Development of Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Pyrenophora graminea in Barley Seeds

Round 1

Reviewer 1 Report

Dear authors,

The manuscript is dealing with a very important issue, and results also contribute to better knowledge and control of P. graminea. However, there are some points that need to be discussed before acceptance for publication. My comments are as follows. 1. You said: "Chemical-treated seeds are only used in limited areas under the new state regulations requiring the elimination of pesticide input for the sustainability of the ecosystem in this region". Using chemicals in seed treatment although provides effective control of seed-borne pathogens it can not be considered as the measure to eliminate pesticide input in the environment. Please, comment. 2. You said: " In addition, all those methods can only be carried out in laboratories, and it isn’t easy to apply those techniques to onsite surveys". LAMP also requires laboratory equipment! Please, be concise with your statements not only in this paragraph, but also throughout the manuscript. 3. You said: " In addition to P. striiformis isolate wheat was identified by Chinese wheat differential host set for differentiating from the pathogen of wheat stripe rust".However, P. striiformis is a causal agent of wheat stripe rust. I suppose that the authors wanted to say something else?   

Author Response

The manuscript is dealing with a very important issue, and results also contribute to better knowledge and control of P. graminea. However, there are some points that need to be discussed before acceptance for publication. My comments are as follows.

1. You said: "Chemical-treated seeds are only used in limited areas under the new state regulations requiring the elimination of pesticide input for the sustainability of the ecosystem in this region". Using chemicals in seed treatment although provides effective control of seed-borne pathogens it can not be considered as the measure to eliminate pesticide input in the environment. Please, comment.

R1: Thank you for the suggestion; as you said, there are problems with our statement. Qinghai is making every effort to build a green and organic agricultural and livestock product export place. To prevent the import of pesticides and maintain the sustainability of regional ecosystems, some areas prohibit using various chemical pesticides including seed dressing. However, there is no clear description of this situation, and the preamble is revised as follows： Page 2 Line 70-75.

2. You said: "In addition, all those methods can only be carried out in laboratories,and it isn’t easy to apply those techniques to onsite surveys". LAMP also requires laboratory equipment! Please, be concise with your statements not only in this paragraph, but also throughout the manuscript.

 

R2: Thank you for pointing out that we were wrong; in this part, we originally intended to describe the relatively simple experimental conditions required for LAMP detection compared with other detection methods. It caused misunderstanding because we did not describe it properly， and as you said, LAMP assay also needs laboratory equipment. However, the experimental methods mentioned in this paper need to enter the laboratory and use professional instruments and equipment to identify diseases. However, the lamp assay can be used for field identification. It has been reported in the literature that an on-field LAMP assay was developed using Buffer C to perform on-site crude DNA extraction and a vacuum-insulated bottle as a temperature-stable, portable container for isothermal amplification under the field environment. Our polished manuscript has been revised as mentioned below： Page 3 Line 93-99.

A novel LAMP assay using hot water in vacuum insulated bottle for rapid detection of the soybean red crown rot pathogen Calonectria ilicicola https://doi.org/10.1007/s13313-022-00855-y

3. You said: " In addition to P. striiformis isolate wheat was identified by Chinese wheat differential host set for differentiating from the pathogen of wheat stripe rust". However, P. striiformis is a causal agent of wheat stripe rust. I suppose that the authors wanted to say something else?

R3: Thank you for your suggestions. Because P. striiformis sometimes infects barley in Qinghai Province, we selected P. striiformis as one of the tested fungal species. The purpose of this section aims to introduce the related information about P. striiformis wheat and use it for LAMP detection to highlight the specificity of the technique for P. graminea.

Author Response File: Author Response.docx

Reviewer 2 Report

Revision of the Manuscript ID: agronomy-2097459 entitled “Development of loop-mediated isothermal amplification assay for the rapid detection of Pyrenophora graminea in barley seeds”

The manuscript addresses the development of a new fast and reliable detection technique for Pyrenophora graminea, namely, a LAMP assay, which is of great importance for plant diseases and especially their early management, which is crucial to reduce the damage caused by this disease. This study is of great interest since it is urgent to develop fast and reliable methods to detect phytopathogens.

However, the manuscript has small weaknesses, and I have some concerns prior to accepting this manuscript to Plant Disease. The authors must address the comments and or justify some putative limitations. In detail below.

a) Title: the title is adequate to the work

b) Keywords: 4 keywords are used. keywords are fine, they are specific to the work, however, 3 of them are already present in the title, and in order to expand the search results of this work in the future I recommend the authors to add or change some of the keywords.

c) Abstract: abstract is well written and addresses the different parts of the work (introduction, materials and methods, results, and discussion/conclusion), which I find to be a good abstract. It also follows the journal guidelines.

d) Abbreviations: Ok

e) Introduction: the introduction is well written, has sufficient good literature background, and at the same time exposes the problem and the urgent need to develop a new diagnostic tool. Objectives are clear. Only a small detail needs to be corrected, namely in line 46: the first time a species is referenced in the main text should be used its full Latin name, in this case, Pyrenophora graminea. Please correct this.

f) Materials and methods: This section is well written and well explained, in order that other researchers can replicate the methods if the case arrives. The experimental design is solid and robust and is enough to validate the LAMP method developed here. I believe that there is a general formatting problem in subsection 2.6, but I believe that it can be an error from the journal, but nevertheless check this.

g) Results: This subsection overall is well written and the images used are appropriate.

h) Discussion: in regards to the discussion, it is a good discussion, well written, but I found it has a lack of references, namely, authors should incorporate in the section more discussion comparing results with other works related to the development of diagnostic techniques. Please improve this.

i) Conclusion: Concluding remarks are adequate.

j) Figures and tables: overall ok.

k) References: references are adequate to the work, and the authors do not use many self-citations, which I find a positive aspect of the manuscript. Of the total of 33 citations, 14 are from the last ten years, which I do not find it adequate, and recommend the authors add more recent information regarding the subject addressed in this work.

In conclusion, this manuscript can be accepted after minor revisions, the authors should address these smaller recommendations.

Author Response

Revision of the Manuscript ID: agronomy-2097459 entitled “Development of loop-mediated isothermal amplification assay for the rapid detection of Pyrenophora graminea in barley seeds”

The manuscript addresses the development of a new fast and reliable detection technique for Pyrenophora graminea, namely, a LAMP assay, which is of great importance for plant diseases and especially their early management, which is crucial to reduce the damage caused by this disease. This study is of great interest since it is urgent to develop fast and reliable methods to detect phytopathogens.

However, the manuscript has small weaknesses, and I have some concerns prior to accepting this manuscript to Plant Disease. The authors must address the comments and or justify some putative limitations. In detail below.

1. a) Title: the title is adequate to the work
2. b) Keywords: 4 keywords are used. keywords are fine, they are specific to the work, however, 3 of them are already present in the title, and in order to expand the search results of this work in the future I recommend the authors to add or change some of the keywords.

R1: Thank you for your comments regarding the keywords, and we have taken your suggestions and made changes to the keywords section: Page1 Line 29-30.

1. c) Abstract: abstract is well written and addresses the different parts of the work (introduction, materials and methods, results, and discussion/conclusion), which I find to be a good abstract. It also follows the journal guidelines.
2. d) Abbreviations: Ok
3. e) Introduction: the introduction is well written, has sufficient good literature background, and at the same time exposes the problem and the urgent need to develop a new diagnostic tool. Objectives are clear. Only a small detail needs to be corrected, namely in line 46: the first time a species is referenced in the main text should be used its full Latin name, in this case, Pyrenophora graminea. Please correct this.

R2: Thank you for pointing this out, this section has been modified. Page1 Line 46.

2. f) Materials and methods: This section is well written and well explained, in order that other researchers can replicate the methods if the case arrives. The experimental design is solid and robust and is enough to validate the LAMP method developed here. I believe that there is a general formatting problem in subsection 2.6, but I believe that it can be an error from the journal, but nevertheless check this.
3. g) Results: This subsection overall is well written and the images used are appropriate.
4. h) Discussion: in regards to the discussion, it is a good discussion, well written, but I found it has a lack of references, namely, authors should incorporate in the section more discussion comparing results with other works related to the development of diagnostic techniques. Please improve this.

R3: Thank you for your valuable suggestions for the discussion section, which has been carefully revised to address your suggestions.

1. i) Conclusion: Concluding remarks are adequate.
2. j) Figures and tables: overall ok.
3. k) References: references are adequate to the work, and the authors do not use many self-citations, which I find a positive aspect of the manuscript. Of the total of 33 citations, 14 are from the last ten years, which I do not find it adequate, and recommend the authors add more recent information regarding the subject addressed in this work.

R4: Thank you for your guidance on the references, which have been revised to address your questions.

In conclusion, this manuscript can be accepted after minor revisions, the authors should address these smaller recommendations.

R5: Thank you for your review.

Author Response File: Author Response.docx

Reviewer 3 Report

minor：

1.Be aware of the canonical writing format of the unit, chemical formula，% and ℃. Line 175, 198, 199, 206, and etc.

2.

Major:

Line 379: figure 8? or figure 7?

Line 407-412: the figure 11 of result 3.5 where is the result of the SCAR primer detection?

Line 413: Result 3.6, I recommand that you’d better show the result of all 30 seeds together.

Accuracy, speed, convenience and cheapness are important indicators to measure the quality of detection method. Nowadays many LAMP assay can reach the  detection limit of fg level, so I think 10 pg may not the limitation of your assay. For economical concern, the chose of the concentrations of Bst DNA polymerase and dNTPs, I think may be 4 U and 0.8 mM is ok.

 

Author Response

1.Be aware of the canonical writing format of the unit, chemical formula，% and ℃. Line 175, 198, 199, 206, and etc.

R1: Thank you for your careful review. The whole article has been checked and revised for significant issues.

2.Line 379: figure 8? or figure 7?

R2: Thank you for the questions and the order of the results section figure has been modified accordingly: Page14 Line 401 and Page15 Line 419.

Line 407-412: the figure 11 of result 3.5 where is the result of the SCAR primer detection?

R3: Thank you for pointing out the shortcomings of the experimental results in this part. The results in the SCAR part were the inferences proposed by the author according to the literature, and no relevant experiment was conducted. Thank you for your questions. This section has been deleted and modified. Page 16 Line 444-461.

Line 413: Result 3.6, I recommand that you’d better show the result of all 30 seeds together.

R4: Thank you for your suggestion and the results of the 30 seed experiments have been presented and modified: Page17 Line 472-480.

Accuracy, speed, convenience and cheapness are important indicators to measure the quality of detection method. Nowadays many LAMP assay can reach the detection limit of fg level, so I think 10 pg may not the limitation of your assay. For economical concern, the chose of the concentrations of Bst DNA polymerase and dNTPs, I think may be 4 U and 0.8 mM is ok.

R5: Thank you for your suggestion on the sensitivity limit. In view of your suggestion, we have indeed found the shortcomings in the experiment. In view of this problem, I would like to explain to you as follows: The sensitivity of LAMP detection has been greatly increased nowadays. The detection limit of 10pg in our experiment is indeed somewhat unreasonable. The sensitivity of LAMP detection can be effectively improved by increasing the purity of primers, and the accuracy of primer purification by HPLC can be improved by 10-100 times compared with page purification. However, as you said, we also considered the practical application, and the use of HPLC would undoubtedly increase the cost, so I chose the ordinary purification method of page, which resulted in the low detection sensitivity of this experiment. We will carefully consider the problem you have raised.

Thank you very much for giving us the best concentration of dNTPs and Bst DNA polymerase. From the economic point of view, it is really best to choose the lowest. However, based on the previous experimental results, experimental stability, and reference to relevant literature, the intermediate concentration was still selected for the follow-up experiments. In the subsequent process of LAMP assay test-kit development, we will carefully refer to your suggestions on the best concentration in consideration of the best economy.

Author Response File: Author Response.docx