Assessing Fungal Plant Pathogen Presence in Irrigation Water from the Rio Grande River in South Texas, USA

Round 1

Reviewer 1 Report

This article entitled: “Assessing Fungal Plant Pathogen Presence in Irrigation Water from the Rio Grande River in South Texas USA” investigates the presence of fungal plant pathogens in the irrigation water from Rio Grande River, located in South Texas USA. The methods and experimental design seem reasonable and sound. However, there are a few suggestions needed to incorporate to improve the quality of this manuscript. I, therefore, suggest minor revision for this manuscript.

Abstract section:

1.         Please underscore the scientific value-added to your paper in your abstract. Your abstract should clearly state the essence of the problem you are addressing, what you did and what you found and recommend. Moreover, abstract needs careful reading and should depict very important results in a precise way. It should have a stronger concluding sentence. That will help a prospective reader of the abstract to decide if they wish to read the entire article.

2.         Please highlight the key findings of your results in abstract section with % difference.

Introduction section:

1.       Please explain the novelty of study i.e., what is known and what need to explore?

2.         At the end of Introduction section, the main objectives of this study should be more detailed presented

3.         What was the hypothesis of current manuscript?

Discussion section

1.         Overall, the authors generated enough data. However, the presentation of those is not scientifically strong. The entire Discussions section is generally weak and must be strengthened by discussing further.   

Conclusions section:

Authors are advised to add conclusion section. The conclusion is generic and fails to provide any improvement in the existing knowledge base. The conclusions can still be improved by providing an analysis of where the current work on adsorbents is focused, what are the remaining gaps in literature where more research should be conducted. It is recommended to use quantitative reasoning comparing with appropriate benchmarks, especially those stemming from previous work. Limitations in the suggested approach should be discussed in the conclusions section. Please add future work as well.

Author Response

Dear editor and reviewers:

Thank you for your letter and the reviewer’s comments on our manuscript entitled " Assessing Fungal Plant Pathogen Presence in Irrigation Water from the Rio Grande River in South Texas USA" (ID: 2439940). Those comments are very helpful for revising and improving our paper, as well as the important guiding significance to other research. We have studied the comments carefully and made corrections which we hope meet with approval. The main corrections are in the manuscript and the responds to the editors and reviewers’ comments are as follow.

Reviewer-1

Introduction section:

1. Please explain the novelty of study i.e., what is known and what need to explore?

Response: Thank you for your comments. There are several methods available for detecting, screening, monitoring, and identification of microorganisms in water samples. However, these methods have many drawbacks.  High-throughput sequencing can overcome these drawbacks and has the potential to be efficiently used for the detection and monitoring of plant pathogens in water. Our research is first report that application ITS amplicon metagenomic method for detecting fungal pathogen in irrigation water. We have addressed the novelty in updated manuscript.

2. At the end of Introduction section, the main objectives of this study should be more detailed presented

Response: Thank you for your comments. Our objective is application of ITS amplicon metagenomic method for detection of fungal pathogen in the Rio Grande River in South Texas USA. We have addressed it in revised manuscript.

3. What was the hypothesis of current manuscript?

Response: the hypothesis of this manuscript is that a metagenomics approach may be useful for identifying potential pathogens in irrigation water.  We have added it in revised manuscript.

Discussion section

 

1. Overall, the authors generated enough data. However, the presentation of those is not scientifically strong. The entire Discussions section is generally weak and must be strengthened by discussing further.

Response: Thank you for your comments. We have revised and re-organized the discussion section in updated manuscript.

Conclusions section:

Authors are advised to add conclusion section. The conclusion is generic and fails to provide any improvement in the existing knowledge base. The conclusions can still be improved by providing an analysis of where the current work on adsorbents is focused, what are the remaining gaps in literature where more research should be conducted. It is recommended to use quantitative reasoning comparing with appropriate benchmarks, especially those stemming from previous work. Limitations in the suggested approach should be discussed in the conclusions section. Please add future work as well.

Response: Thank you for your comments. We have added conclusion section in revised manuscript.

Reviewer 2 Report

 Comments to authors

1.  While the introduction mentions the goal of identifying if water from the RGR carries plant pathogens and if they are transported into farms, it would be helpful to explicitly state the specific research objectives. For example, you could mention the aim of identifying the types of fungal plant pathogens present, assessing their abundance, or comparing fungal populations between the water reservoir and irrigation sites.

 

2. The introduction briefly mentions water quality issues and the potential presence of contaminants in the RGR, but it would be beneficial to provide more specific information and references regarding the types of contaminants or pathogens that have been previously detected in the RGR or similar river systems. This would help to establish the context and importance of the study.

 

3. It would be beneficial to highlight the significance and novelty of the study. For example, you could mention the potential implications of detecting fungal plant pathogens in irrigation water, such as the risk to crop health and yield, the need for improved water management practices, or the potential for early detection and mitigation of plant diseases.

 

4. In the materials and methods, authors stated  that filtration was performed in triplicate for each sampling site, but it doesn't explicitly state whether the three replicate filtrations were combined or treated separately. It would be helpful to clarify this point to ensure consistency and accuracy in the methodology.

 

5. The authors of the study utilized an older version of the Qiime pipeline (Qiime 1) for their analysis, and taxonomic assignment. However, it is important to note that newer versions of Qiime, such as Qiime 2, have been released with improved features and capabilities. In Qiime 2, the default classification database is the SILVA database, which is widely used for microbial 16S and 18S rRNA gene sequences. However, the authors used UNITE release 5.0 for fungal classification, which has a limited number of reference and representative sequences compared to the more recent UNITE release 9.0. Considering the availability of the updated Qiime 2 pipeline and the larger reference database in UNITE release 9.0, it would be advantageous for the authors to reanalyze their data using these newer resources, as it would likely provide more accurate and comprehensive information on fungal diversity.

 

6.  In the results, it was observed that the top phyla represented only a small percentage of the total abundance, with 4.4% and 1.9% for WR and FA samples, respectively. It is important to note that fungal classification may be less accurate compared to bacterial classification due to the limited availability of reference sequences. However, the newer release of the UNITE database (version 9) contains a significantly larger number of sequences, making it more suitable for improved fungal classification compared to the older release (version 5.0), which had a limited number of sequences. Therefore, it is highly recommended that the authors redo the analysis using the updated database and appropriately discuss and interpret the results based on the improved taxonomic classification.

 

7. It is indeed surprising that the authors acknowledged the limitations of classification in their discussion, specifically mentioning the reliability issues with databases such as GenBank and UniProt, but did not utilize an updated and more comprehensive database for their own analysis. It is crucial to ensure the accuracy and reliability of the sequences in the database when conducting metagenomic analyses, especially when targeting specific plant pathogens. Given the availability of newer and improved databases, such as the updated UNITE release, it is strongly recommended that the authors reconsider their choice of database and reanalyze their data for more accurate taxonomic classification and interpretation of results.

 

 

Moderate editing of English language required

Author Response

Dear editor and reviewers:

Thank you for your letter and the reviewer’s comments on our manuscript entitled " Assessing Fungal Plant Pathogen Presence in Irrigation Water from the Rio Grande River in South Texas USA" (ID: 2439940). Those comments are very helpful for revising and improving our paper, as well as the important guiding significance to other research. We have studied the comments carefully and made corrections which we hope meet with approval. The main corrections are in the manuscript and the responds to the editors and reviewers’ comments are as follow：

1. While the introduction mentions the goal of identifying if water from the RGR carries plant pathogens and if they are transported into farms, it would be helpful to explicitly state the specific research objectives. For example, you could mention the aim of identifying the types of fungal plant pathogens present, assessing their abundance, or comparing fungal populations between the water reservoir and irrigation sites.

Response: Thank you for your comments. We have revised it in updated manuscript. And in Table 4, we have compared abundance of several identified fungal pathogen between WR and FA. Just significant difference in OTU456 (Nigrospora oryzae), and labelled by *. We have provided details to explain this label in updated manuscript.

 

 

2. The introduction briefly mentions water quality issues and the potential presence of contaminants in the RGR, but it would be beneficial to provide more specific information and references regarding the types of contaminants or pathogens that have been previously detected in the RGR or similar river systems. This would help to establish the context and importance of the study.

Response: Thank you for your comments. We have added reference in updated manuscript.

 

 

3. It would be beneficial to highlight the significance and novelty of the study. For example, you could mention the potential implications of detecting fungal plant pathogens in irrigation water, such as the risk to crop health and yield, the need for improved water management practices, or the potential for early detection and mitigation of plant diseases.

Response: Thank you for your suggestions. We have revised it following your suggestions.

 

 

4. In the materials and methods, authors stated that filtration was performed in triplicate for each sampling site, but it doesn't explicitly state whether the three replicate filtrations were combined or treated separately. It would be helpful to clarify this point to ensure consistency and accuracy in the methodology.

Response: Thank you for your suggestions. We have revised it.

 

 

5. The authors of the study utilized an older version of the Qiime pipeline (Qiime 1) for their analysis, and taxonomic assignment. However, it is important to note that newer versions of Qiime, such as Qiime 2, have been released with improved features and capabilities. In Qiime 2, the default classification database is the SILVA database, which is widely used for microbial 16S and 18S rRNA gene sequences. However, the authors used UNITE release 5.0 for fungal classification, which has a limited number of reference and representative sequences compared to the more recent UNITE release 9.0. Considering the availability of the updated Qiime 2 pipeline and the larger reference database in UNITE release 9.0, it would be advantageous for the authors to reanalyze their data using these newer resources, as it would likely provide more accurate and comprehensive information on fungal diversity.

 

 

 

6. In the results, it was observed that the top phyla represented only a small percentage of the total abundance, with 4.4% and 1.9% for WR and FA samples, respectively. It is important to note that fungal classification may be less accurate compared to bacterial classification due to the limited availability of reference sequences. However, the newer release of the UNITE database (version 9) contains a significantly larger number of sequences, making it more suitable for improved fungal classification compared to the older release (version 5.0), which had a limited number of sequences. Therefore, it is highly recommended that the authors redo the analysis using the updated database and appropriately discuss and interpret the results based on the improved taxonomic classification.

 

 

 

7. It is indeed surprising that the authors acknowledged the limitations of classification in their discussion, specifically mentioning the reliability issues with databases such as GenBank and UniProt, but did not utilize an updated and more comprehensive database for their own analysis. It is crucial to ensure the accuracy and reliability of the sequences in the database when conducting metagenomic analyses, especially when targeting specific plant pathogens. Given the availability of newer and improved databases, such as the updated UNITE release, it is strongly recommended that the authors reconsider their choice of database and reanalyze their data for more accurate taxonomic classification and interpretation of results.

Response to 5,6,7: Thank you for your comments. We have re-analyzed data using updated database (QIIME 2.0 and UNITE release version 9). We got similar results. However, using updated database, we identified about 500 OTUs for each sample, compared with about 900 OTUs for each sample using version 5.0. In addition, several OTUs can be identified at species lever using version 5.0, while identified at genus level using 9.0. Therefore, we decide to use data analyzed by version 5.0 in this manuscript.

Reviewer 3 Report

General comments:

In this study, the diversity of microorganisms in irrigation water was evaluated using the amplicon-based metagenomics technique. Fungal composition and the presence of fungal plant pathogens that can cause economic damage in agriculture were assessed. The irrigation water that was coming out of an irrigation valve and the water reservoir were the two places where samples were taken.

According to this research, plant pathogens and other microbes can enter or exit the irrigation water system, affecting the microbial population along the way. Farmers can detect risk factors and create countermeasures by understanding the dynamics of plant pathogen transport in irrigation systems.

Considering the importance of global food production, it is of great importance to prevent diseases and damage to crops. I believe that the article makes an important contribution to this issue.

The article is written clearly and concisely. The results are presented in the correct way.

Specific comments

In my opinion, the work would gain in value if the usual microbiological indicators (E. coli, enterococci, C. perfringens) were examined and the relationship between the fungal pathogens tested and the conventional microbiological parameters was presented.

Moreover, a more comprehensive picture of the effects would be obtained by presenting the physico-chemical properties of the water and the environmental factors (air temperature, precipitation, intensity of solar radiation), taking into account their significant impact on the microbial community. These data would be worth sharing irrespective of the fact that sampling was only conducted out once, which is a limitation of this work.

Author Response

Dear editor and reviewers:

Thank you for your letter and the reviewer’s comments on our manuscript entitled " Assessing Fungal Plant Pathogen Presence in Irrigation Water from the Rio Grande River in South Texas USA" (ID: 2439940). Those comments are very helpful for revising and improving our paper, as well as the important guiding significance to other research. We have studied the comments carefully and made corrections which we hope meet with approval. The main corrections are in the manuscript and the responds to the editors and reviewers’ comments are as follow：

In my opinion, the work would gain in value if the usual microbiological indicators (E. coli, enterococci, C. perfringens) were examined and the relationship between the fungal pathogens tested and the conventional microbiological parameters was presented.

Response: Thank you for your comments. In this research, we just detected fungal pathogen in irrigation water, did not detected bacterial pathogen. In the further research, we will study relationship between the fungal pathogens and the conventional microbiological indicators.

 

Moreover, a more comprehensive picture of the effects would be obtained by presenting the physico-chemical properties of the water and the environmental factors (air temperature, precipitation, intensity of solar radiation), taking into account their significant impact on the microbial community. These data would be worth sharing irrespective of the fact that sampling was only conducted out once, which is a limitation of this work.

Response: Thank you for your comments. We have checked parameters of weather on our collecting sample date. And provided the environmental factors such as air temperature, precipitation and humidity in revised manuscript.

Round 2

Reviewer 2 Report

It is quite surprising that when authors reanalyzed the data using the updated UNITE version, they found similar results to the older version. However, what is even more noteworthy is that the authors discovered that the newer version of UNITE reduced the abundance of operational taxonomic units (OTUs) compared to the older version. This finding should be carefully considered. Hence, I would strongly recommend that the authors incorporate these comparison results into the draft and thoroughly discuss them. This will prove invaluable for other researchers and the UNITE community, as it will shed light on the classification issues that need to be addressed.

Minor editing of English language required

Author Response

It is quite surprising that when authors reanalyzed the data using the updated UNITE version, they found similar results to the older version. However, what is even more noteworthy is that the authors discovered that the newer version of UNITE reduced the abundance of operational taxonomic units (OTUs) compared to the older version. This finding should be carefully considered. Hence, I would strongly recommend that the authors incorporate these comparison results into the draft and thoroughly discuss them. This will prove invaluable for other researchers and the UNITE community, as it will shed light on the classification issues that need to be addressed.

Response: Thank you for your comments. We have added these results into updated manuscript and discussed it.