Diversity and Resistance to Thiophanate-Methyl of Colletotrichum spp. in Strawberry Nursery and the Development of Rapid Detection Using LAMP Method

Round 1

Reviewer 1 Report

The MS entitled ‘Diversity, resistance to benzimidazole fungicides of Colletotrichum spp. in strawberry nursery and the development of rapid detection using LAMP method’ by Wu et al. shows the development of a LAMP assay to detect the E198A mutation the b-tubulin associated to Colletotrichum isolates highly resistant to benzimidazole. Authors also identify Colletotrichum species associated to anthracnose symptoms in strawberry seedlings.

The subject is interesting and the MS is well organized. However there are some aspects that must be revised. 

Authors refer that the Colletotrichum isolates that they found are the cause of anthracnose, but they did not perform any tests to confirm that, they did not perform Koch’s postulates. Authors must perform those tests or alternatively make changes in the way it is written. For example, in lines 30-31, authors say that their study indicated that four species were the pathogen of anthracnose but all they did was to identify Colletotrichum isolates in diseased plants. That is not enough to conclude that they are in fact causing the disease. Authors must change many of these statements throughout the MS. Maybe using  ‘isolates associated to anthracnose symptoms’… In addition, authors also mention other studies showing that anthracnose is caused by some Colletotrichum species, when sometimes all those authors did was to show the presence of Colletotrichum species in diseased plants. 

The MS also needs extensive English editing, there are many errors throughout the MS and many sentences are also difficult to understand. For example, ‘Anthracnose-resistant strawberry cultivars are with desirable horticultural qualities adopted to various region are needed’ in lines 465-466 and ‘We detected MBCs-resistance frequency of Colletotrichum spp. isolates, which obtained from strawberry nurseries, was up to 96.2%’ in lines 470-471, to name a few. Authors should rephrase.

Line 104 – Change stepmother to September

Line 107 – Change NaClO to NaOCl

Line 136 – ‘Field symptoms of anthracnose caused by Colletotrichum spp. in strawberry seedling’ please rephrase (anthracnose symptoms…)

Line 215 – ‘Isolates LA18-3 and LA18-2 were collected in 2018, and their sensitivities to benzimidazole were preconfirmed by MIC testing and sequencing’ Please rephrase, sensitivities were not confirmed by sequencing.

Line 232 – all primers should be listed in the table (and change table title to ‘primers used in this study’

The number of seedlings tested (150) is not mentioned in any part of the M&M section, only in the results section. Please correct.

Why did the authors not sequence b-tubulin gene to validate LAMP assay and confirm the 95 isolates with the E198A mutation?

Why did the authors only sequence the 10 negative isolates? It would be interesting to see if the LAMP positive isolates also present the F200Y mutation.

Moreover, authors do not explore much this in the discussion section. E198A mutation is associated to resistance but it was not detected in 4 highly resistant isolates, why?

Author Response

Dear  reviewer,

We would like to thank you for your constructive comments concerning our manuscript (2003184). 

Please see the attachment.

Sincerely,

Jianyan Wu

Author Response File: Author Response.docx

Reviewer 2 Report

The study seems to be suitably done, and the methodology and the results are clearly presented. I think the manuscript is, in general, clear and tables and graphics are properly presented. Nevertheless, some parts of the manuscript have to be adjusted. Specific suggestions and comments are presented below and included in the text.     

General comments:

- Some parts in the text are not easy to read, there are confusing phrases. The grammar and syntax should be revised. The tenses used for some verbs should be revised, especially when the authors are reporting other results due to the fact that those findings are valid in present time. Please check highlighted parts (yellow color) in the manuscript.

- The are some spelling mistakes to check (please see the text).

- The title of the manucript should be revised. 

-  In the abstract, the importance of thiophanate-methyl should be stated briefly.

-The objectives have to be revised. Please specify what MBC fungicide was tested in the study, and the resistance molecular mechanism of the specific MBC fungicide that was evaluated.

- Authors should revise the use of MBC fungicides, Benzimidazole and thiophanate-methyl along the text. Revise when to cite one or the other. In this study, thiophanate-methyl was evaluated; therefore, for the sake of more clarity, it should be specified that the results are related to this particular MBC (Lines 96 to 99) in the results section.

-  Regarding the disease and pathogen, there are some terms that must be revised. Ulcer-like lesion (lines 102 and 137) is not so used in plants and there are more precise terms to describe anthracnose symptoms. In lines 292 and 298 please check the suitable and precise terms to describe the conidia formation on the culture media. Please revise suitable literature related to the disease and the pathogen.

- The authors may explain why as early as 3 days of incubation was a suitable time for this evaluation (line 191) since three days after in vitro incubation Colletotrichum colonies are just starting to grow.  

- In the discussion section, the idea expressed in lines 440 to 442 should be presented in a more open way concerning the use of   MBC fungicides. The problem is not only for farmers but also for the agrochemical industry.

- The results obtained by MIC evaluation and LAMP technique should be discussed (lines 485 to 489). What do the obtained results mean in terms of the contribution of each methodology used? were they similar?, is one supporting the results of the other?, should MIC be omitted in future studies or should be kept as part of this kind of researches. What was the purpose of using both methods, MIC and LAMP, was the main reason the speed to obtain the results?   

- The phrase mentioned in lines 495 to 497 is out of context. The authors should cite the publication or develop the idea better, or cite other published studies; it could be connected to the text presented in lines 440 to 442.

- Authors could look for more suitable ideas as closing text. For example, the authors may go deeper into the obtained results, the methodologies used or the next steps to take in this kind of studies. This suggestion is to emphasize the contribution of the current research.    

Comments for author File: Comments.pdf

Author Response

Dear reviewer,

We would like to thank you for your constructive comments concerning our manuscript (2003184).

Please see the attachment.

Sincerely,

Jianyan Wu

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors have answered all the questions and have made significant changes in the MS.

I would like to add some minor mistakes:

- line 61: '...species of Colletotrichum that causing anthracnose...', remove 'that' or use 'that cause';

- line 68: change 'seedling' to 'seedlings'

- line 78: remove 'in application'

- line 103: remove '.' and change 'And' to 'and'

Author Response

Dear reviewer,

Thank you very much for your suggestions. We have now made the changes accordingly in revised manuscript.  Our point-by-point responses to each comment are presented below.

Point 1: line 61: '...species of Colletotrichum that causing anthracnose...', remove 'that' or use 'that cause';

Response 1: Thank you. We have removed ‘that’. Line 61.

Point 2:. line 68: change 'seedling' to 'seedlings'

Response 2: This is our mistake. We have changed ‘seedling’ to ‘seedlings’. Line 68.

Point 3: line 78: remove 'in application'

Response 3: We have removed ‘in application ’. Line 78. Thanks.

Point 4: - line 103: remove '.' and change 'And' to 'and'

Response 4: We have made the change. Line 103. Thanks.

Sincerely,

Jianyan Wu