Design of Species-Specific Primers for Early Detection of Kretzschmaria zonata, the Causal Agent of Root and Neck Rot of Teak (Tectona grandis)

Round 1

Reviewer 1 Report

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 1:

I reviewed the manuscript entitled “Design of Species-Specific Primers for Kretzschmaria zonata detection, the causal agent of root and neck rot of teak (Tectona grandis)” by Anuar MagañaÁlvarez and collaborators for the publication on Forests (1789721).

 

Kretzschmaria zonata, the causal agent of root and neck rot of teak is one of the most damaging diseases on Tectona grandis and causing a collapse in sap flow leading to mortality. Early detection in asymptomatic plant and fast diagnosis, play a very important role from control of this disease.

In the manuscript was identified a set of specific primers for K. zonata, and I think, the work, is interesting.

Despite my interest in the approach of this problem, the manuscript cannot be accepted in the present form and should be revised (see below).

The initial aim by the authors, Abstrat and Introduction, is:

“…It allows a rapid detection of this pathogen and provides a powerful tool for to identify

asymptomatic plants and take early control measures (Abstract)”.

“…The aim of this work was to design, at least, one set of PCR specific primers for a quickly, early and accurately diagnosis of this pathogen.”, “This molecular tool will facilitate this disease control through elimination of infected but symptomless plants. (Lines 98 and 99 Introduction)”.

I think is not clear. Is not present analysis on asymptomatic material and in the Results are unclear which part of plants (roots, bark, branches ...) could be used for analysis of symptomless samples.

The article is interesting as it can be effective as a diagnostic method and it could reduce time of analysis, but I don't find a direct link with early detection.

 

Response: We agree with the reviewer that we failed to include detection in asymptomatic plants using the developed primers in the manuscript. This work was done as part of the study, but we the authors focused so much on the study to demonstrate the specificity and sensitivity of the specific primers, that we missed adding this other fundamental and complementary part. For this reason, in the revised version of the manuscript that we are now submitting for your consideration, we include both, in Materials and Methods (section 2.7 Kretzschmaria zonata detection on symptomless plants) and in Results (section 3.4 Kretzschmaria zonata detection on symptomless plants), the work we did to test the effectiveness of the primers we developed, in detecting asymptomatic teak plants of the pathogen K. zonata

 

 

The discussion should be integrated.

Response: the discussion was expanded with new elements to cover all the results obtained in the study.

 

 

 

 

I listed below all my major and minor concerns in detail:

 

Title

I think that the title respects the true objective of the article: identify a specific primer pair for the detection of K. zonata.  If you wish you to include the analysis of asymptomatic plants, the title could be modified to emphasize the possibility of detection of K. zonata on asymptomatic samples.

Response: the title was modified to reflect the intention that with these primers it is intended to make an early detection in asymptomatic plants.

 

 

Abstract

- Page 1, line 24-25: Please, check English form for: “…against DNA from” and for “teak

tissue infected”.

- Page 1, line 28: insert a space “k.zonata - specific primer set”;

- Page 1, line 23: Please, “ITS region sequences”.

Response: Abstract was modified and all indicated suggestions corrected.

 

 

Introduction

- Pag. 2, line 78: Please, “The fungus Kretzschmaria zonata (Lév.) P.M.D. Martin”;

- Pag 2, lines 81 and 86: Please, delete (Lév.);

- Pag 2, line 93: I think “early and accurate diagnose”;

- Pag 2, line 97: Please, check the sentence. I think “… early detection of root and neck rot of teak”.

Response: Introduction was modified in order to correct all indicated suggestions.

 

 

Materials and Methods 2.1 Fungal isolation

- Pag. 3, lines 107-111: not clear. Please, Check the sentence. Lesion or plant of trees?

- Pag. 3, lines 107-111: not clear. Please, Check the sentence. Indicate which part of trees?

- Pag 3, line 146: Please, delate 1L

- Pag 3. Line 151: Please, nineteen isolates.

- Pag 3, line 151: I think is important indicate how many and which samples were taken and how many isolates were obtained. The motivation for the choice was then included in the results.

Response: All suggestions were corrected. About how many samples and isolates were obtained, these data are in material and methods section, lines 160-163 and in Result section line 284-287, and there were Eighty-three fungal isolates out of a total of 128 samples. Regarding the motivation for the choice it was include in this section lines 153-155

 

 

 

Materials and Methods 2.2 Genomic DNA isolation

- Pag4, lines 154-155: Please, describe which and how many sample was extracted.

- Pag4, lines 154-155: not clear. Please, rearrange the sentence.

- Pag 4, line 156: Please, “as negative controls”

- Pag4, line 158. Please, “The DNA concentration of each sample”

 Response: All suggestions were corrected. About which and how many sample was extracted, these data are in material and methods section, lines 160-163.

 

Materials and Methods 2.4 Design of Kretzschmaria zonata -specific PCR

- Pag4, line 183: Please, insert a space “k.zonata - specific primer set”;

- Pag 4, line 184: Please, nineteen isolates.

Response: We have a doubt, in this case the reviewer asks us to insert a space, and later in their review asks us to remove it, so we decided to remove it, according to how we have seen it written in other published articles.

 

Materials and Methods 2.5 Kretzschmaria zonata -specific PCR primers amplification

- Pag5, line 209 and 210: Please, delate space “k.zonata - specific primer set”;

- Pag 5, lines 211-214: Please, insert a period.

- Pag 5, lines 211-214: Please, why you used Fusarium sp., Phellinus sp., Colletotrichum

gloeosporiodies and Lasiodiplodia theobromae and the Oomycete Phytophthora sp. as

negative control? In the Introduction are indicated anothers fungal that causes damage on

teak.

- Pag 5. Lines 211-214. Please, provide details of the negative controls used. For example:

have they been isolated from T. grandis?

Response: All suggestions were corrected. These fungal species were used as negative controls precisely because, being also pathogens of teak, it was important to rule out that they were not amplified by the specific primers designed for K. zonata. The negative controls that we used were not necessarily isolated from teak, we used fungal strains isolated from teak, but also from other hosts, that were molecularly identified and characterized, in other studies in our laboratory, because the aim was to demonstrate the specificity of the detection of the developed primers. Also we include a Kretzschmaria deusta collection strain from ATCC, because this species being so close in terms of morphology and hosts to K. zonata we wanted to be sure that it was not amplified by our primers.

 

Materials and Methods 2.6 Kretzschmaria zonata -specific PCR primers sensitivity

- Pag5, line 209 and 229: Please, delate a space “k.zonata - specific primer set”;

- Pag 6, line 231: Please e selects single unit of measure

Response: All suggestions were corrected.

 

Results: 3.1 Fungal Isolates

- Pag. 6, line 237: Please, see the comment in “Materials and Methods 2.1 Fungal isolation”

- Pag. 6, line 239: Please, nineteen isolates.

Response: All suggestions were corrected. The paragraph was rewritten according to the comment on materials and methods.

 

Results: 3.2 Sequence variation in ITS region and Kretzschmaria zonata -specific PCR

primers design

- Pag. 6, lines 249: Please, harmonization of the wording (see comment in Materials and

methods)

- Pag 6, line 251: Please “5.8S-ITS region”

Response: All suggestions were corrected

 

Results: 3.3 Specificity and sensitivity of the K. zonata primer sets

- Pag. 8, line 306: not clear. Please, check which isolates were used

- Pag 8. Line 307: Please indicate which isolate of K.zonate you used. Is a positive control of your sample or is a certificated colony?

- Pag. 8, lines 322-328: not clear. Please, check which isolates were used

- Pag 9, lines 331-332: Please e selects single unit of measure

- Pag 9, lines 337-341: Please e selects single unit of measure

Response: All suggestions were corrected. Isolates used are listed in lines 365 -366. We use a positive control of K. zonata, previously identified and characterized molecularly in our laboratory. As far as we know, there are no certified strains of this fungus in international microbe culture collection and repository such as ATCC (American Type Culture Collection), for this reason it was not possible to include a certified strain of this specie in the study, however, as can be seen in Figure 4, which shows the phylogeny of the isolates considered in the study, all the strains of K. zonata with which we worked showed 100% of identity with the sequences of K. zonata from NCBI database.

 

Discussion

- I recommend a verification and a deepening of discussion and conclusion.

- Pag. 9, lines 343-346: not clear. In the article is not present analysis on asymptomatic

material. I think that is important analysed real sample and indicated which part of tree is

possible used for early detection on asymptomatic material.

- Pag. 9, line 365: not clear. Please, no real samples were included in the analysis.  (See

materials and methods or results). The analysis was performed in silico.

- Pag 10, line 373:  Please e selects single unit of measure

Response: The discussion was broadened and deepened. Of course real samples were included in the analysis, we conducted both analysis in vivo an in silico, see please Material and Methods section lines 211-218, and Results section, lines 362-396 and Figure 5.  The part of tree that is possible to use for early detection on asymptomatic material is the stem base, in the neck, the area close to the root, as was indicated in material and method section (as in lines 250 and 272).

 

 

Conclusions

- I recommend a verification and a deepening of the discussion and the conclusion.

- Pag 10, lines 382: Please e selects single unit of measure

Response: The conclusions were broadened according to all the study.

Author Response File: Author Response.pdf

Reviewer 2 Report

Manuscript forest-1789721 establishes a set of Kretzschmaria zonata species-specific primers for early and rapid diagnosis of teak root and neck rot. In addition, the protocol can prevent this disease by eliminating infected but asymptomatic plants.The work appears to have been performed in an acceptable manner. However, it will be helpful to the readers if the following points are considered and addressed.

Minor concerns

Abstract: Please provide the full name for the first time mentioned

Introduuction. In addition to the rapid and accurate diagnosis that the authors suggest to detect the pathogen that causes this disease, what other measures can be applied to control asymptomatic plants, and avoid the death of trees?

Section 3.1 Fungal Isolates is poorly developed. Describe the methodology in more detail, since the methodology reported in [23] is in Spanish and in [24] it has little information because it is a scientific note.

Discussion. Explain how foresters can take advantage of the method designed in this study, since it requires specialized laboratories with a high economic cost.

Conclusions. I suggest expanding the conclusions

Author Response

Response to Reviewer 2:

Manuscript forest-1789721 establishes a set of Kretzschmaria zonata species-specific primers for early and rapid diagnosis of teak root and neck rot. In addition, the protocol can prevent this disease by eliminating infected but asymptomatic plants. The work appears to have been performed in an acceptable manner. However, it will be helpful to the readers if the following points are considered and addressed.

Minor concerns:

 

1. Abstract: Please provide the full name for the first time mentioned.

Response: at the beginning of the abstract are the full names of the pathogen and the host, in the body of the abstract they appear abbreviated, but because they were already written at the beginning.

 

2. In addition to the rapid and accurate diagnosis that the authors suggest to detect the pathogen that causes this disease, what other measures can be applied to control asymptomatic plants, and avoid the death of trees?

Response: There is no measure that can be applied in asymptomatic trees, for the same reason that no symptoms are observed. According to our field studies and with artificial inoculations, the infection occurs between 3 and 6 months after the first contact of the plant with the pathogen, but even so, the symptoms are not observed, because they are only expressed after 6 to 8 months from the start of the infection and even two years can pass without the symptoms manifesting, and when this occurs, the only control measure that can be taken is the eradication of the tree. Hence the importance of this detection method.

 

3. Section 3.1 Fungal Isolates is poorly developed. Describe the methodology in more detail, since the methodology reported in [23] is in Spanish and in [24] it has little information because it is a scientific note.

Response: In the materials and methods section, at the end of section 2.1, it was added how the observations of the colonies and fungal structures obtained were made (lines 155 to 157). In section 3.1, lines 288 to 297, there were described, in more details, the morphological characteristics observed in the colonies and structures of the K. zonata isolates, and a new reference was added.

 

4. Explain how foresters can take advantage of the method designed in this study, since it requires specialized laboratories with a high economic cost.

Response: It is not a high cost, because the price of a detection is 70 USD per sample, while the alternative is to eradicate the tree, once it shows symptoms, which is a considerable cost due to the long life cycle of the tree, and the investment that it entails in terms of fertilizers, labor and irrigation. And this is multiplied by the trees that can be infected if the diseased one are not eradicated in time.  

 

5. I suggest expanding the conclusions

Response: Conclusions were improve and expanded.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I reviewed the manuscript “Design of Species-Specific Primers for Kretzschmaria zonata early detection, the causal agent of root and neck rot of teak (Tectona grandis)” by Anuar Magaña Álvarez and collaborators for the publication on Forests (1789721) after revision by the authors.

This manuscript is interesting and complete and could be a topic of interest for the Readers of Forests. The manuscript summarizes the sampling and detection strategies for the rapid  detection of Kretzschmaria zonata.

The manuscript has been correctly revised and integrated, in addition, the authors, have integrated the part relating to asymptomatic samples, it is very important for  early detection of K.zonata.

In this new version, after the in-depth analysis of a few paragraphs but above all an in-depth analysis of the data, I think that the article can be considered in the currently version.