Management of Soil-Borne Fungi and Root-Knot Nematodes in Cucurbits through Breeding for Resistance and Grafting

Round 1

Reviewer 1 Report

This review manuscript discusses the state of knowledge and recent advances in host resistance in cucurbit crops to soilborne fungi and plant-parasitic nematodes (namely root-knot nematodes).  The manuscript gives an overview of the pathogen groups, known mechanisms of resistance, and advances in identifying and incorporating genetic resistance, including through traditional breeding, biotechnology techniques, and use of grafting.  

All of my comments on the previous submission of this manuscript have been satisfactorily addressed, thank you.  I have only made the minor additional comments in the list below.  Thank you for the opportunity to review this re-submission.  

• Please double check consistency of "soil-borne" vs. "soil borne".  Particularly line 62 and line 647
• In line 92, the flow of the thought may be improved by replacing "Thus" with "For example" 
• In line 106, "damping-off pathogens inducers of water soaked lesions" would be better changed to "damping-off pathogens that induce water soaked lesions" 
• In line 107, pycnidia do appear as "multiple black specks", but they are more accurately described as the fruiting body structures of the fungus.  This is the same for the perithecia in line 111-112, as these are also fruiting body structures. 
• In line 209, "(PRR)" should be moved to directly after "pathogen recognition receptors"
• In line 270, it would add clarity to specify "Genetic resistance to root-knot nematodes" rather than just "Genetic resistance to nematodes"
• In line 274, the sentence beginning in this line should be rewritten to "Evidence suggests this resistance is not governed by a major dominant gene..."
• In line 316, the word "or" should be changed to "and"
• Line 371-372: are there some citations to support the claim in this sentence?  
• In section 4.1.2.5 (CRISPR), this section could benefit from a brief sentence or two about what CRISPR is and how it is performed in plants/cucurbits.  
• In line 509, insert "it" between "because" and "effectively".  Also in line 510, "an" should be changed to "and" 
• In line 556, do you mean citron melon?  This may need clarification - citron could be confused with the citrus fruit (also in line 558).
• In line 598, insert "to" between "resistant" and "FOM"

Author Response

We have modified the manuscript according to the reviewers' suggestions, with the following minor changes:

Lines 62 and 647: “soil borne” changed to “soil-borne”

Line 92. “Thus” changed to “For example”

Line 106: "damping-off pathogens inducers of water soaked lesions" changed to "damping-off pathogens that induce water soaked lesions" 

Line 107: "multiple black specks" changed to “fruiting body structures of the fungus”

Line 112: “perithecia” changed to “fruiting body structures of the fungus”

Line 210: "(PRR)" moved after "pathogen recognition receptors"

Line 272: “root-knot” added.

Line 276-277: Sentence beginning rewritten to "Evidence suggests this resistance is not governed by a major dominant gene..."

Line 319: "or" changed to "and"

Line 374: one citation added. 

Lines 443-450: New information about CRISPR in section 4.1.2.5 (CRISPR) has been added.

Line 519: "it" added between "because" and "effectively". 

Line 520: "an" changed to "and" 

Lines 566 and 568: “citron” changed to “C. amarus”

Line 608: "to" added between "resistant" and "FOM"

Reviewer 2 Report

Although the MS has been improved, the aspects associated with genetic resistance and Fusarium disease are still poor or they are not well focused having in mind that this MS is a review. There is still an imbalance between the part dedicated to nematodes and grafting and the one referring to soil fungi and genetics.

English should be reviewed being one of the reasons that there are verb tense inconsistences all along the MS.

Thus, the MS needs major changes to be published.

The following comments to author answers in Review Report 2 are:

Review Report 2

1. Host-nematodes interactions should be included in point 2.2. Why it needs to be exposed as different and highlighted?

The section host-nematode interactions have been included in section 2.2 as indicated and former section 3 has been eliminated.

OK

1. The soil-borne fungi in section 2.1 should be treated more extensively. The information about the different pathogens should be more complete, especially when dealing with Fusarium wilt, which is nowadays the most important soil-borne fungus affecting cultivated Cucurbits. In fact, there are problems in some cucurbits species not only because there are many pathogenic species of Fusarium, but because the emerging of new races   in some of them and the overcoming of the resistances obtained, which makes very difficult the development of durable resistance.

This section has been expanded, particularly the part dealing with Fusarium oxysporum since it is the main SBPF affecting cucurbits as indicated by the referee. Lines 80-122 in the new edited ms.

The review of FOM pathotype 1.2 is still poor. There are important and well known papers that are missing. eg; 

Herman R., Perl-Treves R. (2007). Characterization and inheritance of a new source of resistance to Fusarium oxysporum f.sp. melonis race 1,2 in Cucumis melo. Plant Disease. 91, 1180-1186;

Perchepied L., Pitrat M. (2004). Polygenic inheritance of partial resistance to Fusarium oxysporum f.sp. melonis race 1,2 in melon. Phytopathology.94, 1331–1336

1. Section 5 could be titled ‘Methods of control’ including Breeding and Grafting. It would be also desirable make only two divisions under Breeding: Traditional Breeding and Molecular (or Biotechnological) tools?

Section 5 headings and subheadings have been changed, according to referees’ suggestions. The title of this section has been changed to ‘Methods of genetic control’ and breeding and grafting included under this heading. Two divisions have been made under Breeding: Traditional Breeding and Biotechnological tools. Grafting has been divided according to the cucurbit crop.

Please delete Traditional in 4.1 since different technologies helping selection and the development of resistant material in breeding are displayed in the subheadings.

A sentence or paragraph that links 4.1 with 4.1.2 is necessary

Some genetics concepts are not right. The phrase (lines 318-320) ‘The analysis of F1 and F2, obtained by backcross or crossing with pure lines, is common to find out the number of genes involved by resistance segregation, as well as the dominant/recessive nature of the character’ should be changed to ‘Analysis of F1 and F2, and backcrosses obtained by crossing resistant and susceptible genotypes, has allow to find out the dominant/recessive nature and the number of genes involved in the resistance.

The paragraph (lines 325-328) ‘…the response of F1 progenies from susceptible x resistant crossings varied from susceptible to highly resistant (wild agrestis types), providing the susceptible cultivar a recessive resistance inheritance and the resistant cultivar a dominant one‘is not right. There is no paragraph in such paper assessing that the susceptible cultivar provide a recessive resistance inheritance and the resistant cultivar a dominant one.

Please, delete lines 334 and 335.

Lines 336 and 337 should be reviewed: In the paper cited, (Kantoglu Y. et al. 2010), tissue culture and mutation are used to improve tolerance to Fusarium oxysporum f. sp. melonis. Do authors of such paper have obtained material of interest to be used in breeding?

The phrase ‘and it is a necessary……..modifications should be deleted’ (lines 337-338).

 

4. Section 5 should be deeply reviewed, completed and organized. Different concepts and methods are mixed. When reading, omics, molecular markers, transgenics…are mixed mainly in 5.1. eg: why TILLING is treated in traditional breeding? There is not a good separation of the different aspects treated.

Section 5 has been rewritten. New heading and subheadings have been included trying to make it more structured and clearer.

Please delete Traditional in 4.1 since different technologies helping selection and the development of resistant material in breeding are displayed in the subheadings.

A sentence or paragraph that links 4.1 with 4.1.2 is necessary

1. Having in mind the content of the MS, the title should be changed to ‘Methods of control of …’, since the content is not dealing only with resistance.

In this review, we have focused on resistance in agreement with the topic of the special issue of Agronomy “Recent Advances in Genomics, Genetic Resources Evaluation and Breeding of Cucurbitaceae Crops”. The title of the MS has been changed to “Evaluating cucurbitaceous crops for resistance to soil-borne fungi and root-knot nematodes” to reflect more accurately the content of the MS. We feel that “Methods of control…” might not reflect the content of the ms because we have not covered other methods of control such as chemical, biological and cultural methods in the same extent. However, such management practices have been mentioned briefly in the introduction section as also suggested by reviewer 1.

The new title does not fix with the content since the MS is not about the evaluation of cucurbits. Please, try one reflecting the content. eg… Management of resistance to soil-borne fungi and root-knot nematodes in Cucurbits through breeding and grafting? Or something similar? The title should reflect the content of the MS.

1. In section 5.1, a relation of different genes giving resistance to the different fungus is exposed, but no information about the goals achieved by using these genes is given. There is no information about the efficiency of these genes controlling each pathogen… Besides, there is not reference to the existence of genetic resistance to nematodes spp, if any.

A paragraph has been added, explaining the final objective of studies on resistance genes and some practical examples. Lines 330-335 in the new edited ms. However, most of studies have focused in the description of resistance genes, but we have found hardly scientific information about pathogen control efficacies using these genes.

It is difficult to get scientific information of the efficacy of the genes found as well as the genes used by seed companies to develop commercial varieties/hybrids, but for the last point, there are information in web sites (eg. seed companies) where authors could find commercial resistances in different types and species of cucurbits.

Genetic resistance to nematodes in cucurbits crops has not been identified so far to our knowledge with the exception of a M. javanica recessive gene (mj) from C. sativus var. hardwickii  (Walters et al., 1997) but this is not available in commercial cultivars. Resistance to M. incognita in zucchini has been recently identified (Talavera-Rubia et al., 2018) though the genetic base is presently unknown. All this information is included in the manuscript.

OK

1. When describing the resistances to soil-borne pathogens in some paragraphs, there is any information on which pathogen resistance has been found (eg. line 337);  or if the gene/s is/are dominant or recessive (eg. line 278).

The names of the pathogens have been indicated;  R. solani in Line 403 in the new edited ms., Phytophthora melonis in Line 405 in the new edited ms.  The former sentence in L278 has been rewritten as: For M. phaseolina, ...........progenies from susceptible × resistant crossings ........ providing the susceptible cultivar a recessive resistance inheritance and the resistant cultivar a dominant one, Lines 325-328 in the new edited ms..

Attending the paper authors make reference (19), the phrase ‘…providing the susceptible cultivar a recessive resistance inheritance and the resistant cultivar a dominant one’ is not right since the results of such a paper suggest just differences in the genetic basis of the resistance in the different selected sources.

1. One important point to consider when the use of molecular markers is suggested is the phenotyping.  Any mention to how difficult is to phenotype resistance to soil-borne pathogens is exposed in the MS.

Molecular markers subsection has been rewritten extending the examples information and discussing the problems that exist in phenotyping and its relationship with molecular markers.

The paragraph (lines 358-361) should be reviewed and rewritten. In the paper Brotman et al 2005 enriched the genomic region around the Fom-1/Prv resistance locus with RFLP, AFLP, RAPD, and CAPS markers, using two mapping populations that shared a common susceptible parent. Do Brotman et al find any marker that could be useful to select resistance to FOM 0 and 2? The comments of the authors are not complete referring this paper.

The same consideration should be taken in mind with the paper of Tezuka et al (2010)

In general, what should be exposed under the subheading of molecular markers is not only the development of markers carried out by different authors but their utility in the selection and introgression of resistance genes in material of interest.

 

1. Into each section, paragraphs referring to nematodes should be set aside from those referring to soil-borne pathogens. This will make easier the reading.

Paragraphs referring to nematodes appear in a separate graph from those dealing with fungi to improve reading in the revised version.

OK

Among minor fixes:

 Table 4 is not well exposed which makes the content not well understood. Why grafting appears in this table?  Since all markers and methods are described in the text, Table 4 seems to be unnecessary.

We have considered the need of Table 4 and decided not to include this table in the revised version. Table 4 has been removed from the manuscript.

OK

There is no reference to literature in paragraph 317-319.

The reference Guo, S., Zhang, J., Sun, H., Salse, J., Lucas, W. J., Zhang, H., ... & Min, J. (2013). Nature genetics, 45(1), 51-58 has been included. Line 392 in the new edited ms.

OK

In 6.2 a phrase referred to watermelon is included (417-418).

The phrase has been removed. Lines 501-502 in the new edited ms.

OK

Two pathotypes of FOM 1.2 affecting melons are well known, 1.2W and 1.2Y, but no references in the text are referred to them.

The pathotypes of FOM 1.2W and 1.2Y affecting melon have been referred in the revised version (Chikh-Rouhou et al., 2010. HortScience, 45(7), 1021-1025. Line 94 in the new edited ms.

The review of FOM pathotype 1.2 is still poor. There are important and well known papers that are missing (see above)   

What the authors want to expose in phrase 461-463?

We wanted to point out the importance of identifying the pathogens present in a soil, for a proper selection of the rootstock or cultivars to be planted, since some rootstocks are resistant to some pathogens but susceptible to some others. We have rewritten the phrase, Lines 537-539 in the new edited ms.

The following phrase has been included in the abstract: ‘…The importance of proper pathogen diagnosis in the right choice of cultivar or rootstock is highlighted because of the specificity of the response of the cucurbit crops to fungal and nematode species and races…’

OK

The change of nomenclature of C. lanatus citroides is exposed in line 229 , so please, delete in line 473.

Deleted. Line 556 in the new edited ms.

OK

Due to the anatomical characteristics of C. africanus and C. myriocarpus, it seems difficult the use these species as root-stocks of watermelon (lines 503-504). Have the authors any evidences that these root-stocks work with commercial watermelons?

Yes, C. africanus and C. myriocarpus are compatible with commercial watermelon cultivars as the authors reported in a follow-up research. Planting the wild species in advance of the cultivated watermelon optimized stem diameter overcoming the anatomical differences between scion and rootstock which resulted in increased inter-generic compatibility (Pofu et al., 2012).

OK

As a suggestion, in section 7, authors should expose widely the future objectives to be achieved having in mind the actual weakness existing in Cucurbit cultivation facing the pathogens and problems treated in this MS.

The title of the section has been changed to “Conclusions”. Line 642 in the new edited ms.

OK

There are verb tense inconsistences all along the MS

In line 380 sensitive should be replaced by susceptible.

Reference 122 (line 1000) is not right; the correct list of the authors is: Brotman, Y.; Kovalski, I.; Dogimont, C.; Pitrat, M.; Portnoy, V.; Katzir, N.; Perl-Treves, R.

Author Response

Although the MS has been improved, the aspects associated with genetic resistance and Fusarium disease are still poor or they are not well focused having in mind that this MS is a review. There is still an imbalance between the part dedicated to nematodes and grafting and the one referring to soil fungi and genetics.

We have made efforts to further improve the ms, particularly the aspects related to the Fusarium disease and genetic resistance in cucurbits according to the comments of this referee. When planning this review, we discussed the different aspects to be considered which they appear as headings in the MS. These aspects were developed for both, SBPF and RKN. In reviewing the literature, most references on control methods for soil-borne fungi referred to genetic resistance whereas for root-knot nematodes, referred to grafting. For this reason the parts dedicated to fungi and nematodes have different length. Nonetheless, the parts dedicated specifically to the SBPF and to RKN have as a whole approximately similar length, 3867 words for SBPF and 3663 words for RKN. The references listed include nearly twice as many references for fungi than for nematodes. The grafting technology has been widely adopted in some regions (i.e. Mediterranean region) but it is under evaluation as a management option in some other regions (i.e. United Estates). This explains the numerous contributions (references) on this subject in the last decade. For this reason we have emphasized in this review the progress and developments on grafting as it was mentioned in the Introduction section, L77-78 and in Grafting L475-472.

 

English should be reviewed being one of the reasons that there are verb tense inconsistences all along the MS.

The English has been thoroughly reviewed to eliminate the inconsistencies and we apologize for this inconvenience.

The review of FOM pathotype 1.2 is still poor. There are important and well-known papers that are missing. eg; 

Herman R., Perl-Treves R. (2007). Characterization and inheritance of a new source of resistance to Fusarium oxysporum f.sp. melonis race 1,2 in Cucumis melo. Plant Disease. 91, 1180-1186;

Perchepied L., Pitrat M. (2004). Polygenic inheritance of partial resistance to Fusarium oxysporum f.sp. melonis race 1,2 in melon. Phytopathology.94, 1331–1336

Thanks for the input. We have added both references. Citations in lines 96 and 318.

Please delete Traditional in 4.1 since different technologies helping selection and the development of resistant material in breeding are displayed in the subheadings. A sentence or paragraph that links 4.1 with 4.1.2 is necessary

The headings and subheadings in this section have been renumbered as follow: “4.1 Traditional Breeding”, “4.2 Biotechnological tools” with subsections “4.2.1. Molecular markers”, “4.2.2. Omics analyses”, “4.2.3. Mutagenesis and TILLING”, “4.2.4. Transgenic plants”, “4.2.5. CRISPR” and “4.3 Grafting”. We hope that now it is clearer.

Some genetics concepts are not right. The phrase (lines 318-320) ‘The analysis of F1 and F2, obtained by backcross or crossing with pure lines, is common to find out the number of genes involved by resistance segregation, as well as the dominant/recessive nature of the character’ should be changed to ‘Analysis of F1 and F2, and backcrosses obtained by crossing resistant and susceptible genotypes, has allow to find out the dominant/recessive nature and the number of genes involved in the resistance.

Changed to “Analysis of F1 and F2, and backcrosses obtained by crossing resistant and susceptible genotypes, allowed finding out the dominant/recessive nature and the number of genes involved in the resistance”. Lines 322-324.

The paragraph (lines 325-328) ‘…the response of F1 progenies from susceptible x resistant crossings varied from susceptible to highly resistant (wild agrestis types), providing the susceptible cultivar a recessive resistance inheritance and the resistant cultivar a dominant one‘ is not right. There is no paragraph in such paper assessing that the susceptible cultivar provide a recessive resistance inheritance and the resistant cultivar a dominant one.

Modified to: “For M. phaseolina, seven out of 97 accessions of C. melo showed resistance, one cantaloup (Can-NyIsr) and six exotic accessions from Africa, Asia and Eastern Europe (Dud-CUM296Georg, Dud-QPMAfg, Ac-TGR1551Zimb, Con-Pat81Ko, Ag-15591Ghana and Ag-C38Nig) that previously showed resistance to other SBPF [27,118]. The response of F1 progenies from resistant × susceptible crossings varied from susceptible to highly resistant, suggesting differences in the genetic basis of the resistance in the different selected sources. For example, resistance derived from Can-NyIsr seemed to be recessive, as the F1 behave as the susceptible parental, while Ag-15591Ghana and Ag-C38Nig F1 generations suggested dominance of the resistance genes, although further studies with segregant populations are needed to determine the genetic control of each resistance [20]”. Lines 329-337.

Please, delete lines 334 and 335.

Former Lines 354 – 355 have been deleted.

Lines 336 and 337 should be reviewed: In the paper cited, (Kantoglu Y. et al. 2010), tissue culture and mutation are used to improve tolerance to Fusarium oxysporum f. sp. melonis.

We wanted to highlight in vitro culture methods as a tool to be considered in breeding since the manuscript (Kantoglu Y. et al, 2010) proposes a protocol to obtain mutants and also gives some other examples of in vitro tests for soil-borne resistance in cucurbits. Rewritten to: “However techniques such as the in vitro plant tissue culture could be useful when combined with others. For example, tissue culture has been used to produce mutants to improve tolerance to FOM in melon [121] and transformation mediated by Agrobacterium tumefaciens is a tool for producing whole transgenic plants or pathogens [122] or for CRISPR modifications [123].” Lines 348-352.

Do authors of such paper have obtained material of interest to be used in breeding?

They found "two promising DH (doubled-haploid) lines, were selected for resistance/tolerance to Fusarium wilt following inoculations with race 1.2w", but they did not perform subsequent assays with the selected lines in greenhouse conditions.

The phrase ‘and it is a necessary……..modifications should be deleted’ (lines 337-338).

Lines rewritten and moved to “Biotechnological tools” section. Lines 348-349.

Please delete Traditional in 4.1 since different technologies helping selection and the development of resistant material in breeding are displayed in the subheadings.

A sentence or paragraph that links 4.1 with 4.1.2 is necessary

The headings and subheadings in this section have been renumbered as indicated earlier.

The new title does not fix with the content since the MS is not about the evaluation of cucurbits. Please, try one reflecting the content. eg… Management of resistance to soil-borne fungi and root-knot nematodes in Cucurbits through breeding and grafting? Or something similar? The title should reflect the content of the MS.

The title of the manuscript has been changed to “Management of soil-borne fungi and root-knot nematodes in Cucurbits through breeding for resistance and grafting”. Lines 2-4.

It is difficult to get scientific information of the efficacy of the genes found as well as the genes used by seed companies to develop commercial varieties/hybrids, but for the last point, there are information in web sites (eg. seed companies) where authors could find commercial resistances in different types and species of cucurbits.

A sentence has been added: “Despite these advances, only cucurbit cultivars and rootstocks with resistance to some Fusarium and Verticillium pathogens (FOM 0, FOM 1, FOM 2, FON, FOC, FORC, VA) are available commercially for industrial use in greenhouse crops”. It has been added a reference from a Compendium of vegetable varieties available in the European Union: Portagrano (2016). Vademecum de semillas. http://www.portagrano.net/home/. Lines 319-321.

Attending the paper authors make reference (19), the phrase ‘…providing the susceptible cultivar a recessive resistance inheritance and the resistant cultivar a dominant one’ is not right since the results of such a paper suggest just differences in the genetic basis of the resistance in the different selected sources.

The text has been modified to: “For M. phaseolina, seven out of 97 accessions of C. melo showed resistance, one cantaloup (Can-NyIsr) and six exotic accessions from Africa, Asia and Eastern Europe (Dud-CUM296Georg, Dud-QPMAfg, Ac-TGR1551Zimb, Con-Pat81Ko, Ag-15591Ghana and Ag-C38Nig) that previously showed resistance to other SBPF [27,118]. The response of F1 progenies from resistant × susceptible crossings varied from susceptible to highly resistant, suggesting differences in the genetic basis of the resistance in the different selected sources. For example, resistance derived from Can-NyIsr seemed to be recessive, as the F1 behave as the susceptible parental, while Ag-15591Ghana and Ag-C38Nig F1 generations suggested dominance of the resistance genes, although further studies with segregant populations are needed to determine the genetic control of each resistance [20]”. Lines 329-337.

The paragraph (lines 358-361) should be reviewed and rewritten. In the paper Brotman et al 2005 enriched the genomic region around the Fom-1/Prv resistance locus with RFLP, AFLP, RAPD, and CAPS markers, using two mapping populations that shared a common susceptible parent. Do Brotman et al find any marker that could be useful to select resistance to FOM 0 and 2? The comments of the authors are not complete referring this paper.

The sentence “…these markers were more universal than the CAPS markers developed for the same gene (Brotman et al 2005)” comes from Oumouloud et al. 2008; they also commented that “Brotman et al. (2005) have already developed two CAPS makers, which are more tightly linked to Fom-1 than those that were presented in this work”. Refereeing to their CAPS markers, Brotman et al 2005 said that “…they do not separate all the genotypes according to their resistance phenotype. Instead, each horticultural group appears to carry a characteristic haplotype regarding these two closely linked markers…”, so Brotman did not found a useful marker to FOM resistance. However, Oumouloud et al. 2008 concluded with their markers that “The combined use of the identified RAPD and SCAR markers would be very useful in marker assisted selection for introducing resistance to F.o.m races 0 and 2 in melon.”

We have added information clarifying this situation: “… Brotman et al. [125] concluded that their markers are not useful for identifying FOM resistance, while Oumouloud et al. [124] advocated the combined use of RAPD and CAPS markers developed for effective use in MAS for FOM races 0 and 2. Another two CAPS markers were close to Fom-1 gen in melon (from P11 × MR-1), and further analysis correctly predicted the genotype [127].” Lines 372-376.

Also, we have added more information about the Fom-2 gene markers with 2 new references.

Lines new lines 379-383:

a) Wang, Y. H., Thomas, C. E., & Dean, R. A. (2000). Genetic mapping of a fusarium wilt resistance gene (Fom-2) in melon (Cucumis melo L.). Molecular Breeding, 6(4), 379-389.

b) Burger, Y., Katzir, N., Tzuri, G., Portnoy, V., Saar, U., Shriber, S., ... & Cohen, R. (2003). Variation in the response of melon genotypes to Fusarium oxysporum f. sp. melonis race 1 determined by inoculation tests and molecular markers. Plant Pathology, 52(2), 204-211.

“…while others AFLP markers demonstrated a high correlation between the genotype and the resistance phenotype [131]. Further studies using these markers for different germplasm indicated that the expected resistance ratio did not correspond with the inoculation tests, although they were capable of discriminate the homozygosis/heterozygosis genotypes for the Fom-2 gene [132].”

The same consideration should be taken in mind with the paper of Tezuka et al (2010)

“Tezuka et al. 2010” reference have been changed to “Tezuka, T., K. Waki, K. Yashiro, M. Kuzuya, T. Ishikawa, Y. Takatsu, and M. Miyagi, 2009: Construction of a linkage map and identification of DNA markers linked to Fom-1, a gene conferring resistance to Fusarium oxysporum f.sp. melonis race 2 in melon. Euphytica 168, 177—188”. Reference 127.

Information about the markers have been expanded: “… and that there might be different alleles for resistance to races 0 and 2 that originated independently in different lineages of the melon phylogeny”. Lines 377-379.

In general, what should be exposed under the subheading of molecular markers is not only the development of markers carried out by different authors but their utility in the selection and introgression of resistance genes in material of interest.

The text has been rewritten: “…the importance of markers in breeding, when they are highly associated with a trait of interest, is their use as tools to perform marker-assisted selection (MAS) breeding or to be incorporated into resistant genes introgression programs” Lines 359-361.

Among minor fixes:

The review of FOM pathotype 1.2 is still poor. There are important and well-known papers that are missing (see above)   

The works suggested by the reviewer concerning FOM 1.2 have been included in the new version. We think that the number of references for these virulent pathotypes is enough to allow the readers of the ms to get a good understanding of the problem associated to these pathotypes.

In line 380 sensitive should be replaced by susceptible.

It has been replaced, Line 399.

Reference 122 (line 1000) is not right; the correct list of the authors is: Brotman, Y.; Kovalski, I.; Dogimont, C.; Pitrat, M.; Portnoy, V.; Katzir, N.; Perl-Treves, R.

Thank you very much for pointing out. The reference has been changed. New number of the reference is 125.

Reviewer 3 Report

The authors had addressed all the questions and concerns. So, I recommend to accept this manuscript after thoroughly checking the English grammar.

Author Response

The english grammar has been revised again, and some errors have been corrected

Round 2

Reviewer 2 Report

Th MS has been improved mainly in genetics aspects.

Author Response

Dear Reviewer,

Thanks for your comment, in fact we understood that the topic of the special issue deals with genetics.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

This review paper written by Doñas et. al. explores cucurbitaceous resistance against soil-borne fungi and nematode This paper can be accepted for publications after following minor revisions.

 

1. It would be better to discuss some of the points in abstracts from the introduction section. It looks more reasonable to the reader if author gives more specific reasons why plant resistance is important than others.
2. There are other management practices for sustainable agriculture against plant pathogens. For example, biocontrol agents. The authors should mention those practices too in the introduction part.
3. This paper will be more exciting and attract more readers if authors could make a graphical figure about the plant resistance mechanism in cucurbits. It’s not mandatory.
4. Overall, the paper is ready to publish after minor changes.

Reviewer 2 Report

The manuscript is a review on the resistance to diseases caused by soil-borne fungi and to nematodes in Cucurbits. The MS gives actual, enough and interesting information about the incidence and control of nematodes in cucurbit cops; however, the information about the diseases caused by soil-borne fungi is not so well reviewed nor exposed. Thus, the MS needs major changes to be published.

Some changes in the organization of the paper are suggested to make it more suitable to read:

1. Host-nematodes interactions should be included in point 2.2. Why it needs to be exposed as different and highlighted?
2. The soil-borne fungi in section 2.1 should be treated more extensively. The information about the different pathogens should be more complete, especially when dealing with Fusarium wilt, which is nowadays the most important soil-borne fungus affecting cultivated Cucurbits. In fact, there are problems in some cucurbits species not only because there are many pathogenic species of Fusarium, but because the emerging of new races   in some of them and the overcoming of the resistances obtained, which makes very difficult the development of durable resistance.
3. Section 5 could be titled ‘Methods of control’ including Breeding and Grafting. It would be also desirable make only two divisions under Breeding: Traditional Breeding and Molecular (or Biotechnological) tools?
4. Section 5 should be deeply reviewed, completed and organized. Different concepts and methods are mixed. When reading, omics, molecular markers, transgenics…are mixed mainly in 5.1. eg: why TILLING is treated in traditional breeding? There is not a good separation of the different aspects treated.

Having in mind the content of the MS, the title should be changed to ‘Methods of control of …’, since the content is not dealing only with resistance.

In section 5.1, a relation of different genes giving resistance to the different fungus is exposed, but no information about the goals achieved by using these genes is given. There is no information about the efficiency of these genes controlling each pathogen… Besides, there is not reference to the existence of genetic resistance to nematodes spp, if any.

When describing the resistances to soil-borne pathogens in some paragraphs, there is any information on which pathogen resistance has been found (eg. line 337);  or if the gene/s is/are dominant or recessive (eg. line 278).

One important point to consider when the use of molecular markers is suggested is the phenotyping.  Any mention to how difficult is to phenotype resistance to soil-borne pathogens is exposed in the MS.

Into each section, paragraphs referring to nematodes should be set aside from those referring to soil-borne pathogens. This will make easier the reading.

Among minor fixes:

 Table 4 is not well exposed which makes the content not well understood. Why grafting appears in this table?.  Since all markers and methods are described in the text, Table 4 seems to be unnecessary.

There is no reference to literature in paragraph 317-319.

In 6.2 a phrase referred to watermelon is included (417-418).

Two pathotypes of FOM 1.2 affecting melons are well known, 1.2W and 1.2Y, but no references in the text are referred to them.

What the authors want to expose in phrase 461-463?

The change of nomenclature of C. lanatus citroides is exposed in line 229 , so please, delete in line 473.

Due to the anatomical characteristics of C. africanus and C. myriocarpus, it seems difficult the use these species as root-stocks of watermelon (lines 503-504). Have the authors any evidences that these root-stocks work with commercial watermelons?

As a suggestion, in section 7, authors should expose widely the future objectives to be achieved having in mind the actual weakness existing in Cucurbit cultivation facing the pathogens and problems treated in this MS.

Reviewer 3 Report

This review manuscript summarizes research in the area of resistance in Cucurbitaceae crops to soil-borne fungi and nematodes, specifically root-knot nematodes (Meloidogyne spp.).  This manuscript also discusses resistant cultivars for use in nematode management.  This review is timely and important because of the significant crop loss in the Cucurbitaceae due to these pathogens and difficulties in their management, or which host resistance is an important component due to the reduction in use of chemical options for environmental concerns.  

Overall this review is very well written and thorough in its scope.  I have noted below only minor edits at specific locations, noted by the line number.  Thank you for the opportunity to review this manuscript.

Specific comments: 

• In section 2.1, line 67 and line 70, Pythium spp. and Phytophthora species are oomycetes.  Although they are soil-borne, these are fungal-like organisms, not true fungi.  This may need to be stated in section 2.1.  The title of the section could remain "Soil-borne fungi" with this proper note. 
• In section 2.2, the sentence spanning lines 88-90, it would be helpful to include a reference or two supporting this statement.  
• In line 156, the terms "larger" is used.  However, do you actually mean "extensive"?  Larger would imply the galls themselves are physically larger.  Similar in line 157 where the term "smaller" is used.  Perhaps "reduced root galling" or "milder root galling" would work better?  
• In line 159-160, you use the term "tolerance limit".  This sounds similar in concept to the term "damage threshold" or "action threshold", which is commonly used in the literature.  
• At the start of line 199, it would be useful to specify "Plant hormones", instead of just "Hormones"  
• In line 290, please define next-generation sequencing "NGS" here at first mention.  I believe you have this in line 307.  Also "next-generating" here should be "next-generation"  
• The sentence spanning lines 301-303 is a little confusing.  Could this be broken into two sentences, or further clarified?  
• In line 304, simple sequence repeat (SSR) has not been defined in the text prior to using the acronym.  I believe it was defined in the footnotes of Table 4, but would be helpful to re-define in the main text.  
• Also concerning the SSR markers, the statement made around line 304 that "six SSR markers were classified for cucurbit rootstock candidates", is also repeated at line 372.  The sentence at line 372 can perhaps be deleted.  
• In line 481, single quotation marks are needed around the cultivars here
• Table 5 - please indicate which root-knot nematode species was assessed.  Also the footnote for this table should be "not significant"