PONE-D-23-21640Exploring the Interplay Between the Core Microbiota, Physicochemical Factors, Agrobiochemical Cycles and Resistome in the Soil of the Historic Tokaj Mád Wine RegionPLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

Reviewer #5: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: Yes

Reviewer #5: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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Reviewer #1: REVIEW OF MANUSCRIPT (PONE-D-23-21640) ENTITLED „EXPLORING THE INTERPLAY BETWEEN THE CORE MICROBIOTA, PHYSICOCHEMICAL FACTORS, AGROBIOCHEMICAL CYCLES AND RESISTOME IN THE SOIL OF THE HISTORIC TOKAJ MÁD WINE REGION” BY REMENYIK J. ET AL.

The soil microbial community plays a pivotal role in the growth and vigor of cultivated plant species, so the research aim is sensible and well-articulated. Soil samples, originated from ten vineyards of the Tokaj-Mád wine region were analyzed and compared based on a metagenomics research strategy. Characteristics of the soil microbial community were evaluated at three depths and at two-time points. The first question to come to light is related to the sampling strategy; authors indicated that altogether 60 soil samples were collected, nevertheless one sample consisted of 15-20 subsamples: 1) How the subsamples were pooled, soil or community DNA were combined? 2) Why the percentages of top, middle and deep soil samples differ? 3) What is the reason of soil sample number discrepancy mentioned in the text and S1 Fig. and Table (60 vs 42 respectively)?

In order to correctly investigate the qualitative diversity and the community structures of the samples, rarefaction and/or rank abundance curves generation is critical. 4) Please demonstrate/ validate that the number of OTUs are saturated in all group samples to get enough sequencing depth. 5) By the way, for some reason almost all of the figures and their labels are not sharp in the pdf version of the manuscript, however downloaded tiff files were legible. 6) Please pay more attention to the proper preparation of the figure and table legends; do not use incorrect, inconsistent or unexplained abbreviations in the figures, or such that are not common knowledge.

A few comments about the applied techniques and their presentation. The performed methods and techniques meet the current technological standards and are suitable for answering the scientific questions formulated in the objectives. Shotgun metagenomics is a powerful approach

to assess microbial composition, diversity, and functional potential, 7) however, it is hard to believe that “direct analysis of individual reads rather than contigs”, would provide more reliable results, especially in the case of short reads (2 × 151 bp). 8) As for the quantity of reads generated from the samples – 20 million reads per sample – it highly questionable that such a uniform high-quality read count could be correct. 9) Although it's a small thing, MagNA Lyser Instrument indicates oscillation frequency (speed) in revolutions per minute (rpm) not in G-force. Nevertheless, these are minor remarks, not affecting unfavorably the value of the work presented. The results and discussion section nicely follows the logic of the materials and methods. It mostly presents the findings clearly. In general, the writing style of the manuscript is proper, and the text contains only a few typographic errors and ambiguous phrasing, however personally I would like to see a much more intense and detailed result comparison with previous knowledge during the discussion.

Reviewer #2: Reviewer Comment for Manuscript PONE-D-23-21640: Exploring the Interplay Between the Core Microbiota, Physicochemical Factors, Agrobiochemical Cycles and Resistome in the Soil of the Historic Tokaj Mád Wine Region

Summary of the studies:

Shotgun metabarcoding was used in the assessment of microbial-terroir in the Tokaj-Mád vineyards of Hungary. The mutual interactions between soil diversity, functional potential and soil chemistry were investigated across the 0 - 90 cm soil layers (0 - 30 cm, 31 - 60 cm and 61 - 90 cm). The results from 60 samples indicated that fungal phyla, Ascomycota, Basidiomycota, and Mucoromycota were operating at 97% operational taxonomic units. By investigating the correlation between the 26 biological activities and the soil physico-chemical parameters, the authors found that soil Na (both plant available and non-available), similar to K2O and P2O5 contents, stimulated most of the agrobiochemical cycles studied, except linoleic acid metabolism, alkaloid and anthocyanin biosynthesis. Soil bacterial diversity was positively correlated with the vast majority of the examined soil physico-chemical characteristics, while fungal diversity was negatively correlated with these characteristics. The pH and CaCO3 content were inversely proportional to the 26 agrobiochemical cycles investigated with the exception that highly calcareous (i.e., alkaline) soils favoured the biosynthesis of alkaloids and isoflavonoids. From the discussion, the natural conditions, altitude, bedrock, micro- and mezzo-climatic factors, and concentration of trace elements in the soil render the Mád wine region suitable for making single-vineyard wines, thus contributing to some of their distinctive characteristics and superb quality. The authors observed that temporal factors exerted a marked influence on soil bacterial community composition. Fungal compositions showed a higher correspondence due to ripening confirms the general observation that community resilience increases with diversity. This also suggests that diverse soil fungal symbiomes are more resistant to external interventions. The authors conclude that, understanding the contribution of soil microbial communities to soil agrobiochemical cycles is critical for predicting terrestrial ecosystem feedbacks.

General Remarks:

This is an interesting and well written manuscript. The title is appropriate for the content of this article. The abstract is concise and accurately summarizes the essential information of the paper. The introduction provides an excellent, generalized background of the topic and this gives the reader an appreciation of the wide range of applications to the technology (nucleic acid extraction and shotgun sequencing) used for this research. The authors collected a unique dataset and the data were analyzed following laid down scientific methodology. The experimental design is standard and appropriate for this study. The authors provided adequate information especially in the materials and methods to make this research experiment capable of been reproduced by a capable research scientist.

The main strength of this paper is that it addresses an interesting topic and it contributes to our understanding on the importance of soil resistome studies and the associated antibiotic resistance genes (ARG) dissemination mechanisms. The manuscript also finds a novel solution based on a carefully selected set of rules and procedures and finally provides a clear solution to the research questions asked. Literature cited in the manuscript, are relevant for this study. The figures and tables are well organized, easy to interpret and follows a systematic and a logical order. It improves our understanding of the importance of the spatial variability of soil resistomes and how ARG depends on microbial community structure as well as its functional potential.

Generally, the study is of great significance and contributes extensively to our understanding of soil microbial communities to soil agrobiochemical cycles is critical for predicting terrestrial ecosystem feedbacks in the study area. I recommend this manuscript (PONE-D-23-21640) for publication after some corrections have been taken into account. Thus, to make the manuscript more reader friendly to an international audience, I suggest that the authors, if possible, should rephrase the title of the manuscript, some sentences in the manuscript and also include the significant level (e.g., 0.05, 0.01, 0.001) and the p - values (e.g., p < 0.03), if possible, into their findings where significant or not significant and/or where positive or negative corelation (r = 0.00; p -value) is reported in the manuscript.

Specific Remarks:

Pg. 3

Line 83 - 84: Metabolites produced by the rhizobiome are important contributors to the quality, fragrance and distinctive personality of the products (5,6).

Please replace “personality” with “quality”.

Pg. 4

Line 86 - 88: The adaptation abilities of the microbiota depend on community plasticity. Network analysis-based approaches can help in better understanding the intricate nature of microbe‒microbe and microbe-community interactions.

Please rephrase the above sentence as: The adaptation abilities of the microbiota depend on community plasticity. Network analysis-based approaches can help understand the intricate nature of microbe‒microbe and microbe-community interactions.

Pg. 10

Line 244 - 245: At the phylum level, 90.0% of the Bacteria and 70.0% of the Fungi OTUs were present in our sample population.

Please edit as: “At the phylum level, 70% and 90% of Fungi OTUs and Bacteria were present in our sample population, respectively”.

Pg. 11

Line 255 - 256: Comprehensive comparisons of the soil samples of the Tokaj-Mád vineyards were made based on the hierarchical clustering of the bacterial and fungal 100% core microbiota (Fig. 5).

Please rephrase the above sentence as: Comprehensive comparisons of the soil samples of the Tokaj-Mád vineyards were made based on the hierarchical clustering of the bacterial and fungal’s 100% core microbiota (Fig. 5).

Pg. 12

Line 279 - 280: Verrucomicrobia (0.066% - 9.25%)

Please rewrite as: “Verrucomicrobia (0.66% - 9.25%)”.

Pg. 14

Line 321: Fig. 6: Soil bacterial community changed significantly due to ripening.

Suggestion: Please do insert the significant level (e.g., p-value = 0.05, or 0.01, or 0.001) and if possible, the significant value (e.g., 0.03).

Pg. 14

Line 326 - 328: Based on an investigation of the soil core, the bacterial alpha diversity was significantly lower in the preharvest samples than in the postveraison samples.

Please insert the significant level and the p-value after the significantly (eg, p < 0.05, p = 0.03). The authors should take not and insert the significant values (p = 0.03) and the significant level (p < 0.05) in their script. This will enrich the findings of this research as well as prevent readers from searching for these in the tables and figures.

Pg. 17

Line 397: Microbial biological activities such as nitrogen

Please rewrite as: “Microbiological activities such as nitrogen…………….”

Pg. 18

Line 433: …..the levels were significantly higher only in relation to the lowest layer (61-90 cm).

Please insert the significant level and/or the p-value.

Pg. 21

Line 513-514: In Mád, a relatively dry August and September is conducive to grapes achieving full biological ripeness before the onset of overripening before the berries begin to shrivel.

Please rephrase as: “In Mád the relatively dry climate in August and September is conducive for grapes achieving full biological ripeness before the onset of overripening and before the berries begin to shrivel”

Pg. 22

Line 525-526: Rhizopodaceae and Mycosphaerellaceae have been found to become more influential throughout the ripening period.

Rewrite as: “Rhizopodaceae and Mycosphaerellaceae have been found to be more influential throughout the ripening period”

Figures and Tables

Dear authors, please increase the resolution of the figures and tables. For example, Figures 2A, and 2B, Figures 7A, and 7B, Figures 9D, and 9F.

Thank you.

Johnny Kofi Awoonor

(Research Scientist: CSIR-Soil Research Institute, Kumasi. Ghana)

Reviewer #3: The problem is very nice and interesting. Soil microbes and vine production must be correlated in some points.

The several sampling point resulted a huge database with an enormous potential to give answers for questions. But the qestions should be more focused and the area of investigations should be reduced.

Current questions and aims in the manuscript:

“thoroughly characterize the soil microbiota of the historic Tokaj-Mád wine region”

It is more or less was characterized, but in this form it does not say too much. Very general. No Comparison. No conclusion.

“We aimed to retrieve the hidden microbial patterns that might be descriptive of the terroir of the Mád vineyards, including the four historic vineyards (Szent Tamás, Király, Betsek, Nyúlászó). NGS was used to describe the molecular barcodes of the historic vineyard microbiotas, which are the hallmarks of the interrelated dynamic physical and chemical properties of the vineyard soil.”

A lat of data and figures, but no answer for this question. Is it differ in any way from other soils, other vineyards soils?

“We aimed to characterize the 100% core soil microbiota and the related agrobiochemical cycles and to identify factors inducing compositional shifts. Our further aim was to investigate the multifarious relationship between the biogeochemical processes and the metagenomic functional potential of the vineyard soil by carrying out correlation analyses between the physicochemical characteristics and the functional potential of the microbiota.”

An existing significant correlation in the sampled data set not necessarily mean cause and effect relationship:

“Soil Na (both plant available and nonavailable), similar to K2O and P2O5, stimulated most of the agrobiogeochemical cycles investigated, except linoleic acid metabolism and alkaloid and anthocyanin biosynthesis.”

There is correlation. What stimulates what, it cannot be obvious according to this data matrix.

“We also wanted to investigate whether the ripening of the plant exerts any effect on the soil core bacterial and fungal microbiotas over the growing season.”

Seasonal changes can be the effect of ripening, as it can be the effect of several other things: temperature, drought, rain, weeds – where there weeds or cover plants?... It is not an established conclusion.

“Finally, we planned to investigate the ARG reservoirs of the Mád vineyards. We believe that our data will emphasize the importance of soil resistome studies and facilitate the discovery of possible ARG dissemination mechanisms.”

This is a very different question. It would worth an absolutely separated manuscript. With a more focused data analysis and discussion. In this extent it is impossible, as we did not get answers to the question.

General comments: methods, data analysis, figure legends often incomplete or not clear. The resolution of some figures are very poor, some of them unreadable.

Recommendation: this data set absolutely valuable. I am sure, a more focused question with the appropriate data analysis could results answers, but a reconsideration, a focused analysis with less data and/or factors and rewrite is necessary. In this form it is incomprehensible for the readers.

Reviewer #4: The manuscript by Remenyik and colleagues is focused on the metataxonomic analysis of several vineyards in the Tokaj region. Although of potential interest the work should be shortened. The Summary suffers from serious defects as well as the discussion in which numerous statements are made that are not supported by any reference or evidence and should be properly reviewed. Some comments to try to improve the manuscript are mentioned next.

- Major comments

� Abstract should be improved. The authors begin by mentioning the fungal microbiota, with a very brief mention of the bacterial microbiota and again insist on other aspects related to the fungal microbiota. My recommendation is to give more weight in the abstract to the description of the bacterial microbiota and that there be a clearer common thread when describing the main results of the work.

� Materials and Methods section. Some critical information is missed.

+ Lines 583-586. The description of the types of soils should be moved to Result section.

+ Lines 587-589. How the soils samples were collected, specially at 31-60 and 61-90 cm depth?. Authors should indicate an exact and complete methodology of soil sampling.

+ Lines 591-595. A more complete description of the methods used to analyze physico-chemical characteristics of soils should be made, including references.

+ Lines 615-617. All the primers used in the Shotgun sequencing process for both bacteria and fungi should be exactly reported.

+ How the sequencing data were processed?. Were they filtered and trimmed?. Any quality analysis was carried out?. This information is important.

� Results section:

+ Lines 130 to 134: Authors indicate the number of reads identified as Bacteria, Archaea, Eukaryotes and Virus. How they identified all these taxons?. We do not have any idea about the primers used for sequencing.

+ A subsection reporting the physicochemical characteristics of each of the 10 soil plots analyzed is missed.

+ Lines 137-138. Authors mention 42 bulk soil samples, but in Materials and Methods section they indicate to have taken “Altogether 60 samples were collected from 0-30 cm, 31-60 cm, 61-90 cm depth zones. One sample consisted of 15-20 subsamples picked between vines in two rows from each zone. Sixty-two percent of the samples were topsoil samples (0-30 cm), 18% of the samples were middle soil (31-60 cm), and 20% of the samples were from the bottom (61-90 cm)”. I am totally confused. Which these 42 samples are?. Not the topsoil samples since the 62% of 60 samples are 37.2 samples (which is impossible). Please, can you clarify this point?.

+ The subsection “The soil food web of the Tokaj-Mád vineyards” leads to confusion (how is it possible that at the species level, as the authors claim, fungi do not exist in the core microbiota?; lines 248-249), does not contribute anything significant to the study and should be removed.

+ Lines 414-415. The authors state “Soil microbiota-associated antibiotic resistance genes are known environmental pollutants posing a potential threat to public health”. What evidence do the authors have to make this statement? Antibiotic resistance genes are a normal part of the genome of countless microorganisms, including the antibiotic-producing microorganisms themselves, so they cannot be considered "environmental pollutants" in any way. This phrase should be removed. Also for what is affirmed in lines 416-417 “Fungal antimicrobial compounds favor the accumulation of bacterial antibiotic-resistant factors”. Any evidence?.

� Discussion section. Throughout the discussion section many claims are made that are not supported by evidence or references (i.e lines 469; lines 462-465, lines 529-530, lines 40-541and many others). A careful review of this section should be carried out in order to indicate references supporting many of the statements made.

- Minor comments

� Line 48: “representing 97 of fungal operational taxonomic units”.

� Line 52. “Soil core bacteria changed markedly due to ripening”: What did that change consist of?. Please, describe.

� Line 139. S1 Table. There is no legend for the table that indicates which soil samples these data correspond to (I assume that they must be the 42 top soil samples but it is not clear for a general reader of the manuscript). Please, correct.

� Lines 161-162. According to what it is observed in Figure 1E Streptomyces taxon is not the dominant one in Betsek sample. Please, correct.

� Lines 198-200. Authors state that “In general, bacterial diversity typically exerted a positive effect, while fungal diversity showed rather a negative effect on the soil chemical parameters measured in this study”. How the authors deduce this statement?. It could be also the opposite: the physico-chemical parameters of soils exert a positive or negative effect on microbial communities. Please, change.

� Lines 248-249. The statement indicating that “at the species level the 100% core microbiota did not contain fungal species” is particularly strange.

� Lines 414-415. The authors state “Soil microbiota-associated antibiotic resistance genes are known environmental pollutants posing a potential threat to public health”.

� Line 469. Any evidence or reference supporting this statement?.

Reviewer #5: The research article by Remenyik et al. entitled “Exploring the Interplay Between the Core Microbiota, Physicochemical Factors, Agrobiochemical Cycles and Resistome in the Soil of the Historic Tokaj Mád Wine Region characterize the soil microbiota of the historic Tokaj-Mád wine región. By NGS the authors retrieve the hidden microbial patterns that might be descriptive of the terroir of the Mád vineyards. The work reports timely and interesting data contributing to deepen current knowledge on grapevine soil microbiome. Overall, the paper was well written, and the data look quite convincing. However, some points need clarification.

The accession number(PRJNA909960) was not found in SRA.

- Section “Six clusters were identified on the basis of the physicochemical properties of the bulk soil samples”

If I understood well, you collected 42 soil samples for physicochemical analysis and 60 for sequencing. How many samples were obtained from each of the 10 sites?

About the six soils clusters (SPP), what was the criteria for defining the 6 clusters. Because looking at the tree in figure s1, more than 6 groups could be defined.

Fig. 1B. Why in the analysis were 8 of the ten vineyards? (Nyúlászó and Szilvás are missing).

L 147-148 “Principal coordinate analysis (PCoA) was performed to investigate clustering according to the soil physicochemical properties (Fig. 1C) and possible correspondence between the microbial taxonomy of the historical vineyards and the other (Fig. 1D).”

Fig. 1C, Why the in the PCoA of soil properties most of the points are in the same coordinate of the axis 1 if the soils clearly present different features. Fig. 1D, It is not clear what is the point of the figure, or what are you showing.

L 155-157 “Remarkable intra-vineyard alterations were observed in the 20 most abundant fungal genera especially in the case of Szent Tamás, Betsek and Sarkad”

Were are shown this results?

L 158-160 “…bacterial genus Bradyrhizobium which are beneficial to plant development and physiology due to the excretion of the polyhydroxylated steroid phytohormone (e.g. brassinosteroids) production and nitrogen fixation…”

Bradyrhizobium only fix nitrogen in symbiosis with leguminous plants.

L 160-162 “The root colonizing rhizobacteria Streptomyces conferring pathogen resistance dominated in Betsek.”

In Nyúlászó and Szilvás the level of Streptomyces are bigger than in Betsek (Fig.1E)

- Section Pronounced community taxonomy shifts according to soil physicochemical properties.

L 184-185 “It was shown that in terms of clades, the largest differences 185 appeared among clusters SPP2, SPP3 and SPP6.”

Where is this information?

Fig.2A, More information is needed in figure caption to interpret the tree. What are the colored circles in the cladogram?

- Section The soil food web of the Tokaj-Mád vineyards

L 234-236 “On average, Bacteria (98.61% ± 5.43%) and Archaea (1.20% ± 1.02%) dominated in the samples. Virus (0.010% ± 0.022%) were less frequent than Fungi (0.15% ± 0.21%), Algae (0.021% ± 0.08%) and Protists (0.007% ± 0.0021%).”

Protists are less abundant than virus.

Fig.4A, why the same % is represented by different size?

- Section The 100% core soil microbiota

Fig. 5

L 292-295 “The scaling is determined through referencing scientific literature. If the relative occurrence of a taxon exceeds 75%, it falls under the strong category. If it surpasses 50%, it is considered moderate. If it exceeds 25%, it is classified as weak, and if it is below 25%, it is labeled as low”.

Percentage of what, the total count of bacterial OTUs in that sample?

- Section The most remarkable taxonomic shifts were observed due to ripening of the grapevine

L 299-302 “In the case of bacteria, spectacular differences in pattern dynamics were observed in community diversity, resulting in two clusters (postveraison – cluster 1, preharvest – cluster 2) with different spatial ordinations.”

Did you did any statistical test support this conclusion?

- Section Network analyses elucidate the complex dynamics of the soil fungal interactome

Fig.7A.

L 365-367 “In all cases, the size of a node represents the degree of the OTU, reflecting the number of connections that it has to other community members in the network.

All the nodes presented the same size. What does it mean the different colors in the figure?

- Section Deciphering of the soil resistome of Tokaj-Mád vineyards

Do the authors know what type of the fertilization the different vineyards use? The animal manure could incrase the ARG.

Fig.9E.

Why the 3 showed PCoA presented the same variation in the axis 1?

- Section Materials and methods

L 638-639. “The results were presented via principal coordinates analysis (PCoA) and nonmetric multidimensional scaling (NMDS) analysis.”

The NMDS is only mentioned in this section.

L 639-640 “To depict the variance between different groups, the Adonis test together with principal coordinates analysis (PCoA) was used.”

What were the results of the test? They are not mentioned throughout the manuscript.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

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Attachments

Attachment

Submitted filename: Review on manuscript.pdf

Attachment

Submitted filename: PLoSONE PONE-D-23-21640 Reviewer Comment - November 2023.pdf

PONE-D-23-21640R1Exploring the Interplay Between the Core Microbiota, Physicochemical Factors, Agrobiochemical Cycles in the Soil of the Historic Tokaj Mád Wine RegionPLOS ONE

Dear Dr. Paholcsek,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The changes made in addressing the reviewers comments are appreciated and have resulted in a manuscript that is close to meeting the threshold for acceptance. Please closely consider the comments from reviewer 5 and address them in a revised manuscript.

Please submit your revised manuscript by Apr 01 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Theodore Raymond Muth

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #5: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #5: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #5: No

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #5: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #5: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors satisfactorily answered the questions and complied with the requested changes, so I consider the revised version of the manuscript suitable for publication.

Best regards

Reviewer #2: The authors have done a great job on the subject matter and I wish them all the best in this field of study.

Reviewer #5: On reading through the "new version” of the manuscript, I was made aware of the many changes that have been made explain the observed points. But not all concerns reported in my previous evaluation report have been addressed carefully.

L 147-148 “Principal coordinate analysis (PCoA) was performed to investigate clustering according to the soil physicochemical properties (Fig. 1C) and possible correspondence between the microbial taxonomy of the historical vineyards and the other (Fig. 1D).” Fig. 1C, Why the in the PCoA of soil properties most of the points are in the same coordinate of the axis 1 if the soils clearly present different features. Fig. 1D, It is not clear what is the point of the figure, or what are you showing.

In the case of Figure D, we would like to emphasize that, concerning the taxonomic composition of soils, the points representing the highlighted vineyards are closely situated, indicating a similarity based on taxonomy. However, compared to other vineyards, there are significant differences. With this, our goal is to demonstrate that the microbial fingerprints of the highlighted vineyards could be distinguished from those of the other areas. We also added a sentence to the discussion please see lines: 522-524. “Concerning the taxonomic composition of vineyard soils, it can be estimated that microbial fingerprints of the historic vineyards are distinguishable from those of other areas in the Tokaj-Mád wine region, a distinction that arises from the exhibited taxonomic similarities.”

The authors didn’t answer my question about the Figure 1C.

L 155-157 “Remarkable intra-vineyard alterations were observed in the 20 most abundant fungal genera especially in the case of Szent Tamás, Betsek and Sarkad” Were are shown this results?

Yes, please see Fig1/F. Szent Tamás, Betsek and Sarkad Fungal compositions are remarkably different from other vineyards.

What you can see in figure 1f is the inter-vineyards variation and not intra-vineyards

L 299-302 “In the case of bacteria, spectacular differences in pattern dynamics were observed in community diversity, resulting in two clusters (postveraison – cluster 1, preharvest – cluster 2) with different spatial ordinations.” Did you did any statistical test support this conclusion?

No, this is based on the clusters in PCoA plot.

In PCoA plot to define valid cluster a statistical test should be done

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Reviewer #1: No

Reviewer #2: No

Reviewer #5: No

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Attachments

Attachment

Submitted filename: PLoSONE Manuscript Review-Jan 2024.pdf

Exploring the Interplay Between the Core Microbiota, Physicochemical Factors, Agrobiochemical Cycles in the Soil of the Historic Tokaj Mád Wine Region

PONE-D-23-21640R2

Dear Dr. Paholcsek,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Theodore Raymond Muth

Academic Editor

PLOS ONE

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