarified that the assumptions of normality and homogeneity of variance were checked prior to ANOVA (Page 8, Section 2.7, Lines 188–193).

In addition, we clearly defined the experimental unit in the Pathogenicity Testing section, specifying that each Fusarium isolate was inoculated onto three independent M. sextelata ascocarps as biological replicates (Page 8, Section 2.6, Lines 176–177).

 

Comments 2:

Virulence interpretation: Since you note differences in frequency and virulence (e.g., common vs. highly virulent taxa), consider adding one paragraph explicitly discussing how growers should interpret “high frequency” vs. “high virulence” for monitoring priorities (even if you keep recommendations conservative).

Response 2:

We appreciate this valuable suggestion. In the present study, the pathogenicity assays were designed to confirm whether the identified Fusarium species are pathogenic to morel ascocarps, rather than to quantitatively compare virulence among species. Accordingly, one representative isolate per species was tested, and we acknowledge that intraspecific variation in pathogenicity may occur among isolates from different geographic origins.

To avoid overinterpretation, we revised the manuscript to remove statements implying comparative differences in virulence among species and limited our conclusions to pathogenicity confirmation (Pages 12–13, Section 3.4, Lines 301–305). We agree that evaluating the relationship between isolation frequency and virulence is an important topic and will be addressed in future studies with expanded experimental designs.

 

Comments 3:

Practical management link: Your conclusions already point toward diagnostics and integrated management; you could strengthen impact by adding 3–5 sentences on actionable monitoring (e.g., when/where to sample, what loci are most informative for routine identification) without expanding experiments.

Response 3:

We thank the reviewer for this constructive suggestion. We have revised the Discussion section to strengthen its practical relevance by adding specific, actionable recommendations for disease monitoring and management, including sampling timing, potential inoculum sources, and the importance of accurate molecular diagnostics (Page 14, Section 4, Lines 368–387).

Comments 4:

Figures/tables usability: Ensure that key figures (sampling map; phylogenies) have fully self-contained captions (what colors/symbols mean, what loci were concatenated in each tree), consistent with what you describe in Methods.

Response 4:

We revised Figures 2, 3, and 4 to ensure that all captions are fully self-contained and clearly explain symbols, colors, and the concatenated gene loci used in each phylogenetic analysis.

Additional clarifications regarding sampling locations and sites where no Fusarium was detected have been incorporated into the text and figure legends (Page 8, Section 3.1, Lines 210–214; Page 9, Lines 220–224).

The phylogenetic methods and concatenated gene regions used in Figures 3 and 4 remain consistent with those described in the Methods section (Page 5, Section 2.5, Lines 149–153), and no discrepancies remain between figures and methodology.

 

Comments 5:

Sentence length and redundancy: Several passages are long and can be split for clarity, especially in the Introduction and Discussion.

Response 5:

We thank the reviewer for this suggestion. To improve clarity and readability, we revised several long sentences in the Introduction by splitting them into shorter, more concise statements.

Specifically:
• Page 2, Lines 64–66: The sentence on metabarcoding limitations was split into two sentences to improve clarity.
• Page 2, Lines 74–77: The concluding sentence of the Introduction was revised for conciseness and precision.

In addition, the Discussion section was thoroughly revised to reduce redundancy and to use shorter, more focused sentences throughout.

 

Comments 6:

Consistency in terminology/abbreviations: Use one form consistently (e.g., “white mold disease (WMD)”) and avoid redefining. Keep species names italicized throughout.

Response 6:

We have standardized terminology and abbreviations throughout the manuscript. Redundant redefinitions of abbreviations (e.g., white mold disease [WMD] and potato dextrose agar [PDA]) have been removed, and each abbreviation is now defined only at its first occurrence. In addition, all species names have been checked and are consistently italicized throughout the manuscript.

 

Comments 7:

Minor grammar and style issues: Ensure consistent tense in Methods, consistent hyphenation (e.g., “north-central”), and uniform wording for symptoms (e.g., “villous mycelial overgrowth”).

Response 7:

We have corrected minor grammar and style issues across the manuscript to ensure consistency. Specifically, hyphenation has been standardized (e.g., “north-central”), punctuation has been unified (e.g., “B–M” and “5’–3’”), and wording for symptom descriptions has been made uniform (e.g., “villous mycelial overgrowth”).

In addition, based on reviewer feedback, symptom descriptions were removed from Section 2.1 (Field survey and sampling) to avoid repetition, ensuring consistent and concise presentation of symptoms elsewhere in the manuscript.

 

Comments 8:

Precision in phrasing: A few statements would read more scientifically with hedging (e.g., “suggests/indicates”) unless directly supported by data.

Response 8:

We agree with the reviewer and revised phrasing where appropriate to improve scientific precision. For example, on Page 1 (Line 38), “have demonstrated” was replaced with “indicated that.” We also reviewed the manuscript to ensure that definitive language is used only where conclusions are directly supported by the data. If there are any remaining issues that require correction, please kindly let us know.

 

In addition to the above changes, we also: (1) removed the redundant sentence: "The frequency of isolation for each Fusarium species was calculated as a proportion of the total recovered isolates" (2.7 Data Analysis section); (2) replaced ”To evaluate the pathogenicity of Fusarium isolates associated with white mold disease (WMD) in cultivated morels, representative isolates were selected based on geographic origin and colony morphology“ with ” To determine whether the 12 Fusarium species isolated from diseased Morchella ascocarps were all pathogenic, pathogenicity verification was carried out according to Koch’s postulates. One strain from each of the 12 Fusarium species was selected as a test isolate (representative isolates were selected based on geographic origin and colony morphology, Supplementary Table 2)“(2.6. Pathogenicity testing section, lines 168–172); (3) updated the corresponding author's email address from “458560969@qq.com” to zhangcl88@ccu.edu.cn; (4) added “Supplementary Table 1” after "features" at line 109.

 

Author Response File: Author Response.docx