The Mycobiota of High Altitude Pear Orchards Soil in Colombia

Round 1

Reviewer 1 Report

The authors here described the soil microbiota of a pear orchard in Colombia. Their results are interesting since it is the first report of a soil microbiome of a pear orchard.

However, the authors do not look into the important portion of the soil microbiome: Arbuscular Mycorrhizal Fungi. They do not mention if they detected any presence of AMF. They did not observe any AM fungi in their soil samples? The difference in the P composition in the two sites might be related to this as well. This information is critical for the paper since this is a soil microbiome paper and AMF consists of an important part of the soil microbiome on all agricultural sites. 

Also, the authors do not describe the collection site well enough. Were there any other plant species grown in these orchards? If so, how does this affect the fungal biodiversity? Authors should provide more information about these sites in the materials and methods. Also, was there any application of herbicides/pesticides in these different orchards?  What was the status of pear trees in the research site when the soil was collected? What developmental stage were the trees at? Was the developmental stage of the trees on two sampling sites the same?

I asked these questions when I reviewed this paper for MDPI biology, however; it seems like they are not addressed in the new manuscript. this is why I mentioned them here one more time. 

 

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors,

Attached is the manuscript with comments, which I hope you find helpful.  Overall, the manuscript is improved from last time, but it was hard to tell what text you now intend to include due to all the strikethrough formatting.  

Here are my main concerns (which are still related to my initial thoughts on version 1 and are further expanded on in comments on document):

-Lack of clarity in how the stated methods (especially statistical) are appropriate for the objective of the study (which is still a bit unclear).  What is the question that the statistical methods aim to uncover?  

-A major guild of fungi (the Glomeromycota) is missing from the analysis.  This was a question I had in round one and you appear to have looked into it and found that some off the sequences actually match to Glomeromycotan sequences in another database.  However, why not include this data in the results, instead of only mentioning this in the discussion.  The issue here is that, between this paper and several others cited (I think by this same research group) that uses the same primer set (which largely precludes identification of these ecologically significant fungi), we are adding to the (likely false) notion that arbuscular mycorrhizal fungi are absent from Columbian agricultural land.  There is solid evidence that the methods used (primer set and databases) miss this group of fungi (https://doi.org/10.3389/fenvs.2019.00173).  This should be at least made clear up front and an additional primer set should be used in future studies if the true mycobiota of agricultural soils (especially rootzones!) are to be reported accurately.  It is possible that the very abnormally high rates of fertilizers used (in excess of 250 kg/ha of P each year by my calculation!) could inhibit mycorrhizal fungi in these orchards.

-The manuscript was very difficult to read because it was a PDF with strikethrough formatting and I think some of the strikethroughs were actually new paragraphs (e.g. information about fertilizers in methods, Glomeromycota in discussion) meant to be added and not removed.

 

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2 Comments

 

Point 1: Attached is the manuscript with comments, which I hope you find helpful. 

Response 1: You can find all the answer to your comments in the PDF attached.

Point 2: Overall, the manuscript is improved from last time, but it was hard to tell what text you now intend to include due to all the strikethrough formatting. 

Response 2: Unfortunately, there was an uploading error of the system. After the first revision, we modified the manuscript according to the reviewers comments, but, in the version you saw, the parts we added are the ones you saw crossed out, while the ones we cancelled were solid. We reported this error to the editor as soon as we saw these last revisions, and we are now doing this round of revisions on the correct version of the manuscript. We are very sorry for the inconvenience. We modified ample parts of the text according to your revisions already the first time and you did not had the chance to see it because it was crossed out (as you noticed in your comments).

 

Point 3: Lack of clarity in how the stated methods (especially statistical) are appropriate for the objective of the study (which is still a bit unclear).  What is the question that the statistical methods aim to uncover? 

Response 3: We added some background information about the present situation of the department of Boyacá regarding the socio-economical situation and deciduous fruit growing and we narrowed down the aim of our study: “The aim of the present research was to provide an accurate description of the mycobiota present in the bulk soil of two different high altitude pear orchards in the Colombian Andes, one in a university experimental farm in Soracà and one in a private producer farm in Nuevo Colón. These results will provide a global picture of the soil fungal community associated with this cultivation in Colombia and it will be useful for future reference and soil management for the experimental farm. Moreover, since still little is known on soil fungi in Colombia, our data will contribute to the ongoing process of uncovering Colombian agricultural soil mycobiota” (lines 104-113). Having clarified our aims, we also added the particular aims of our statistical analysis, that is to to detect any differences among the pear orchard in the experimental farm (SR) and the two plots of the private fruit producer (NC-A, NC-B) (lines 224-226).

 

Point 4: A major guild of fungi (the Glomeromycota) is missing from the analysis.  This was a question I had in round one and you appear to have looked into it and found that some off the sequences actually match to Glomeromycotan sequences in another database.  However, why not include this data in the results, instead of only mentioning this in the discussion.  The issue here is that, between this paper and several others cited (I think by this same research group) that uses the same primer set (which largely precludes identification of these ecologically significant fungi), we are adding to the (likely false) notion that arbuscular mycorrhizal fungi are absent from Columbian agricultural land.  There is solid evidence that the methods used (primer set and databases) miss this group of fungi (https://doi.org/10.3389/fenvs.2019.00173).  This should be at least made clear up front and an additional primer set should be used in future studies if the true mycobiota of agricultural soils (especially rootzones!) are to be reported accurately.  It is possible that the very abnormally high rates of fertilizers used (in excess of 250 kg/ha of P each year by my calculation!) could inhibit mycorrhizal fungi in these orchards.

Response 4: We added a sentence in the Results (lines 282-283) to highlight this unexpected results. The possible explanation and hypotheses of this result are in the Discussion (lines 399-418). We expanded the paragraph in the discussion since the last revisions and we added some information about the extensive study of Glomeromycota in Colombian soils and the different hypotheses that could have contributed to the fact we did not find any in our fungal community study. We also added some recommendations for future fungal communities studies for not running the same risk, recommendations we will for sure follow in our future studies.

 

Point 5: The manuscript was very difficult to read because it was a PDF with strikethrough formatting and I think some of the strikethroughs were actually new paragraphs (e.g. information about fertilizers in methods, Glomeromycota in discussion) meant to be added and not removed.

Response 5: You are indeed right, all the strike-through information was the added information and the solid one was the old version. As we explained above, there was an system error in the upload of the manuscript. We signaled it to the editor and are very sorry for the inconvinience. Now you can see the right version of the manuscript with all the intended modifications.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Dear Authors,

I have attached a PDF with highlighted comments, which include both grammatical as well as content suggestions and questions.  Overall, my main questions surround the design, purpose, and question the study seeks to address.  It is not clear from reading the manuscript why you chose to sample two pear orchards if the purpose of the study was to uncover Columbian soil biodiversity.  You might consider re-orienting the focus to be on documenting soil fungal biodiversity in Columbian pear orchards or agricultural soils, etc., because I would imagine you would get a very different picture of soil fungal biodiversity, assemblage, and dominance of different groups were you to sample different undisturbed habitats.  I think the paper would be stronger if you could describe why you would look at a pear orchard when seeking information about soil fungal biodiversity when we know that cultivation (tillage, pesticides, decreased plant diversity) decreases soil fungal diversity in many cases.

It is unclear, based on the stated purpose, why the study compares soil fungi between these two orchards.  Instead is the goal to see how soil properties or orchard management alter soil fungi?  But if that were the goal, you still have the problem of multiple factors.  I thought you might also be interested in the relationship between soil edaphic factors and soil fungal communities, but the data are probably too sparse (only three sites and three samples at each site) to analyze for that.  

Finally, it is surprising given all the other studies that look at soil in orchards and vineyards that there is no report of any Glomeromycota in the fungal community, when these fungi are some of the most ubiquitous in soils, including agricultural soils.  I wonder if some of the bioinformatics somehow filtered them out or if the BLAST somehow mis-assigned those reads to Mortierellomycota (a sister clade, right?).  If this is really the case, it should probably be mentioned in the discussion, because it is pretty surprising.  I also recommend uploading your sequences to a database so that others who aim to add to this work on Columbian soil biodiversity can easily use them.

Please see comments in line on PDF.  Thank you.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1 Comments

Point 1: Overall, my main questions surround the design, purpose, and question the study seeks to address.  It is not clear from reading the manuscript why you chose to sample two pear orchards if the purpose of the study was to uncover Columbian soil biodiversity.  You might consider re-orienting the focus to be on documenting soil fungal biodiversity in Columbian pear orchards or agricultural soils, etc., because I would imagine you would get a very different picture of soil fungal biodiversity, assemblage, and dominance of different groups were you to sample different undisturbed habitats.  I think the paper would be stronger if you could describe why you would look at a pear orchard when seeking information about soil fungal biodiversity when we know that cultivation (tillage, pesticides, decreased plant diversity) decreases soil fungal diversity in many cases. It is unclear, based on the stated purpose, why the study compares soil fungi between these two orchards.  Instead is the goal to see how soil properties or orchard management alter soil fungi?  But if that were the goal, you still have the problem of multiple factors.  I thought you might also be interested in the relationship between soil edaphic factors and soil fungal communities, but the data are probably too sparse (only three sites and three samples at each site) to analyze for that.  

Response 1: We expanded the Introduction with the agricultural, economic and social background of the department of Boyacá (Colombia), where the two sampled orchards lay. We also narrowed down and detailed the aims of the work. Although fruit cultivation in still quite limited in the region and mostly cultivated in very small farms with no infrastructure, it is of the upmost importance, both for the farmers and for the internal market. In this background, the work of the experimental farm of the Project Deciduous of the University “Fundación Universitaria Juan de Castellanos” (JDC) is very important for the economic and social advancement of the area. In this frame, we collaborated with the experimental farm and a pear private producer in Nuevo Colón to provide a global picture of the soil fungal community associated with this cultivation in Colombia so that it could be useful for future reference and soil management for the experimental farm. Moreover, regarding the soil fungal biodiversity of Colombia, since so little is known about it at the moment, our data will go to implement what is already known, especially about agricultural soils. We hope one day to have the occasion to study the mycobiota of Colombian pristine areas, that for sure will host a much wider fungal biodiversity than agricultural soils.

Point 2: Finally, it is surprising given all the other studies that look at soil in orchards and vineyards that there is no report of any Glomeromycota in the fungal community, when these fungi are some of the most ubiquitous in soils, including agricultural soils.  I wonder if some of the bioinformatics somehow filtered them out or if the BLAST somehow mis-assigned those reads to Mortierellomycota (a sister clade, right?).  If this is really the case, it should probably be mentioned in the discussion, because it is pretty surprising.  I also recommend uploading your sequences to a database so that others who aim to add to this work on Columbian soil biodiversity can easily use them.

Response 2: As the reviewer pointed out, the total absence of Glomeromycota in our samples is unexpected. We decided to proceed with a manual control of random obtained sequences, comparing them with the Mycobank database (https://www.mycobank.org/page/Pairwise_alignment) and we found out that some of the unassigned sequences were actually identified as beloning to Glomeromycota. As a consequence, we think that some of the biodiversity especially regarding this phylum could be hidden among the sequences that resulted as unassigned when compared with the database UNITE v.8.2. We added this explanation to our Discussion.

As already stated in the “Data Availability Statement” at the end of the manuscript, our data are openly available available in the NCBI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) under the BioProject Number PRJNA748561.

Point 3: Please see comments in line on PDF. 

Response 3: All the comments in the PDF were taken into consideration, the manuscript was changed accordingly. Our answers to your comments can be found in the PDF attached, while the changes we made are in the new version of manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

I think it is interesting to see the first report from the fungal biodiversity of Colombian soils. Authors here analyzed the soil samples of three different pear orchard sites and check the biodiversity of fungi in these soils. Their results are interesting however, this manuscript can be improved with some minor edits. Further comments are provided below:

The simple summary and abstract sections are very similar to each other.

Introduction:

• Authors should provide more information about why they choose pear as a system to study. Is pear economically important for Colombia?

Materials and Methods:

Description of the research site:

• Authors should provide information about the climate conditions of the region. What is the annual rain situation? Did the authors check the soil conditions in the region? Please provide more information on the study site`s climate, soil conditions, and rain regime.
• What was the status of pear trees in the research site when the soil was collected? What developmental stage were the trees at? Was the developmental stage of the trees on two sampling sites the same?

Statistics: why did researchers use the Wilcoxon test for the statistical analysis of samples? Researchers should use the ANOVA model for statistical analysis of their results, including information from their field setup. This will provide a better analysis of their results and yield more accurate results.

Phylogenetic Analysis: What are the parameters and tree models used in this analysis to generate the fungal tree? Since the tree is essential to this paper, more information about how the tree was generated should be provided.

 

Results and Discussion:

The authors observe a big and significant difference in the P content of soil samples taken from two different sites. However, they do not interpret this result in their discussion. Can this be related to the composition of the fungi they have? It would be good to add an interpretation of this result in the discussion.

The authors do not mention AM fungi in their results at all. They did no observe any AM fungi in their soil sampleS? The difference in the P composition in the two sites might be related to this as well.

Were there any other plant species grown in these orchards? If so, how does this affect the fungal biodiversity? Authors should provide more information about these sites in the materials and methods. Also, was there any application of herbicides/pesticides in these different orchards?

The authors observed significant differences between the two NC sites in the composition of the fungal species. Even though these samples were taken from the same orchard, they only share around 1/3 of fungi species. How do the authors interpret this result?

I think authors should put more emphasis on the fungal species which were not shared between different sites. It would be interesting if there was a chart or diagram somehow showing these species or mentioning it.

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

The results of this study revealed that the detected local  mycobiome in Colombian bulk soils from high altitude orchards  contained many species not earlier detected. Comparison with similar referenced studies (Landinez_Torres et al. ) indicated that the diversity of the mycobiome in each orchard may be a local characteristic.

According to my understanding this study exhibit novelty and potential of practical importance,  specially as the new species may be  producers of useful bioactive compounds. 

However , there is one thing that I did not understand: In the abstract the authors claims that they analysed culturable and nonculturale fungi. This sentence confused me because I did not fing anything about cultured fungi, or how big proportion of the detected mycobiome was metabolically active in the soil?. May be the authors could explain/ discuss this. It is also possible that I am am stupid and simply did not understand the message of this sentence.

Author Response

Please see the attachment

Author Response File: Author Response.docx