Host Suitability of Lettuce and Bean Germplasm for Meloidogyne incognita and M. javanica Isolates from Spain

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript contains both information on resistance of germplasm  to  2 Meloidogyne species and differences in virulence of populations of these 2 species. The focus of the paper is the resistance and differences in virulence is hardly mentioned. Therefore, I think that this has a good practical  purpose, but needs more scientific reflection. However, all the information is there. By rewriting, especially the discussion, but also by giving more information/background on the populations used in the materials and methods, the scientific depth can be improved. The discussion also lacks critical reflection on own results (methodology used, large variation and perhaps unexpected results..) and mainly compares with other publications and talks about genetic background of the germplasm, the latter without a link with own results. I recommend to describe that it is relative resistance that is assessed (not resistance, Pf/Pi) and adapt terminology where needed. Explain why you prefer to test relative resistance.

The experiments were very well organized and performed. The methodology is well described. A repeat of the screening in pots could have answered some questions (especially the large variability in reproduction between populations: virulence differences or coincidence?) or could have confirmed results. There is a high variability in egg count, although this is not uncommon in nematological tests, despite 10 replicates. If the authors have preliminar knowledge about the virulence, origins of the populations or other aspects of the populations, that can explain these observations, they should be mentioned.

Detailed remarks to improve the paper:

I suggest to take bean and snap bean together and refer to bean germplasm to avoid confusion. At certain places in the text snapbean is included when talking about beans (e.g. line 19), sometimes not.

Mention the species in the tables, not only populations. Although it is said they do not make a difference (data not shown) for the results presented here, it is handy for the reader.

Usually resistance is evaluated by considering the value of Pf/Pi (<1 indicates resistance). What is described here is relative resistance (comparing with Regina 71 and Bolinha). Are these general standards in the breeding for lettuce and beans? or only for your lab?

Some populations hardly multiply on Regina or Bolinha- please discuss this:  reasons, can cultivars still be evaluated based on these numbers, … This is mentioned in lines 237-240 but needs more attention. Is this due to differences in virulence or coincidence or experimental error? Should these populations be used for future resistance screening? Which populations do you recommend for (future) resistance screening? How can methodology be improved?

I wonder if there are enough data to calculate a in the graphs. A lower Pi (around 100 J2) would have been useful. How well do the data fit the regression (tables 6 and 8)? The graphs clearly show that Pf/Pi is higher than 1, which strictly means all cultivars multiply RKN and are not resistant, but some allow less multiplication than the standard, so the relative resistance is good.

Lines 221-227 are not very clear to me and I wonder what the added value is of this information. What is incomplete penetrance?

Is the race known of the used populations? Mention this in discussion or table 11 (if known) because the discussion talks about races and the link with results cannot be made. Discussion refers to species in literature, but the populations  and not species are mentioned . It would make it easier for the reader to know which populations belong to which species (although they are in table 11).

The comparisons using percentages is often used but is not very informative I think, and can be left out (e.g. lines 92-95).

Lines 375-377 needs some explanation: where/what is the “parasitic capacity” described? Are these the data not shown?

References:

Some are not in English and I wonder if they can be easily accessed?

Brown is about entomopathogenic nematodes

Pedroche_ check author: De Waele, D.

Not all references are peer reviewed publications.

Need to correct:

Definition of RI under the tables: “number of eggs on the tested cultivar as a percentage of those on the susceptible cultivar”

Cornell is mentioned in title of Table 7 but not in table.

mesh is not a recognized scientific unit, use µm

correct numbering of pages and subtitles (4.4 and 4.5)

Comments on the Quality of English Language

Some expressions are not completely correct English. E.g. eggs prodcued on ... (not in), line 333 about should be proportional to or devided by  (not about). 

 

Author Response

Dear reviewer,

 

Thank you for reviewing our manuscript. The answers to your comments are below and the changes proposed were included in the manuscript

I suggest to take bean and snap bean together and refer to bean germplasm to avoid confusion. At certain places in the text snapbean is included when talking about beans (e.g. line 19), sometimes not.

Answer: Snap has been removed

Mention the species in the tables, not only populations. Although it is said they do not make a difference (data not shown) for the results presented here, it is handy for the reader.

Answer: RKN species have been included in the Tables

Usually resistance is evaluated by considering the value of Pf/Pi (<1 indicates resistance). What is described here is relative resistance (comparing with Regina 71 and Bolinha). Are these general standards in the breeding for lettuce and beans? or only for your lab?

Answer: One indicator of plant resistance is the Pf/Pi ratio but it does not consider the effect of the Pi used in the experiment. As Pf is Pi dependent, the Pf/Pi ratio can vary depending on the Pi used in the experiments. Then, another indicator is used in this study, the reproduction index because the Pf in the putative resistent cultivar is relativized to that on the susceptible one at a given Pi. In fact, “resistance to nematodes is most often defined based on the effects on nematode reproduction, with resistant plants supporting lower levels of reproduction than reproduction on susceptible plant genotypes (Roberts, 2002; Cook and Starr, 2006)” Starr and Mercer 2009 (Root-knot nematodes). The RI is also used for the detection of virulent nematode populations, those with RI >50%.

This criteria is used for several authors from Taylor 1967 to date, for example:

-Hadisoeganda, W.W.; Sasser, J.N. Resistance of tomato, bean, southern pea, and garden pea cultivars to root-knot nematodes based on host suitability. Plant Dis. 1982, 66(2), 145-150

-Karuri, H. W., Olago, D., Neilson, R., Mararo, E., & Villinger, J. (2017). A survey of root knot nematodes and resistance to Meloidogyne incognita in sweet potato varieties from Kenyan fields. Crop protection, 92, 114-121.

Gómez-Rodríguez, O., Corona-Torres, T., & Aguilar-Rincón, V. H. (2017). Differential response of pepper (Capsicum annuum L.) lines to Phytophthora capsici and root-knot nematodes. Crop protection, 92, 148-152.

Molinari, S., & Baser, N. (2010). Induction of resistance to root-knot nematodes by SAR elicitors in tomato. Crop Protection, 29(11), 1354-1362.

Seid, A., Fininsa, C., Mekete, T. M., Decraemer, W., & Wesemael, W. M. L. (2017). Resistance screening of breeding lines and commercial tomato cultivars for Meloidogyne incognita and M. javanica populations (Nematoda) from Ethiopia. Euphytica, 213, 1-15.

Celik, I., Sogut, M. A., Ozkaynak, E., Doganlar, S., & Frary, A. (2016). Physical mapping of NBS-coding resistance genes to the Me-gene cluster on chromosome P9 reveals markers tightly linked to the N gene for root-knot nematode resistance in pepper. Molecular Breeding, 36, 1-7.

 

Some populations hardly multiply on Regina or Bolinha- please discuss this:  reasons, can cultivars still be evaluated based on these numbers, … This is mentioned in lines 237-240 but needs more attention. Is this due to differences in virulence or coincidence or experimental error? Should these populations be used for future resistance screening? Which populations do you recommend for (future) resistance screening? How can methodology be improved?

                Answer: Regina and Bolinha are the susceptible cultivars for comparison. There were differences in nematode reproduction on the susceptible cultivars, indicating that both the genetic background of the RKN isolate and that of the plant play an important role in the plant-nematode interaction. For example, the RKN isolates Curas and P Almeria showed little reproduction on lettuce cv. Regina but much more on bean cv. Bolinha. Then, the characterization of plant germplasm resistance level to a wide number of RKN populations or isolates provides a broad view of the possible response in field conditions since the result of the plant-nematode interaction is dependent on the genetic background of both plant and RKN isolate. Consequently, all the RKN isolates are useful to foresee the putative response of the plant germplasm in Spain. The methodology could be improved by increasing the number of repetitions

I wonder if there are enough data to calculate a in the graphs. A lower Pi (around 100 J2) would have been useful. How well do the data fit the regression (tables 6 and 8)? The graphs clearly show that Pf/Pi is higher than 1, which strictly means all cultivars multiply RKN and are not resistant, but some allow less multiplication than the standard, so the relative resistance is good.

Answer: Plants were cultivated in ten plots with different Pi. The lowest Pi in plots cultivated with lettuce was 0.436 J2/cc of soil, and that in those cultivated with bean was 0.362 J2/cc of soil. The wider range of data the better for estimating a, E and M, but in field conditions is not easy to have a wider range of densities. Then, the calculation of the a and E parameters provides additional information on the performance of the plant germplasm. No regression analysis was conducted.

Lines 221-227 are not very clear to me and I wonder what the added value is of this information. What is incomplete penetrance?

Answer: Is a genetic concept to indicate that a given character is not expressed in all members of the population.

Is the race known of the used populations? Mention this in discussion or table 11 (if known) because the discussion talks about races and the link with results cannot be made. Discussion refers to species in literature, but the populations  and not species are mentioned . It would make it easier for the reader to know which populations belong to which species (although they are in table 11).

Answer: The authors do not know the race of the RKN isolates, it is not a common practice in our research group. In fact, the races of RKN have increased from those postulated by Taylor and Sasser, and some works have demonstrated that by increasing the number of crops used to differentiate races, additional races can be described. So, in our opinion and considering the crops used to determine the RKN races, it is not practical information for growers in our agronomical conditions. Moreover, the differential test is time-consuming.

The comparisons using percentages is often used but is not very informative I think, and can be left out (e.g. lines 92-95).

Answer: In our opinion, the comparisons using percentages are interesting to know the relative value of a given parameter or the frequency of a given resistance level and to provide additional information to that contained in the tables and figures.

Lines 375-377 needs some explanation: where/what is the “parasitic capacity” described? Are these the data not shown?

Answer: That sentence was removed

Some are not in English and I wonder if they can be easily accessed?

Answer: Yes, all the non-English can be easily accessed

Brown is about entomopathogenic nematodes

                Answer: The reference was wrong. The corrected one is (which was changed): Brown, C.R.; Mojtahedi. H.; Santo, G.S,; Willamson, V.M.. Effect of the Migene in tomato on reproductive factors of Meloidogyne chitwoodi and M. hapla.J. Nemat. 1997, 29, 416–419

Pedroche_ check author: De Waele, D.

                Answer: The author’s name has been corrected

Not all references are peer reviewed publications.

                Answer: Although not all the references are peer-reviewed, they are relevant to the manuscript

Definition of RI under the tables: “number of eggs on the tested cultivar as a percentage of those on the susceptible cultivar”

                Answer: The definition has been changed

Cornell is mentioned in title of Table 7 but not in table.

                Answer: Cornell was deleted

mesh is not a recognized scientific unit, use µm

Answer: mesh has been changed to µm

correct numbering of pages and subtitles (4.4 and 4.5)

Answer: The numbering has been corrected

Some expressions are not completely correct English. E.g. eggs prodcued on ... (not in), line 333 about should be proportional to or devided by  (not about). 

Answer: The manuscript has been carefully revised by a native English speaker who is a language professional.

Your sincerely,

Ariadna Giné

Corresponding author

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript title “Host suitability of lettuce, bean and snap bean germplasm to Meloidogyne incognita and M. javanica isolates from Spain” is shown well and has scientific worth. In this study, the lettuce, bean and snap bean susceptibility was checked against different M. incognita and M. javanica isolates from Spain.

This MS needs minor improvements. My comments for authors are as follows:

1-      In abstract line 12: Please start the sentences like this “In this study, the host suitability of” or “this study highlights the host suitability of”

2-      Line 17-21: the sentence “Lettuce cv. Grand Rapids and Salinas 88 were resistant to the most nematode isolates in the pot experiment but performed as slightly and moderately resistant, respectively, under plastic greenhouse at increasing Pi. Regarding beans, Aporé was resistant to the majority of the Meloidogyne isolates whereas Macarrão Atibaia and Ouro Negro were slightly resistant and Cornell 49242 was susceptible in pot experiment” is very long and confusing. Please split this sentences and re-write it.

3-      Add 1-2 concluding lines in the end of the abstract.

4-      Conclusion lines: 379-381 should be re-written for easy understanding and sentence sequence flow.

 

5-      Table 3, and Table 7, in these tables don’t use “,” to separate the decimals i.e. 8,00 ± 0,38. Write like this 8.00 ± 0.38. The same issue is in line 120 P < 0,05. Write link this P < 0.05

Comments on the Quality of English Language

Some English sentences should be re-written for easy understanding and sentence sequence/flow.

Author Response

Dear reviewer two,

 

Thank you for reviewing our manuscript. The answers to your comments are below and the changes proposed have been included in the manuscript

 

In abstract line 12: Please start the sentences like this “In this study, the host suitability of” or “this study highlights the host suitability of”

Answer: This study highlights the host suitability of was added

Line 17-21: the sentence “Lettuce cv. Grand Rapids and Salinas 88 were resistant to the most nematode isolates in the pot experiment but performed as slightly and moderately resistant, respectively, under plastic greenhouse at increasing Pi. Regarding beans, Aporé was resistant to the majority of the Meloidogyne isolates whereas Macarrão Atibaia and Ouro Negro were slightly resistant and Cornell 49242 was susceptible in pot experiment” is very long and confusing. Please split this sentences and re-write it.

Answer: These are already two sentences

Add 1-2 concluding lines in the end of the abstract.

Answer: These results play an important role in increasing the effective and ecofriendly Meloidogyne management strategies. was added

Conclusion lines: 379-381 should be re-written for easy understanding and sentence sequence flow.

Answer: The sentence was changed to: This study confirmed the resistance of lettuce cultivars Grand Rapids and Salinas 88 to M. incognita and M. javanica isolates from Spain in all the experiments.

Table 3, and Table 7, in these tables don’t use “,” to separate the decimals i.e. 8,00 ± 0,38. Write like this 8.00 ± 0.38. The same issue is in line 120 P < 0,05. Write link this P < 0.05

Answer: , were changed to .

 

The English quality has been improved by a native English speaker who is a language professional

Your sincerely

Ariadna Giné

Corresponding author

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled "Host Suitability of Lettuce, Bean and Snap Bean Germplasm to Meloidogyne incognita and M. javanica isolates from Spain" assesses the resistance of three lettuce cultivars and four bean cultivars to root-knot nematodes (RKNs) in both pot assays and greenhouse experiments. The manuscript is appropriately designed and presents reliable results. However, I have concerns about the novelty of the research, as most of the plant materials selected in this study have previously been reported to exhibit resistance against RKNs. As mentioned in the discussion section, it would be worthwhile for the authors to investigate the resistance of these plant germplasms against different RKN species in crop rotation sequences.

Specific comments

Page2 line 49, please add new relevant literature to support the fact that temperature affects the function of R gene-mediated nematode resistance in plants

The Discussion section need extra revision. Please provide more proper discussion about the optimal of use of these resistant germplasm under field conditions.

Comments on the Quality of English Language

The manuscript is well written. 

Author Response

Dear Reviewer,

 

We would like to thank you for all your comments and suggestions. Those will improve the manuscript for publication in Plants Journal.

Page2 line 49, please add new relevant literature to support the fact that temperature affects the function of R gene-mediated nematode resistance in plants

Answer: Reference have been added

de Carvalho, L.M.; Benda, N.D.; Vaughan, M. M., Cabrera, A. R., Hung, K., Cox, T., ... & Teal, P. E. Mi-1-mediated nematode resistance in tomatoes is broken by short-term heat stress but recovers over time. J. Nematol. 2015, 47(2), 133.

Verdejo-Lucas, S.; Blanco, M.; Cortada, L.; Sorribas, F.J.  Resistance of tomato rootstocks to Meloidogyne arenaria and Meloidogyne javanica under intermittent elevated soil temperatures above 28 C. Crop Prot. 2013, 46, 57-62.

This sentenece “ some resistance genes fail at constant high soil temperatures [10]” havent changed by “ some resistance genes, like Mi 1.2 gene in tomato, fail at constant soil temperatures above 28ºC [10] but not when high soil temperature peaks during the day (Verdejo-Lucas et al., 2013) because the expression is recovered with time as soil temperature decrease (de Carvalho et al., 2015).

 

The Discussion section need extra revision. Please provide more proper discussion about the optimal of use of these resistant germplasm under field conditions.

                Answer: Discussion have been improved

 

Also, the manuscript has been carefully revised by a native English speaker who is a language professional.

 

Your sincerely,

Ariadna Giné

Corresponding author

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Line 56: “and when virulent RKN populations or species occur [1113-153]” -- make this a separate sentence. Now it is not fluent to read after the text about the temperature.

Line 61: I suggest to leave out “increasing”, this aspect is for the next sentence. Host status is determine by Pf and Pi, increasing or not.

Line 62: “The Pi, in the absence of limiting  factors, increases over time, and the multiplication rate (Pf/Pi) is at a maximum at low Pi values". I suggest to leave out “proportionally” or explain proportionally to ... (what?).

Lines 252-255 is not clear. Some words can/should be left out or added:

“In our study, greater differences in reproduction between RKN isolates on the susceptible cultivar Regina 71 were observed than on the resistant cultivars Grand Rapids or Salinas 88, indicating that both the genetic background of the RKN isolate which can play an important role on IN the plant-nematode interaction [37-38].”

OR did you mean:

“In our study, greater differences in reproduction between RKN isolates on the susceptible cultivar Regina 71 were observed than on the resistant cultivars Grand Rapids or Salinas 88, indicating that both the genetic background of the RKN isolate and that of the cultivar can play an important role on IN the plant-nematode interaction [37-38]

Line 255 “In fact, the RKN isolate Curas was virulent to both lettuce cultivars, probably because of its poor reproduction on the susceptible cultivar Regina 71.” This is not the right conclusion or way to say, Curas did not multiply more on  GR and Salinas 88 than on other isolates, but it was due to its poor reproduction on the standard Regina 71 and the use of the concept of RELATIVE resistance that the two cultivars GR and Salinas were classified as susceptible. Hence this is an artifact of the resistance evaluation method, not a virulent population.

I propose to write: “For example, the RKN isolate Curas reproduced poorly on all lettuce cultivars, including the susceptible cultivar Regina 71.”

I leave it up to the authors to write that low reproduction on the standard susceptible cultivar can lead to wrong classification of the tested cultivar. Some reflection on methodology would have been  good; a discussion calls for (critical) evaluation of methods and results obtained. Keep in mind for the next manuscript!

Comments on the Quality of English Language

Some sentences can still be improved for the English language. E.g. “Moreover, it was assessed the response of these cultivar..” should be “Moreover, the response of these cultivar was assessed…”

There are some typos, and placement of words in sentences can be better. 

Author Response

Dear Reviewer 1,

Thanks again for your suggestions which will increase the quality of our manuscript.

Line 56: “and when virulent RKN populations or species occur [1113-153]” -- make this a separate sentence. Now it is not fluent to read after the text about the temperature.

The sentence has separated. The second sentence has been replaced by: Also, the repeated cultivation of resistant tomato cultivars carrying Mi 1.2 gene, may allow for the development of virulent RKN populations or species

Line 61: I suggest to leave out “increasing”, this aspect is for the next sentence. Host status is determine by Pf and Pi, increasing or not.

Increasing has been removed

Line 62: “The Pi, in the absence of limiting  factors, increases over time, and the multiplication rate (Pf/Pi) is at a maximum at low Pi values". I suggest to leave out “proportionally” or explain proportionally to ... (what?).

Proportionally has been removed

Lines 252-255 is not clear. Some words can/should be left out or added:

 “In our study, greater differences in reproduction between RKN isolates on the susceptible cultivar Regina 71 were observed than on the resistant cultivars Grand Rapids or Salinas 88, indicating that both the genetic background of the RKN isolate and that of the cultivar can play an important role on the plant-nematode interaction [37-38]

The sentence has been changed as the reviewer proposed

Line 255 “In fact, the RKN isolate Curas was virulent to both lettuce cultivars, probably because of its poor reproduction on the susceptible cultivar Regina 71.” This is not the right conclusion or way to say, Curas did not multiply more on  GR and Salinas 88 than on other isolates, but it was due to its poor reproduction on the standard Regina 71 and the use of the concept of RELATIVE resistance that the two cultivars GR and Salinas were classified as susceptible. Hence this is an artifact of the resistance evaluation method, not a virulent population.

I propose to write: “For example, the RKN isolate Curas reproduced poorly on all lettuce cultivars, including the susceptible cultivar Regina 71.”

            The sentence has been changed as the reviewer proposed

I leave it up to the authors to write that low reproduction on the standard susceptible cultivar can lead to wrong classification of the tested cultivar. Some reflection on methodology would have been  good; a discussion calls for (critical) evaluation of methods and results obtained. Keep in mind for the next manuscript!

            Thanks for the suggestion, and we will consider for a next manuscript.

Some sentences can still be improved for the English language. E.g. “Moreover, it was assessed the response of these cultivar..” should be “Moreover, the response of these cultivar was assessed…”

The sentence has been changed as the reviewer suggested