Analysis of Soil Fungal Community Characteristics of Morchella sextelata Under Different Rotations and Intercropping Patterns and Influencing Factors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled Analysis of Soil Fungal Community Characteristics of Morchella sextelata under Different Rotation and Intercropping Patterns and Influencing Factors is written well. There are some points for consideration and improvement of this manuscript.

1. In the abstract part clear cut hypothesis is missing. First provide information about, why you want to conduct this study, after that provide other information.
2. Introduction of the study lacking some points like Different crops in rotation or intercropping systems may introduce unpredictable changes in soil microbial communities. Discuss all these in introduction part.
3. Current knowledge on how these systems influence fungal pathogens and beneficial fungi in Morchella sextelata soils is insufficient. Add these in detail.
4. Methodology part is missing explanation for why certain crops (ginger, grape and mulberry) were chosen for rotation/intercropping.
5. Why sampling has done 3-5cm depth only?
6. Mention the biological replicates also.
7. In ITS sequencing, PCR conditions (cycles, annealing temperature etc.) should be listed.
8. Specify the version of QIIME2 and other software.
9. Indicate the total number of samples sequenced to clarify.
10. Why were both PERMANOVA and ANOSIM used?
11. There is no mention of whether metadata (temperature, humidity, soil moisture, etc.) was recorded. Add this.
12. No information about how sequencing data was archived.
13. Results and discussion section are written well. Add some more recent studies to make it more valuable.

 

Author Response

Thank you for your suggestions, the following is the reply：

 

Comments 1:In the abstract part clear cut hypothesis is missing. First provide information about, why you want to conduct this study, after that provide other information.

Response 1:In the abstract, this section is added. ' Morchella rotation and intercropping is a new and efficient ecological planting mode, which not only contributes to economic growth, but also promotes the sustainable development of agriculture and has high ecological benefits '

 

Comments 2.Introduction of the study lacking some points like Different crops in rotation or intercropping systems may introduce unpredictable changes in soil microbial communities. Discuss all these in introduction part.

Response 2. In introduction part, it is shown that “M.esculenta rotation, intercropping and intercropping can allow other crops to decompose the secretions of the last M.esculenta mycelium and establish a microbial flora suitable for the growth of M.esculenta mycelium. However, due to the complexity of the microbial system, the interaction between different crops and M.esculenta may form dominant colonies in the soil, thereby inhibiting the growth of other microorganisms, and the harmful microorganisms that feed on specific crop roots and M.esculenta hyphae will also accumulate in large quantities, which may eventually lead to a decrease in microbial diversity. Therefore, different crops in rotation or intercropping systems may bring unpredictable changes to soil microbial communities.”

 

Comments 3.Current knowledge on how these systems influence fungal pathogens and beneficial fungi in Morchella sextelata soils is insufficient. Add these in detail.

Response 3. In introduction part, it is shown that “The mechanism of continuous cropping obstacle of Morchella is very complex, which may be related to the imbalance of soil microbial flora and the increase of harmful microorganisms. It has been reported that the diseases caused by continuous cropping obstacles are generally closely related to the microbial community structure of the soil . The main reason for the high incidence of diseases caused by continuous cropping of crops is the reduction of beneficial microorganisms and the accumulation of pathogens.”

 

Comments 4.Methodology part is missing explanation for why certain crops (ginger, grape and mulberry) were chosen for rotation/intercropping.

Response 4.Ginger, grapes and mulberry trees are planted all year round in this area. We take measures according to local conditions and start sowing in the same block among the soil, grapes and mulberry trees.

 

Comments 5.Why sampling has done 3-5cm depth only?

Response 5.The hyphae of M.esculenta are usually distributed from the soil surface to the shallow layer ( 3-10 cm ), which is the key area for the interaction between hyphae and soil microorganisms. The depth of 3-5cm is closer to the active area of mycelium, which can reflect the characteristics of microbial community directly related to the growth of Morchella.

 

Comments 6.Mention the biological replicates also.

Response 6.In paragraph 1.1, it is shown that ' In order to reduce the experimental error, the soil physical and chemical properties and high-throughput sequencing were repeated six times. '.

 

Comments 7.In ITS sequencing, PCR conditions (cycles, annealing temperature etc.) should be listed.

Response 7.PCR conditions have been added to the paragraph 1.3 is, PCR conditions : pre-denaturation at 98 °C for 30 s ; denaturation at 98 °C for 10 s, renaturation at 58 °C for 30 s, extension at 72 °C for 1 min, 28 cycles ; extension at 72 °C for 2 min.

 

Comments 8.Specify the version of QIIME2 and other software.

Response 8.Using the lllumina NovaSeq 6000 version, which has been modified in paragraph 1.4

 

Comments 9.Indicate the total number of samples sequenced to clarify.

Response 9.In paragraph 1.1, it is shown that “The total number of sequencing samples was 30.”

 

Comments 10.Why were both PERMANOVA and ANOSIM used?

Response 10.A single statistical method may be biased due to different assumptions or calculation methods. PERMANOVA focuses on explaining the variance between groups, while ANOSIM ranks the similarity between side recombinations. The combination of the two can reflect the characteristics of community differences more comprehensively.

 

Comments 11.There is no mention of whether metadata (temperature, humidity, soil moisture, etc.) was recorded. Add this.

Response 11.In paragraph 1.1, it is shown that “The local temperature is 27 °C, the air humidity is 70 %, and the soil humidity is 80 %.”

 

Comments 12.No information about how sequencing data was archived.

Response 12.ITS raw data has been uploaded to NCBI, the accession number is PRJNA1234878.This paragraph has been added in the text

 

Comments 13.Results and discussion section are written well. Add some more recent studies to make it more valuable.

Response 13.The latest research has been supplemented in the discussion, respectively for these :

Li Xuesong et al.found that the dominant microbial groups in the soil of diseased Morchella were Paecilomyces, Fusarium, Trichoderma, etc., which were all related groups of macrofungal pathogens.

In addition, Song et al.found that there was a significant change in soil pH between diseased M.esculenta and healthy M.esculenta during the cultivation of M.esculenta, and pH was related to the community structure and composition of fungi.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

 

The manuscript entitled “Analysis of Soil Fungal Community Characteristics of Morchella sextelata under Different Rotation and Intercropping Patterns and Influencing Factor” was submitted by Feng (2025) to Agriculture. This study aimed to identify disease-associated microorganisms in Morchella sextelata cultivation soils and provide a theoretical basis for mitigating succession barriers in practical production.

The manuscript presents a important study on the fungal community dynamics in soils cultivated with Morchella sextelata under different cropping patterns. The study is significant for sustainable mushroom cultivation, soil microbial ecology and potential rotation constraints.

However, several areas require improvement, particularly regarding clarity, language accuracy, methodological transparency, and discussion coherence. Below are detailed comments on various sections of the manuscript.

For example: the keywords that are used are in the title and this cannot happen.

The abstract lacks clarity in some sections. Sentences should be reworded to improve readability.

L21 Please modify by "Our results indicate that Morchella sextelata cultivation leads to a significant decline in soil fungal diversity compared to uncultivated soils."

Introduction:

Please, clearly define the gap. While the text discusses the importance of Morchella sextelata, it does not explicitly state how this study differs from or builds upon previous research.

 

Materials and Methods

The manuscript states that soil was collected from 3–5 cm depth. It is a layer

Provide a map of the area

 

Do not put dates, but rather how long it was after sowing

Characterize the soil before the experiment

Include citations to the methods used

Mention the specific version of QIIME2 used.

Provide details on filtering thresholds for sequence quality and taxonomic classification confidence levels

 

Results

Some paragraphs are overly descriptive. Summarizing key results concisely would improve readability.

L59 Please modify by: Among cropping systems, fungal diversity was lowest in GRS and highest in FS, as indicated by the Chao1, ACE, Shannon, and Simpson indices

Figures lack proper captions and do not always include statistical significance indicators (e.g., p-values). For clarity, letters or asterisks should denote significant differences between treatments.

Cite the figures in the text;

Please, improve the tables

 

Discussion

The discussion is somewhat repetitive, often restating results instead of providing deeper interpretations.

Please, make a comparison with previous studies. How do these findings align with or contrast with existing literature?

Improve the section of soil physicochemical properties should be discussed more critically:

For example, why did pH show a negative correlation with Fusarium? Could this be linked to known pH-dependent fungal growth mechanisms?

The manuscript states that nitrate nitrogen increased in most cropping systems but not in VIS. However, no clear explanation is given. Please, give propose a hypothesis.

Given these limitations, my recommendation is to Major Revisions the manuscript.

 

Best regards,

Author Response

Thank you for your suggestions, the following is the reply：

 

Comments 1. the keywords that are used are in the title and this cannot happen.

Response 1.The keyword fungal community was changed to pathogenic fungi

 

Comments 2.The abstract lacks clarity in some sections. Sentences should be reworded to improve readability.

Response 2.It has been modified.

 

Comments 3.L21 Please modify by "Our results indicate that Morchella sextelata cultivation leads to a significant decline in soil fungal diversity compared to uncultivated soils."

Response 3.It has been modified.

 

Comments 4.Please, clearly define the gap. While the text discusses the importance of Morchella sextelata, it does not explicitly state how this study differs from or builds upon previous research.

Response 4.In order to illustrate that this study is based on previous studies, this large section is added.‘The mechanism of continuous cropping obstacle of Morchella is very complex, which may be related to the imbalance of soil microbial flora and the increase of harmful microorganisms. It has been reported that the diseases caused by continuous cropping obstacles are generally closely related to the microbial community structure of the soil . The main reason for the high incidence of diseases caused by continuous cropping of crops is the reduction of beneficial microorganisms and the accumulation of pathogens.M.esculenta rotation, intercropping and intercropping can allow other crops to decompose the secretions of the last M.esculenta mycelium and establish a microbial flora suitable for the growth of M.esculenta mycelium. However, due to the complexity of the microbial system, the interaction between different crops and M.esculenta may form dominant colonies in the soil, thereby inhibiting the growth of other microorganisms, and the harmful microorganisms that feed on specific crop roots and M.esculenta hyphae will also accumulate in large quantities, which may eventually lead to a decrease in microbial diversity. Therefore, different crops in rotation or intercropping systems may bring unpredictable changes to soil microbial communities.’

 

Comments 5.The manuscript states that soil was collected from 3–5 cm depth. It is a layer

Response 5.The hyphae of M.esculenta are usually distributed from the soil surface to the shallow layer ( 3-10 cm ), which is the key area for the interaction between hyphae and soil microorganisms. The depth of 3-5cm is closer to the active area of mycelium, which can reflect the characteristics of microbial community directly related to the growth of Morchella.

 

Comments 6.Provide a map of the area

Response 6.

 

Comments 7.Do not put dates, but rather how long it was after sowing

Response 7.It has been modified to Soil samples were collected five months after sowing

 

Comments 8.Characterize the soil before the experiment

Response 8.In paragraph 1.1, it is increased that the area is sandy loam soil, and the air permeability and water permeability are good. Ginger, grape and mulberry are planted all year round, and we take measures according to local conditions.

 

Comments 9.Include citations to the methods used

Response 9.Using five-point sampling method

 

Comments 10.Mention the specific version of QIIME2 used.

Response 10.Using the lllumina NovaSeq 6000 version, which has been modified in paragraph 1.4

 

Comments 11.Provide details on filtering thresholds for sequence quality and taxonomic classification confidence levels

Response 11.In paragraph 1.4, it is increased that ‘The specific processing steps are as follows : Remove reads containing low-quality bases ( mass value ≤ 15 ) exceeding a certain proportion ( default set to 40bp ) ; remove reads with a certain proportion of N bases ( default is set to 10bp ) ; remove reads that overlap with the Adapter exceeding a certain threshold ( default is set to 10bp ) ’

 

Comments 12.Some paragraphs are overly descriptive. Summarizing key results concisely would improve readability.

Response 12.It has been modified.

 

Comments 13.L59 Please modify by: Among cropping systems, fungal diversity was lowest in GRS and highest in FS, as indicated by the Chao1, ACE, Shannon, and Simpson indices

Response 13.has been modified in paragraph 2.1

 

Comments 14.Figures lack proper captions and do not always include statistical significance indicators (e.g., p-values). For clarity, letters or asterisks should denote significant differences between treatments.

Response 14.Some inappropriate chart names have been modified, and the box diagram is used for fungal diversity analysis, which is more intuitive.

 

Comments 15.Cite the figures in the text;

Response 15.It has been modified.

 

Comments 16.Please, improve the tables

Response 16.The table has been modified.

 

Comments 17.The discussion is somewhat repetitive, often restating results instead of providing deeper interpretations.

Response 17.The discussion has been further elaborated upon.

 

Comments 18.Please, make a comparison with previous studies. How do these findings align with or contrast with existing literature?

Response 18.A lot of literature has been added to the discussion section to compare with the existing results.

 

Comments 19.Improve the section of soil physicochemical properties should be discussed more critically:

For example, why did pH show a negative correlation with Fusarium? Could this be linked to known pH-dependent fungal growth mechanisms?

The manuscript states that nitrate nitrogen increased in most cropping systems but not in VIS. However, no clear explanation is given. Please, give propose a hypothesis.

Response 19.Thank you very much for your advice, has made major changes to the article, especially in the discussion, please help me take a look again.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Here are the comments regarding your manuscript titled  “Analysis of Soil Fungal Community Characteristics of Morchella sextelata under Different Rotation and Intercropping  Patterns and Influencing Factors”. Although the manuscript has three clear objectives, it is not presented appropriately. This also reflects a lack of attention to detail in the preparation of an academic work. 

Introduction:

I would recommend starting by explaining what type of plant Morchella is, followed by a discussion of its composition and cultivation methods

Metodology:

None of the methodologies mentioned, whether for physicochemical soil analysis or molecular analysis or licensed software, include references. If these methodologies were developed by the authors, this should be explicitly stated in the manuscript.

Results:

In the results section, specifically in sections 3.1 and 3.2, the subtitles indicate fungal abundance and diversity. However, section 3.1 does not present results on abundance; it only includes findings on fungal diversity. Therefore, the title should be revised accordingly.

Although Figure 1 is subdivided, another Figure 1 appears later, unrelated to the previous one. This should be reviewed, along with the sequential numbering of the subsequent figures and how they are referenced in the text.

Although an analysis of predicted functions based on ITS information is conducted (section 2.7), this methodology is not described in the corresponding section.

The title of Figure 8 should be modified to clearly indicate what is actually being presented. As it stands, its title is too similar to those of Figures 1 and 2.

Discussion:

Unfortunately, in my personal opinion, it is not possible to assess the relevance of the discussion of the results without the supporting citations.

There is no specific conclusions section. The final paragraph of the discussion, although it provides a brief summary of the main findings, does not serve as a conclusion based on the three research objectives presented in the introduction. This section should be further developed to appropriately present the main conclusions and projections of the study.

Comments for author File: Comments.pdf

Author Response

Thank you for your suggestions, the following is the reply：

 

Comments 1.I would recommend starting by explaining what type of plant Morchella is, followed by a discussion of its composition and cultivation methods

Response 1.In the first paragraph of the introduction has been added ‘Morchella spp is a rare edible and medicinal fungus of Ascomycota, which is mainly divided into black type ( M.angusticeps ) and yellow type ( M.esculenta ). Its honeycomb fruiting body has unique morphological characteristics and is widely distributed in the northern temperate deciduous forest area. Molecular systematics studies have confirmed that the genus contains more than 120 cryptic species, showing significant geographical differentiation characteristics.’

 

Comments 2.None of the methodologies mentioned, whether for physicochemical soil analysis or molecular analysis or licensed software, include references. If these methodologies were developed by the authors, this should be explicitly stated in the manuscript.

Response 2.Thank you for the suggestion, the references have been added

 

Comments 3.In the results section, specifically in sections 3.1 and 3.2, the subtitles indicate fungal abundance and diversity. However, section 3.1 does not present results on abundance; it only includes findings on fungal diversity. Therefore, the title should be revised accordingly.

Response 3.The titles of 3.1 and 3.2 have been revised

 

Comments 4.Although Figure 1 is subdivided, another Figure 1 appears later, unrelated to the previous one. This should be reviewed, along with the sequential numbering of the subsequent figures and how they are referenced in the text.

Response 4.The chart position has been modified, and the icon number is also corresponding to the good.

 

Comments 5.Although an analysis of predicted functions based on ITS information is conducted (section 2.7), this methodology is not described in the corresponding section.

Response 5.I 've added a method in paragraph 2.7

 

Comments 6.The title of Figure 8 should be modified to clearly indicate what is actually being presented. As it stands, its title is too similar to those of Figures 1 and 2.

Response 6.It has been modified.

 

Comments 7.Unfortunately, in my personal opinion, it is not possible to assess the relevance of the discussion of the results without the supporting citations.

There is no specific conclusions section. The final paragraph of the discussion, although it provides a brief summary of the main findings, does not serve as a conclusion based on the three research objectives presented in the introduction. This section should be further developed to appropriately present the main conclusions and projections of the study.

Response 7.Thank you very much for your suggestion, and the discussion section has been significantly revised. It includes a more in-depth explanation of some of the content, adding a part of the previous research to compare with the existing research, all of which are discussed more critically, such as why PH is negatively correlated with Fusarium, PH-dependent fungal growth mechanism, etc., and adding some recent research to make the article more valuable. I hope you can look again and give more valuable advice.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Considering the improvements made to your work and the incorporation of the suggested revisions, I am inclined to recommend to the editor the acceptance of the manuscript in its current form.

Author Response

Thank you for your suggestions, the following is the reply：

 

Comments 1:The authors should integrate data on the rhizosphere microbial communities, soil physicochemical properties, and Morchella yield under crop rotation and intercropping modes to provide insightful conclusions that can guide production practices. At the very least, preliminary conclusions should be summarized.

Response 1:Specific practical guidance has been given in the last paragraph of the article. That is, grape intercropping cultivation interferes with nitrogen metabolism due to root exudates, resulting in abnormal accumulation of nitrate nitrogen, which induces specific enrichment of Paecilomyces, and ecological regulation needs to be achieved through nitrate nitrogen supplementation. Mulberry intercropping cultivation promoted the proliferation of Fusarium due to soil acidification, and the application of lime to adjust pH could block the niche advantage of pathogenic bacteria. The rotation cultivation of ginger increased the damage due to excessive phosphorus and potassium and synergistic pathogenicity. It was necessary to combine element leaching with the introduction and optimization of biological antagonistic bacteria.

 

Comments 2:Regarding soil physicochemical properties and fungal communities, it is essential to determine whether the cultivation mode alters soil physicochemical properties, thereby affecting microbial communities, or if it changes microbial communities, which in turn influence soil physicochemical properties. The authors should reference previous studies to establish this logical relationship and draw corresponding conclusions.

Response 2:Referring to the classical cascade model of ' agricultural management → soil chemical environment → microbial ecology → crop health ', the logic of this study is explained in the last paragraph of the discussion.

 

Comments 3:The manuscript contains several formatting inconsistencies, such as the failure to italicize Latin scientific names. Additionally, Latin names should be abbreviated upon their second and subsequent mentions, e.g., Morchella sextelata should be abbreviated as M. sextelata.

Response 3:All Latin names have been italicized, and Latin names have also been abbreviated for the second time and later.

 

Comments 4:The clarity of figures has not been significantly improved, particularly in Figures 5, 7-9.

Response 4:Picture 5, 7-9 has been modified and is now clearer.

 

Comments 5:The map suggested by the reviewers does not appear in the main text, and the labels on the map are in Chinese. Given the excessive number of figures, it is recommended that some be moved to the supplementary materials.

Response 5:Thank you very much for your suggestion. Because we have no property rights to the map, the use of the picture needs to go through complex procedures, and the map given above cannot be published publicly, so it is only placed in the reply, and I am very sorry that the map cannot be included in the article. We have described the test site in detail in the test plan : latitude 28.884132 ° N, longitude 120.387460 ° E, altitude 326 m

Author Response File: Author Response.docx