Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Specific and Sensitive Detection of Mycocentrospora acerina (Hart.) Causing Round Leaf Spot Disease in Sanqi (Panax notoginseng)
Round 1
Reviewer 1 Report
The paper is well written, well organized, and absolutely clear about how the authors conducted the research. Given the great urgency of gathering information on plant diseases and the benefit that this kind of novel approach can bring to the state of the art, the study could be of certain interest to the target of Horticulture.
Considering these premises, I recommend the paper for publication in its present form.
Author Response
Response to Reviewer Comments
Point 1: The paper is well written, well organized, and absolutely clear about how the authors conducted the research. Given the great urgency of gathering information on plant diseases and the benefit that this kind of novel approach can bring to the state of the art, the study could be of certain interest to the target of Horticulture. Considering these premises, I recommend the paper for publication in its present form.
Response 1: We appreciate for the reviewers’ warm work earnestly, and look forward to hearing your response to this revised submission.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Lan et al., developed a new LAMP procedure for the leaf round spot disease pathogen of Panax notoginseng. Although there are some areas where I have minor questions and suggestions, I think the manuscript is in a good shape. Please see below for detailed comments.
Line 19: remove “It was found that”
Line 23: “… assay successfully detected…”
Line 24: remove “used as”
Line 48: water stains? Maybe water-soaked lesion?
Line 49: Chestnut????
Line 54-: …”generally cause loss of 20-50%, and 100% loss has been reported with serious outbreaks”??
Line 62: …”determine the pathogenicity”
Line 64: …”such a morphology-based identification”
Line 61: Based on the way the authors wrote the next sentences, Koch’s postulate may or may not be relevant. They are mixing the identification and proof of pathogenicity. I,e., these several sentences need to be organized to be clear.
Line 70-: I often see this argument to justify LAMP, but I am not sure the economical constraints still exist with regular PCR. You can find very economical thermocyclers (less than $1,000) nowadays. Plus, water path or heater than can be set to target temperature ranges can be as expensive as a cheap thermocycler. In addition, you need to consider the cost of 4 to 6 primer sets, and gel imaging, etc, which are additional costs. I would argue that there are other benefits of LAMP.
Line 198: ”… for a range of times to determine…”
Line 280: In the materials and methods section, the authors mentioned about stopping with 80C for 10 min. Did you use a different method to make sure it can be done without the stopping temperature? Please clarify.
Line 296: ” … that the highly sensitive LAMP assay developed in this study was …”
Author Response
Response to Reviewer 2 Comments
Point 1: Line 19: remove “It was found that”.
Response 1: “It was found that” has been removed.
Point 2: Line 23: “… assay successfully detected…”
Response 2: The sentence has been revised.
Point 3: Line 24: remove “used as”.
Response 3: “used as” has been removed.
Point 4: Line 48: water stains? Maybe water-soaked lesion?
Response 4: Yes, “water stains” unsuitable, and has been replaced by “water-soaked lesion”.
Point 5: Line 49: Chestnut????
Response 5: It is chestnut-color.
Point 6: Line 54-: …”generally cause loss of 20-50%, and 100% loss has been reported with serious outbreaks”??
Response 6: We accepted the reviewers’ suggestions.
Point 7: Line 62: …”determine the pathogenicity”.
Response 7: We accepted the reviewers’ suggestions.
Point 8: Line 64: …”such a morphology-based identification”
Response 8: We accepted the reviewers’ suggestions.
Point 9: Line 61: Based on the way the authors wrote the next sentences, Koch’s postulate may or may not be relevant. They are mixing the identification and proof of pathogenicity. I,e., these several sentences need to be organized to be clear.
Response 9: Yes, the Koch’s postulate is not relevant in the text. We accepted the reviewer’s suggestions, and the sentence---“and then determine the pathogenicity according to Koch's postulate (inoculation of the pathogen on the host plant, isolation and identification after the occurrence of the disease, and comparison with the original strain)” has been deleted in the revised manuscript, and the sentences also have been organized.
Point 10: Line 70-: I often see this argument to justify LAMP, but I am not sure the economical constraints still exist with regular PCR. You can find very economical thermocyclers (less than $1,000) nowadays. Plus, water path or heater than can be set to target temperature ranges can be as expensive as a cheap thermocycler. In addition, you need to consider the cost of 4 to 6 primer sets, and gel imaging, etc, which are additional costs. I would argue that there are other benefits of LAMP.
Response 10: We agree with the reviewers. With the development of science and technology, thermocyclers are becoming cheaper (economic) than before. The price of a simple thermocyclers is less than $1,000, but still higher than the price of simple water path or heated (less than $300). Although LAMP requires extra costs such as 4-6 primers and gel imaging, it still has many advantages, such as simple operation, short amplification time (within 1 hour), strong specificity and high sensitivity, which have been discussed in the text (Line 77-87; Line 291-297). Thanks again for the reviewers' suggestions.
Point 11: Line 198: ”… for a range of times to determine…”
Response 11: Has been revised
Point 12: Line 280: In the materials and methods section, the authors mentioned about stopping with 80C for 10 min. Did you use a different method to make sure it can be done without the stopping temperature? Please clarify.
Response 12: Yes, in the preliminary experiment, we used spectrophotometer and agarose gel electrophoresis to analyze the concentration of LAMP amplification products. And the results showed that LAMP could amplify DNA without stopping temperature. In order to evaluate the sensitivity of LAMP, the LAMP reaction mixture was heated at 80 °C for 10 min to inactivate the Bst DNA polymerase. So, LAMP can’t work after treated on stopping temperature for 10 min.
Point 13: Line 296: ” … that the highly sensitive LAMP assay developed in this study was …”
Response 13: Has been revised.
Author Response File: Author Response.pdf
Reviewer 3 Report
This is a fairly routine LAMP assay development study, but it seems to be carried out thoroughly and may be useful in practical testing for the disease of interest. I have only two concerns that require revisions:
1) The conclusion that the “LAMP assay can be used as a tool for early diagnosis” (e.g., lines 319-320), although stated in many similar papers, is overly naïve. While the assay may be sufficiently sensitive, sampling for detection becomes a major bottleneck at low disease levels early in the epidemic. For example, the number of samples required to detect disease reliably at a low incidence and especially at the latent stage may be prohibitive. The authors should add a brief paragraph at the end of the Discussion, outlining how a sampling strategy for use with this assay can be developed.
2) The two asymptomatic samples that tested positive (lines 258-259) require follow-up work to determine that they were in fact latently infected, instead of being a false positives. This kind of determination requires artificially inoculated leaves whereby leaves with and without inoculation are tested during the latent period to determine accuracy of detection. Without this additional evaluation, doubts about these two positive detections will linger.
Minor points:
4: Is “leaf round spot” really the correct common name of the disease? In common English usage this should be “round leaf spot” (similar in usage, for example, to angular leaf spot or zonate leaf spot). This may need to be corrected throughout the manuscript.
4, 11, 32: Mention in the title, abstract, and Introduction that the plant host is also known as Chinese ginseng.
61-64: Koch’s postulates are not used for routine pathogen identification. They are used for first reports.
92-96: This summary of results does not belong in the Introduction.
Additional minor points are indicated in editing mode in the attached PDF file.
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 3 Comments
Major points:
Point 1: The conclusion that the “LAMP assay can be used as a tool for early diagnosis” (e.g., lines 319-320), although stated in many similar papers, is overly naïve. While the assay may be sufficiently sensitive, sampling for detection becomes a major bottleneck at low disease levels early in the epidemic. For example, the number of samples required to detect disease reliably at a low incidence and especially at the latent stage may be prohibitive. The authors should add a brief paragraph at the end of the Discussion, outlining how a sampling strategy for use with this assay can be developed.
Response 1: The authors think that the reviewers’ opinions are scientific and reasonable. Although the established LAMP assay may be sufficiently sensitive in this study, the DNA amount of pathogens in the early latent stage may be lower than the detection limit of the developed LAMP assay, so than pathogens cannot be detected by LAMP. So, the conclusion that “LAMP assay can be used as a tool for early diagnosis” may be not unscientific. The authors have accepted the reviewers’ opinions, and changed the conclusion to "The LAMP assay can be as a tool for diagnosis ….”. The word (“early”) had been deleted. In addition, the author had added a brief paragraph in the discussion section to summarize the sampling strategy of LAMP for the early diagnosis of Panax notoginseng round leaf spot. The brief paragraph for sampling strategy [Although the established LAMP assay is sufficiently sensitive and has potential application in the early diagnosis of P. notoginseng round leaf spot, the DNA amount of the pathogen (M. acerina) in the early latent stage may be lower than the detection limit of the LAMP assay, which often lead to that the pathogen cannot be detected by the LAMP assay. Therefore, once the P. notoginseng leaves appear similar symptoms of round leaf spot, the seemingly healthy leaves (no less than 20, as much as possible) around the symptomatic leaves should be collected and detected by LAMP assay to determine whether the asymptomatic leaves have been latent infected by M. acerina].
Point 2: The two asymptomatic samples that tested positive (lines 258-259) require follow-up work to determine that they were in fact latently infected, instead of being a false positives. This kind of determination requires artificially inoculated leaves whereby leaves with and without inoculation are tested during the latent period to determine accuracy of detection. Without this additional evaluation, doubts about these two positive detections will linger.
Response 2: In order to confirm the accuracy of LAMP assay, it is necessary to use some reliable methods to determine that the samples with positive LAMP analysis are indeed infected by target pathogen (M. acerina), rather than false positive.
In this study, we used traditional tissue-isolation method to isolate target pathogen (M. acerina) in leaf samples, and the fungus was preliminarily identified based on the morphological characteristics.
The results showed that all symptomatic leaves and two of ten asymptomatic leaves exhibited positive reactions. Meanwhile, M. acerina was successfully isolated from all positive leaf samples (data had not showed in this study). All of the leaf samples found positive by the traditional tissue-isolation method were also positive by LAMP.
The authors have added the fungus isolation method and the results in the revised manuscript.
Minor points:
Point 1: 4: Is “leaf round spot” really the correct common name of the disease? In common English usage this should be “round leaf spot” (similar in usage, for example, to angular leaf spot or zonate leaf spot). This may need to be corrected throughout the manuscript.
Response 1: “leaf round spot” has been corrected to “round leaf spot” throughout the revised manuscript.
Point 2: 4, 11, 32: Mention in the title, abstract, and Introduction that the plant host is also known as Chinese ginseng.
Response 2: Thank you for the reviewers’ suggestions. Panax notoginseng is one of the Chinese ginseng. The author thinks that it is better not to add Chinese ginseng before Panax notoginseng.
Point 3: 61-64: Koch’s postulates are not used for routine pathogen identification. They are used for first reports.
Response 3: We agree with the reviewers’ point. The sentence----“Koch's postulates (inoculation of the pathogen on the host plant, isolation and identification after the occurrence of the disease, and comparison with the original strain)” has been removed.
Point 4: 92-96: This summary of results does not belong in the Introduction.
Response 4: We agree with the reviewers’ point. The summary of results in the introduction has have been deleted.
Point 5: Additional minor points are indicated in editing mode in the attached PDF file.
Response 5: We appreciate for the reviewers’ warm work earnestly, and revise the manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
I checked above that presentation of the results "must be improved" because Fig. 2 was missing from the PDF. I assume this was an oversight on the part of the technical editor.
Beyond that, I appreciate the authors’ efforts to revise their manuscript and address my comments. Regarding major point 1 raised in my previous review, my concern was more about the low disease incidence at early stages of the disease (requiring a very large number of samples to find positives) and less so about the detection limit. I edited the section in the Discussion accordingly. My second major point was addressed satisfactorily by the authors.
The attached PDF of the manuscript contains a few minor text edits, in addition to the change mentioned above. The paper is acceptable with minor revisions (plus add Fig. 2 back in), and I do not need to see it again during the next cycle.
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 3 Comments
Point 1: I checked above that presentation of the results "must be improved" because Fig. 2 was missing from the PDF. I assume this was an oversight on the part of the technical editor.
Response 1: Fig.2 has been added in the revised manuscript.
Point 2: Beyond that, I appreciate the authors’ efforts to revise their manuscript and address my comments. Regarding major point 1 raised in my previous review, my concern was more about the low disease incidence at early stages of the disease (requiring a very large number of samples to find positives) and less so about the detection limit. I edited the section in the Discussion accordingly. My second major point was addressed satisfactorily by the authors.
Response 2: We misunderstood the meaning of major point 1 proposed by the reviewer and mistook it as the detection limit. Thank the reviewer for his/her comments on the modification of major point 1. We had accepted the reviewers' suggestions in the revised manuscript.
Point 3: The attached PDF of the manuscript contains a few minor text edits, in addition to the change mentioned above. The paper is acceptable with minor revisions (plus add Fig. 2 back in), and I do not need to see it again during the next cycle.
Response 3: Thanks again for the reviewers’ modifications and suggestions for this manuscript. The author had made minor revisions according to the reviewers’ suggestions.
Author Response File: 
Author Response.docx
