Enzymatic and Molecular Identification of Meloidogyne Species in Tomato Orchards in Paraguay

Round 1

Reviewer 1 Report

The manuscript entitled Biochemical and molecular identification of meloidogyne species in tomato orchards in Paraguay by Resquín-Romero et al. This work has great significance and contribution to root-knot nematode control in Paraguay. However, the authors needed to make major changes in the language, content and experimental design of the article before it could be published in the journal Agronomy.

1.       Lines 30-31: It could be changed to: The SCAR primers (incK14F/incK14R) were used to amplify specific fragments for M. incognita....... Grammar elsewhere in the article should be check all.

2.       Introduction: Add some previously reported literature on tomato root-knot nematode identification methods from elsewhere.

3.       Lines 90-97: pictures of second instar larvae and males can be added.

4.       The text content of Lines 125-128 is duplicated in Lines 94-97.

5.       Lines 108-123: A pair of universal primers (TW81/AB28 or D2A/D3B) could be added to amplify the sample to verify the quality of the DNA.

6.       Figure 1: There is no need to put pictures of the process of making samples in the article.

7.       The results order should correspond to the order in Materials and Methods. Thus the results order should be morhological, biochemical and molecular characterization.

8.       Please check that Figure 6 is not cited in the article

9.       The pixels of Figure 3, Figure 4 and Figure 5 are too low, the authors should change the clear picture.

10.   The content of this study is not sufficient to suggest that the root knot index of different regions can be added.

11.   The language of the article needs to be refined and polished, and the structure of the article needs to be more logical.

Author Response

Please, check the answer in the attached PDF

Author Response File: Author Response.pdf

Reviewer 2 Report

In my opinion, this is a interesting study that offers important information about Meloidogynespecies for the first time in Paraguay. However, the manuscript has quite a lot of deficiencies mainly in the scientific description in different issued along the paper. In 

The title is a bit too wide in relation with the work that was performed; ie., talking about Biochemical identification could involves not only enzyme profiles; but could means composition and structural proteins, secondary metabolite production and solubilization, among others. I think the title should be more specific and focused to the use of Esterase phenotype profile.  

Introduction section, there is a lack of general information about methodological strategies used; ie., Esterase phenotype profile and molecular procedures to identify Meloidogyne species. Some additional information could be useful for many readers.

In Materials and Methods section the information provided is, since my point of view a bit too poor and more scientifically detailed information should be included. It is important to be more precise in the description of the strategy to using SCAR markers in the molecular identification and in the Esterase phenotype profile. For instance, no details about the protein concentration, volume, etc. that were used was mentioned.  

In Results section in Figure 4 The fact that in two wells on the gel showed the presence of two bands about 399 and 670 bp, could be dilucidated by sequencing the PCR products separately.  

It is not clear if the length size of the PCR product is an indicative of the Meloidogynespecies. I think that the aligning step after sequencing, should be necessary to confirm the species based on the NBCI records about the Meloidogynespecies and the high similarity with the previously reported isolates of this nematode is crucial for an accurate identification.   On the other hand, counting with a positive control with the Meloidogyne species could be also necessary.   In Figure 3, indicating clearly what product was used in every well in the gel would be very helpful for readers. 

Figure captures are not in the correct position that match with their figure.

Regarding the use of Isozymes the information is not clear. Why did you use Isozymes? Could you briefly explain why you used isozymes? A general and brief comment about using isozymes in the nematode identification in the Introduction section and in Methodology could be very useful for readers.  

It is not clear if you used a kit for identification of these isozymes.   

Additionally, I found that some cites don´t match with the paragraph. Could you check them carefully, please? 

More comments are marked in the attached file.

In my opinion there is quite a large number of details in this manuscript that make it unsuitable to be published in Agronomy.

My decision is: Reject

Comments for author File: Comments.pdf

Author Response

Please, check the answers in the attached documents.

Thanks for your suggestions

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript ‘BIOCHEMICAL AND MOLECULAR IDENTIFICATION OF Meloidogyne SPECIES IN TOMATO ORCHARDS IN PARAGUAY’, by Resquín-Romero and collaborators, describes a molecular characterization of several Paraguayan Meloidogyne populations hosted on tomato. The authors claimed the study as the first molecular characterization with Paraguayan nematode samples.  The experiments seem to be well conducted, however the manuscript presentation needs to be improved in order to be published in ‘Agronomy’. Therefore, the manuscript cannot be accepted in the present form needing a careful revision.

Issues

1.      The esterase profile for M. incognita and M. javanica should be described in the text. Is the band intensity important? Authors claim in Figure 3 that samples tagged as M.sp2 have an atypical profile. For me, they look like a faint M. incognita profile, as the samples have less nematode tissue, and, consequently, less protein, and resulting in a week staining. Also, why there is not a positive control for M. incognita in the image? Which samples (district) are represented in the gel? Finally, Figure 3 is not cited in the text.

2.      Did the authors tested the SCAR primers with the species that present the atypical pattern? This should confirm the esterase results.

3.      For me, Figures 2 and 6 should be removed. They add no relevant information. Indeed Figure 6 are even not cited in the text. Material should be just described in the text.

4.      Describe in the text the main differences between perineal patterns of M. incognita and M. javanica. Also, authors should add a column in the table 1 identifying the perineal pattern to integrate morphological with molecular results. Without these information Figure 5 is useless.

5.      Table 2 should be named as table 1 since it is cited before the actual table 1, at material and methods section.

Minor issues

-          Line 26, change "prevalence" for "present".

-          Lines 38-39, I suggest updating this information, if possible. 2017 is already five years ago.

-          Line 67, change "hyoigaea" for "hypogaea".

-          Line 95, change "bright orchard" for "bright field".

-          Line 102, change Sacarose for Sucrose. What is “saccharose o glycerol”? Did you mean “sucrose and glycerol”?

-          Line 103, change “syringe Hamilton Syringe – 10 @ l.” for “Hamilton Syringe – 10 ml or µl”

-          Line 107, change “DNA extraction” for “DNA extraction and SCAR genotyping”.

-          Line 112, Put the reference in the correct citing format.

-          Lines 124 to 128 should be removed because it is already present in section 2.2 (lines 92-97).

-          In table 2 the reference for M. incognita SCAR primers is different from the cited in the text (Line 140)

-          Line 208, change “patter” for “pattern”.

-          Reference 12 is not cited in the text.

-          Reference 35 is cited in the text; however, it is not in the reference list.

Author Response

Please, check the answers in the attached documents.

Thanks for your suggestions

Author Response File: Author Response.pdf

Reviewer 4 Report

The authors of this study present their result of their survey of Paraguay tomato orchards where they identified root-knot nematode species using 3 different techniques – SCAR PCR, traditional morphological identification, and esterase isozyme phenotypes. Root-knot nematodes found in agricultural samples in Paraguay are not usually identified to species this manuscript presents a nice summary of the root-knot nematode species found in Paraguay on tomato which will be useful for growers in creating management strategies.

The manuscript in general is very well written and easy to follow and provides a very nice background to the reader of agriculture in Paraguay. However I feel like there is room for improvement in clarifying the methods, results, and discussion to make the manuscript more robust. I have tried to highlight them below.

Abstract – line 27 –I believe there is a comma in 39.13 when should be period

Line 85 – were the same amount of root material taken from each plant? Was the whole root system taken? Could there be a bit more info about the field conditions – was it common to find plants exhibiting symptoms vs healthy plants? It may have been better to randomly sample the field vs selecting only symptomatic plants – so that you could talk about prevalence within in the fields sampled.

It would be nice to have a more clear break down of how the root system was divided for each identification method – how many females went to morphological id, how many to esterase phenotyping, and how much of the root system was used for egg extraction – was it the same for each sample?

Section 2.3 – were no controls of m. incognita or other RKN used?

Section 2.4 – how many eggs per a sample? Were the same number of eggs used for genomic dna extraction from each sample?

Line 140-145 – did pcr and enzymatic results match for each sample tested? For how many females from each sample? Did perineal patterns match with other identification techniques?

Table 1 – This could be expanded to include the individual females from each sample and their results for each methodology, such as the frequency of species identification with each method. Additionally in figure 4 it is not clear what the sample codes 1,2,3,4,5,6,7,8 belong with each sample.

Figure 3 – each lane of the gel should have a sample identification along with the nematode species identified

Figure 5 – what samples do these belong to? It would be nice to include an image that represents each sample district to give an idea of the variation that is seen in morphological id.

Figure 6 – more detail in a) description – what type of symptoms are present

Line 182 – this is a slightly overstatement as only symptomatic samples with galling were taken and from limited number of fields in the regions.

Line 184 - Were rkn found in the same regions as in Lopez-Nicora et al. as they were in this study? It might be nice to highlight more specifics where the two studies agree.

There could be more expansion on the atypical esterase phenotypes that were observed both in the discussion section and the results section – what was atypical about these results? Is there any literature that contains similar results for other nematodes or other Meloidogyne species?

Author Response

Please, check the answers in the attached documents.

Thanks for your suggestions

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The paper has been improved; however some issues have to be attended  before the paper be able to be recommended to be published. 

Page2, lines 53-54. I understand perfectly well the importance of identifying the different Meloidogyne species; However, authors establish that the identification of Meloidogyne species is essential for the proper management of the nematode. On this regard, authors could be more specific. The issue here, is that pesticides or even sustainable strategies of control are the same for the different Meloidogyne species! or is there a specific strategy to control de different Meloidogyne species? 

On the other hand, the objective needs to be improved. 

The objective should not be to use enzymatic and molecular methods to identify Meloidogyne species...

perhaps could say something like:

The objective of this study was to identify Meloidogyne species from small-scale orchards in seven tomato-producing departments in Paraguay using enzymatic and molecular methods.

4. Discussion section 

Again, how the RKN species identification helps to implement adequate management strategies?

If you have this response please, insert a brief explanation; otherwise, this is not a justification to identify the different Meloidogyne species. 

If you have some references about differences in the pathogenicity of the different Meloidogyne species; then, identifying the different species of this genus would be totally justified. Likewise, if the different species of Meloidogyne have different levels of resistance or susceptibility to pesticides or other strategies of control, then identifying the different species is totally justified. If you do not have these answers you could propose that future works focused to identify differences in pathogenicity and pesticide resistance should be done to establish better strategies of control. 

Since my point of view this is am important manuscript that should be published; although, this small details should be fixed. 

My decision is: Minor Revision

Author Response

Response to Reviewer 2 Comments

Dear reviewer, after your suggestions on our manuscript, we believe it has improved substantially, we thank you.

Point 1: Page2, lines 53-54. I understand perfectly well the importance of identifying the different Meloidogyne species; However, authors establish that the identification of Meloidogyne species is essential for the proper management of the nematode. On this regard, authors could be more specific. The issue here, is that pesticides or even sustainable strategies of control are the same for the different Meloidogyne species! or is there a specific strategy to control de different Meloidogyne species?.

Response 1: Done, it has been included in the text.

Point 2: On the other hand, the objective needs to be improved.

The objective should not be to use enzymatic and molecular methods to identify Meloidogyne species...

perhaps could say something like:

The objective of this study was to identify Meloidogyne species from small-scale orchards in seven tomato-producing departments in Paraguay using enzymatic and molecular methods…

Response 2: Done, I has been included in the new version.

Point 3: Discussion section

Again, how the RKN species identification helps to implement adequate management strategies?

If you have this response please, insert a brief explanation; otherwise, this is not a justification to identify the different Meloidogyne species.

If you have some references about differences in the pathogenicity of the different Meloidogyne species; then, identifying the different species of this genus would be totally justified. Likewise, if the different species of Meloidogyne have different levels of resistance or susceptibility to pesticides or other strategies of control, then identifying the different species is totally justified. If you do not have these answers you could propose that future works focused to identify differences in pathogenicity and pesticide resistance should be done to establish better strategies of control.

Response 3: Done, it has been included in the manuscript.

Reviewer 3 Report

Just change the tables' order. Table 2 should be renamed Table 1 if it is mentioned first, and vice versa. The figures should be renumbered appropriately since Figure 2 has been removed.

Author Response

Response to Reviewer 2 Comments

Dear reviewer, we thank you very much for your suggestions.

Point 1: Just change the tables' order. Table 2 should be renamed Table 1 if it is mentioned first, and vice versa.

Response 1: It has been done

Point 2: The figures should be renumbered appropriately since Figure 2 has been removed.

Response 2: Done. We check it, thank you