A Fast and Sensitive Enzyme-Mediated Duplex Exponential Amplification Method for Field Detection of Bursaphelenchus xylophilus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes an enzyme-mediated method for the identification of the pinewood nematode (PWN), Bursaphelenchus xylophilus.  Although there are several molecular diagnostic methods suitable for the identification of this important pest, field diagnostic tools are still limited and required. Therefore a protocol for the rapid and reliable identification of this pest that can be conducted in the field is relevant. However, it was not clear to me if the protocol described in the manuscript can be conducted in the field, since there are steps that require lab equipments, such as a metal bath instrument, a microscope to count the nematodes, and the Baermann funnel technique, which takes 24h, etc.

In the Discussion section, the authors say that the protocol “enables rapid detection (<30 min) and does not require specialized equipment”. Please clarify that in the Abstract and in the end of the Introduction, as well as in the M&M. For example, were the nematodes counted Only to prove the sensitivity of the method? Or is it required to count the nematodes? As a manuscript describing a protocol, I recommend that the protocol should be better described.

If the protocol can be used in the field, I recommend the publication of the manuscript, with minor suggestions for the text. Mainly, please clarify in the text and especially in the abstract that the protocol can be conducted in the field and what equipments will be necessary to collect the samples.

Does the protocol require a DNA extraction from a certain number of nematodes? Can the DNA extraction protocol be conducted in the field or it requires lab equipments, including a microscope?

Please make sure that all scientific names of species are in italics.

Please provide a complete and informative title for tables and figures, in a way that the reader does not need to go back to the main text to understand their contents.

Author Response

Comment 1:“Although there are several molecular diagnostic methods suitable for the identification of this important pest, field diagnostic tools are still limited and required. Therefore a protocol for the rapid and reliable identification of this pest that can be conducted in the field is relevant. However, it was not clear to me if the protocol described in the manuscript can be conducted in the field, since there are steps that require lab equipments, such as a metal bath instrument, a microscope to count the nematodes, and the Baermann funnel technique, which takes 24h, etc.”

Response 1:Thank you very much for your valuable comment. We agree that field applicability must be clearly demonstrated. We have now clarified in the Abstract (Page 1, Lines 16–19), Introduction (Page 2, Lines 66–69), and Materials and Methods (Page 4, Lines 94–95 and 139–140) that the protocol is designed for field use. A portable dry block heater and lightweight fluorescence detector are used in place of laboratory equipment. The Baermann funnel and microscope were used only for comparative sensitivity analysis, not as part of the proposed detection procedure. This has been clearly stated in the revised version.

Revised text in Abstract (Page 1, Lines 16–19):

“This protocol enables rapid detection (<30 min) of B. xylophilus in the field using a portable dry block heater and a compact fluorescence detector, without the need for laboratory equipment such as a microscope or Baermann funnel.”

 

Comment 2:“In the Discussion section, the authors say that the protocol ‘enables rapid detection (<30 min) and does not require specialized equipment’. Please clarify that in the Abstract and in the end of the Introduction, as well as in the M&M.”

Response 2:We appreciate this suggestion and have clarified in all the recommended sections. Specifically, we updated the Abstract, end of the Introduction (Page 2, Lines 66–69), and Materials and Methods (Page 4, Lines 94–95) to emphasize that only a dry block heater and a simple fluorescence detector are needed. These are compact and battery-powered, thus suitable for on-site application.

 

Comment 3:“For example, were the nematodes counted only to prove the sensitivity of the method? Or is it required to count the nematodes?”

Response 3:Thank you for pointing this out. We have clarified this in Materials and Methods (Page 4, Lines 108–110) that nematode counting was performed only to evaluate the sensitivity of the method in controlled conditions. For field application, sample preparation does not require nematode counting or microscopy.

 

Comment 4:“As a manuscript describing a protocol, I recommend that the protocol should be better described.”

Response 4:We agree. In response, we have revised Section 2.6 and 2.7 (Pages 6–7) to improve the clarity and structure of the protocol. Each step (sample collection, DNA extraction, reaction setup, and fluorescence detection) has been described more clearly. Figure 1 was also updated to depict all equipment required in the field, including the dry block heater and fluorescence device.

 

Comment 5:“If the protocol can be used in the field, I recommend the publication of the manuscript, with minor suggestions for the text. Mainly, please clarify in the text and especially in the abstract that the protocol can be conducted in the field and what equipment will be necessary to collect the samples.”

Response 5:Thank you. We have now explicitly stated in the Abstract (Page 1, Lines 16–19) and Materials and Methods (Page 4, Lines 94–95, 139–140) that the equipment required includes a battery-powered drill for sawdust collection and a portable dry block heater/fluorescence detector for detection. These devices are compact and suitable for on-site deployment.

 

Comment 6:“Does the protocol require a DNA extraction from a certain number of nematodes? Can the DNA extraction protocol be conducted in the field or it requires lab equipment, including a microscope?”

Response 6:Thank you for this relevant question. As clarified in the revised Section 2.4 (Page 5, Lines 114–117), the DNA extraction method is simplified and suitable for field conditions. It does not require centrifugation or a microscope. The detection sensitivity is high enough that even trace nematode DNA in sawdust samples can be detected.

 

Comment 7:“Please make sure that all scientific names of species are in italics.”

Response 7:Thank you. We have carefully reviewed the manuscript and corrected all instances where scientific names (e.g., Bursaphelenchus xylophilus, Botrytis cinerea) were not italicized.

 

Comment 8:“Please provide a complete and informative title for tables and figures, in a way that the reader does not need to go back to the main text to understand their contents.”

Response 8:We appreciate this helpful suggestion. We have revised the captions of all figures and tables (Figure 1–8; Table 1–2) to be self-contained and informative. For example, Figure 7 now describes the sample types and results of specificity testing more clearly.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Newly submitted paper ‘A fast and sensitive enzyme-mediated duplex exponential amplification method for field detection of Bursaphelenchus xylophilus’ by  Kai Guo and his co-authors is an example of the scientific research directed to the practical needs of the very important part of economic relationships – international trade.  The wood is a valuable object of the international trade and taking into account the packaging of commercial goods into boxes made of wood or wide use of wood pallets in the shipping procedures this material is probably one of the most important in such types of human activity. It is well known for nematologists that pine wood originating from some Asian countries and North America (and now even Western Europe) can contain the life cycle stages of so-called pine wilt nematode (PWN), belonging to the species Bursaphelenchus xylophilus. It is important to note that other species of the same genus are not demonstrating visible impact on the economic activity. Detection of these nematodes in the wood material crossing international borders is a crucial problem of everyday life. Nematodes can be extracted from wood saw dust by some simple methods (as filtration in the funnels – Baermann method), but identification of the stages obtained is not always decisive, when based on the morphological features only. Powerful approaches based on the PCR amplification of the nematode DNA are developed, but the majority of these demand the collection of samples on the spot, transportation of these latter to the equipped lab and quite long in-lab process of DNA extraction, preparation of the ingredients for PCR amplification, visualization of results in gel electrophoresis and final analysis of obtained amplicons.  

          What the authors of this submission propose? Using only dry block heater and simplistic fluorescence detector, it is possible to arrange all the procedure of PNW detection just near the imported wood logs or boxes. PCR ingredients are prepared before and lyophilized, then DNA is extracted from sawdust and the PCR reaction is arranged just on the point of custom control. Species-specific primers and probes with attached fluorescent markers are used and fluorescent detector can reveal the results of successful amplification.

          To check the sensitivity and specificity of the method proposed authors used four populations of B. xylophilus and eight populations of other Bursaphelenchus  species. Only samples with the first species resulted in positive amplification, whereas no amplification was observed in the other populations. The sawdust from four species of pines were used. What is important, is that the lower limit of sensitivity was about 1/500 (!) of that of a single adult nematode per reaction. It is very promising fact.

 

This submission is very valuable addition to the PWN studies and can be published after minor editorial changes.

E.g. I am not sure that the term ‘metal bath’ (e.g. line 94-95  Metal bath’)is a correct one to describe dry block heater. In my understanding, the term ‘metal bath’ is something from my childhood life in the village.

Also on the line 139-140 in the phrase: “incubated for 20 min at the optimal temperature (42 °C) 139 using the portable EmDEA instrument”. Please explain, if you mean just the same dry block heater or some ANOTHER equipment, not depicted on the Fig. 1?

Also, the word processing of the text was not always correct. E.g. hyphenation signs are left in some words in the middle of the line:

37 - Ameri-ca

109 - re-sultant

131 - pri-mers

217  spe-cies

 

The Latin binomial on the line 73 - Botrytis cinerea – please italisize.

In the References. It is not clearly indicated in the “Instructions for authors” of Horticulturae, nevertheless, (probably?!)  the initials of the Editors in such references as,

Hooper, D.; Evans, K. Extraction, identification, and control of plant-parasitic nematodes. In: Plant Parasitic Nematodes In Temperate Agriculture. Evans K, Trudgill D, Webster J (eds) CABI, Wallingford 1993, pp. 1–60.

Leal, I.; Allen, E.; Humble, L.; Green, M.; Rott, M. Application of Conventional PCR and Real-Time PCR Diagnostic Methods for Detection of the PineWood Nematode, Bursaphelenchus xylophilus in Wood Samples from Lodgepole Pine. In: Pine Wilt Disease: A Worldwide Threat to Forest Ecosystems. Mota MM, Vieira P (eds) Springer, Netherlands, 2008, pp 197-210.

are to be presented as Evans, K., Trudgill, D., Webster, J. – i.e. with commas and dots.

Author Response

Comment 1:“What the authors of this submission propose? Using only dry block heater and simplistic fluorescence detector, it is possible to arrange all the procedure of PNW detection just near the imported wood logs or boxes…”

Response 1:We thank the reviewer for the clear summary of our study. In the revised manuscript, we have further clarified that while the dry block heater and fluorescence detector form the core of the field detection kit, additional minimal accessories (e.g., a battery-powered drill for sawdust collection and pre-packed DNA extraction kits) are also part of the workflow. These components are now visually depicted in the revised Figure 1, and a complete list is provided in the Materials and Methods section (Page 4, Lines 93–95).

 

Comment 2:“E.g. I am not sure that the term ‘metal bath’ (e.g. line 94-95 Metal bath) is a correct one to describe dry block heater.”

Response 2:Thank you for pointing out this terminology issue. We have replaced all instances of “metal bath” with the correct term “dry block heater” throughout the manuscript, including Lines 94–95, to avoid misunderstanding.

 

Comment 3:“On lines 139–140, the phrase: ‘incubated for 20 min at the optimal temperature (42 °C) using the portable EmDEA instrument’ — please clarify if this refers to the same dry block heater or another device.”

Response 3:This is a valid point. The term “portable EmDEA instrument” in this context refers to an integrated dry block heater with built-in fluorescence detection function. We have clarified this in the revised manuscript at Line 140 and in Figure 1 caption, where we specify that the portable device serves both heating and fluorescence detection purposes.

 

Comment 4:“Word processing of the text was not always correct. E.g. hyphenation signs are left in some words in the middle of the line: 37 - Ameri-ca; 109 - re-sultant; 131 - pri-mers; 217 - spe-cies.”

Response 4:Thank you for your careful proofreading. We have corrected all erroneous hyphenations in the manuscript, including “Ameri-ca”, “re-sultant”, “pri-mers”, and “spe-cies”. We also performed a full check to ensure that similar issues do not remain elsewhere in the text.

 

Comment 5:“The Latin binomial on line 73 - Botrytis cinerea – please italicize.”

Response 5:Thank you. We have corrected this and ensured that all Latin binomials (including Botrytis cinerea and Bursaphelenchus xylophilus) are properly italicized throughout the manuscript.

 

Comment 6:“In the References... initials of the Editors in such references as... should be presented as ‘Evans, K., Trudgill, D., Webster, J.’ – with commas and periods.”

Response 6:Thank you for this detailed observation. We have revised the relevant references according to standard format, ensuring that editor names appear as “Evans, K., Trudgill, D., Webster, J.” with proper punctuation and spacing, and verified other entries for consistency.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The article is devoted to solving a practically important issue – developing a new method for detecting the most important pine pest – Bursaphelenchus xylophilus.

The introduction to the article provides a good argument for the necessity and purpose of the method, its practical importance for international timber trade and forest protection at the local level. The method is a practically important addition to the existing laboratory methods for analyzing wood for infestation with B. xylophilus.

The second column of Table 1 should contain the authors' names and the year of description (in accordance with the International Code of Zoological Nomenclature). The last column should indicate the author's name in accordance with the International Code of Botanical Nomenclature. The same applies to all plant and animal species mentioned in the text of the article (after their first mention).

Table 2 should be transformed into a more understandable and generalized form. For example, in the form of columns – plant species, in the form of rows – geography of the province and country, and in the cells – characteristics of the process specified in the first 7 columns of table 2. I do not insist on this form of data presentation. It is important that readers do not see the primary data, but analyze the primary results already processed by the authors.

The discussion is very superficial, it does not allow for a deep understanding of the features of the method, assessing the prospects for its application, comparing the features of this method for B. xylophilus with similar methods for other types of nematodes – parasites of plants and animals. A superficial analysis of the problem is unacceptable.

Author Response

Comment 1:“The second column of Table 1 should contain the authors' names and the year of description (in accordance with the International Code of Zoological Nomenclature). The last column should indicate the author's name in accordance with the International Code of Botanical Nomenclature. The same applies to all plant and animal species mentioned in the text of the article (after their first mention).”

Response 1:Thank you for this important suggestion. We have revised Table 1 to include the authors and year of description for all nematode species, as required by the ICZN. For plant species, we now cite the author of the scientific name at first mention in the text and have aligned with the International Code of Nomenclature for algae, fungi, and plants (ICN). These updates can be found on Page 5, Table 1, and throughout the Results section (Pages 7–9).

 

Comment 2:“Table 2 should be transformed into a more understandable and generalized form. For example, in the form of columns – plant species, in the form of rows – geography of the province and country, and in the cells – characteristics of the process specified in the first 7 columns of table 2.”

Response 2:We appreciate the reviewer’s concern about data readability. While we acknowledge the benefit of summarizing data in a more concise matrix form, we retained the current structure of Table 2 because it clearly shows the number of samples processed by traditional and EmDEA methods, highlighting the advantages of our approach. However, we have improved the formatting and caption (Page 8, Table 2) to enhance clarity and emphasized processed results rather than raw data.

 

Comment 3:“The discussion is very superficial, it does not allow for a deep understanding of the features of the method, assessing the prospects for its application, comparing the features of this method for B. xylophilus with similar methods for other types of nematodes – parasites of plants and animals. A superficial analysis of the problem is unacceptable.”

Response 3:Thank you for this insightful comment. We have substantially revised the Discussion section (Pages 10–11) to provide deeper insights into the strengths and limitations of the EmDEA method. We now compare our approach with conventional PCR and LAMP methods, highlighting its suitability for field use. We have also added a paragraph discussing potential adaptation of this technique for detecting other nematode species, including animal-parasitic nematodes, as part of future development and application scenarios.

 

Revised text (Discussion section):

“Compared with conventional PCR and isothermal methods such as LAMP, the EmDEA assay exhibits higher sensitivity, faster amplification speed, and better resistance to field inhibitors. This makes it especially suitable for point-of-entry quarantine detection. In future studies, the core design principles of EmDEA may be extended to detect animal-parasitic nematodes or soilborne pathogens, provided species-specific primers and probes are developed.”

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I hope you will agree that the authors have completely ignored my comments. I cannot approve the publication of this article. I believe that the repeated ignoring of my comments indicates that the authors do not understand modern principles of presenting scientific information in journal articles.

1. Table 2 contains primary data. The processed data in this form cannot be published. 2. The discussion of the article (lines 278-315) is very superficial, the authors ignored my comments.

Author Response

Comment 1: “Table 2 contains primary data. The processed data in this form cannot be published.”

Response 1: We sincerely thank the reviewer for this valuable suggestion. In the revised manuscript, Table 2 has been carefully reviewed and reformatted to present grouped and summarized results by region and host species. Individual-level or batch-level raw data have not been included. Instead, only the number of positive and negative detections for each method is shown, along with brief explanatory notes. We hope this revised format better reflects processed data and meets the publication standards. Thank you again for your helpful comment.

 

Comment 2: “The discussion of the article (lines 278–315) is very superficial, the authors ignored my comments.”

Response 2: We sincerely thank the reviewer for the insightful and valuable suggestion. In response, we have carefully revised and expanded the discussion section to enhance its depth and clarity. Specifically, we incorporated a more detailed comparison between EmDEA and other amplification methods (PCR, qPCR, LAMP, and RPA), and highlighted the practical advantages of EmDEA in terms of rapid operation, minimal equipment requirements, and suitability for on-site application. Additionally, we described a stepwise protocol illustrating how EmDEA can be integrated into field diagnostics and quarantine workflows. To support the revised discussion, we have also added 10 new references. The revised content has been marked in blue in the manuscript. We truly appreciate your comments, which helped us improve the manuscript.

Author Response File: Author Response.pdf