“Secreted in Xylem” Genes (SIX Genes): Relationship to the Aggressiveness of Fusarium oxysporum f. sp. albedinis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please look at the attached PDF. I made several suggestions for the authors.

In the abstract, the phrase "with SIX12 and SIX13 consistently detected" is missing the detail of which isolates have the two genes. Are these two genes in all of the isolates or only in the aggressive isolates? Please add this important detail. If the two genes are only found in the aggressive isolates, then the phrase is probably not needed.

The methods for the multiple correspondence analysis and the hierarchical tree needed to be added to section 2.

Figure 6. If possible, I suggest to capitalize percentage.

Figure 7. How are the authors certain that they obtained Foa from diseased leaves? There were possibly other fungi in the diseased leaves.

I think that the authors should consider that the presence of a SIX gene in the genome of Foa does not guarantee that the gene will be expressed during infection of its host. Perhaps there is some data in the literature about this idea that could be added to the discussion.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The quality of the English in the manuscript is fair. I made a number of suggestions in the attachment.

Author Response

Reviewer 1

1. The reviewer asked to connect the following sentence to the last paragraph on page 9: ‘Two distinct groups could be found: the first contained the aggressive isolates (MRB2, Sam22, CHS and AIA), and the second had hypo-aggressive and moderately aggressive isolates.’.

 

We have connected the sentence to the last paragraph of page 9 as highlight.

 

The reviewer found that the level of sodium hypochlorite (40%) is very high

Indeed, we used a high concentration of sodium hypochlorite to ensure complete sterilization of the leaf surface, because the leaves we harvest are usually much infected. However, we counterbalance this high level of sodium hypochlorite with a relatively short-time treatment.

 

2. The reviewer noted that in the abstract, the phrase "with SIX12 and SIX13 consistently detected" is missing the detail of which isolates have the two genes. Are these two genes in all of the isolates or only in the aggressive isolates?

We have now clarified in the abstract that these two genes were found exclusively in all aggressive isolates. This important detail has been added to ensure accurate representation of our findings.

 

3. The reviewer asked that the methods for the multiple correspondence analysis and the hierarchical tree needed to be added to section 2.

We have now added a detailed description of both analyses to the Methods section as highlighted in the manuscript.

 

4. The reviewer suggested that Figure 6. If possible, capitalize percentage.

We have revised the figure accordingly, ensuring that “Percentage” is now capitalized in all relevant labels for consistency and improved presentation.

 

5. The reviewer raised a concern about the certainty of isolating Fusarium oxysporum f. sp. albedinis (Foa) from diseased leaves, noting the possible presence of other fungi.

We thank the reviewer for this important point. To address this, we have clarified in the revised manuscript that the re-isolated fungus from wilted leaves was morphologically characterized and identified based on colony features and microscopic examination, which matched the characteristics of the originally inoculated Foa isolate. Furthermore, molecular confirmation using specific primers targeting Foa was performed to ensure that the re-isolated organism was indeed Foa. This additional methodology detail has been added to the relevant section to strengthen the reliability of our results.

 

5. The reviewer suggested that we should consider that the presence of a SIX gene in the genome of Foa does not guarantee that the gene will be expressed during infection of its host. Perhaps there is some data in the literature about this idea that could be added to the discussion.

We thank the reviewer for this thoughtful and important observation. We agree that the presence of a SIX gene does not necessarily imply its expression during host infection. In response, we have revised the Discussion section to include this consideration and cited relevant literature that supports this point (see highlight).

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigated the relationship between the pathogenicity of different strains of Fusarium oxysporum f. sp. albedinis (Foa) and the distribution of SIX genes within these strains. The findings revealed that strains with stronger pathogenicity harbored a greater number of SIX genes. This research provides a meaningful foundation for understanding the pathogenic mechanisms of Foa. However, the study has the following limitations: (1) the data compilation is overly simplistic, and (2) the data analysis lacks depth.

In the methodology, Figures 1 and 2 are not directly relevant to the results of this paper and do not need to be included in the main text. Additionally, Figure 7 is not referenced in the article. It is recommended that Figure 8 be presented in color. Figures 5 and 6 could be merged into a single figure, while Figure 3 suffers from low resolution and lacks a scale bar.

Another issue is whether the genome of Foa has been sequenced and published. Are there differences between the sequences of SIX genes in Foa and those of Six genes in F. oxysporum f. sp. lycopersici? Do the SIX gene sequences among different Foa strains exhibit significant variations or discernible patterns? These questions could be addressed through single-gene sequencing and bioinformation analysis.

Author Response

Reviewer 2

The reviewer commented that Figures 1 and 2 are not directly relevant to the results of this paper and do not need to be included in the main text. Additionally, Figure 7 is not referenced in the article. It is recommended that Figure 8 be presented in colour. Figures 5 and 6 could be merged into a single figure, while Figure 3 suffers from low resolution and lacks a scale bar.

We thank the reviewer for the detailed suggestions on figure organization and presentation.

We have now moved Figures 1 and 2 to the Supplementary Materials section and labelled them as Supplementary Figures 1 and 2, respectively.

 

Figure 7 has now been removed from the manuscript, as it was not referenced in the main text and is no longer necessary.

 

We presented the figure 8 in colour as suggested by the reviewer.

Regarding Figures 5 and 6, we acknowledge the suggestion to merge them. However, we prefer to keep them as separate figures to maintain clarity and avoid overloading a single figure with too much information. This separation improves readability and allows each analysis to be interpreted more easily on its own.

Finally, Figure 3 has been replaced with a higher-resolution version, and a scale bar has been added to ensure proper visualization and interpretability.

We appreciate the reviewer’s feedback, which has helped improve the overall presentation of our figures.

 

2. The reviewer mentioned: Another issue is whether the genome of Foa has been sequenced and published. Are there differences between the sequences of SIX genes in Foa and those of SIX genes in oxysporum f. sp. lycopersici? Do the SIX gene sequences among different Foa strains exhibit significant variations or discernible patterns? These questions could be addressed through single-gene sequencing and bioinformatic analysis.

We agree that comparing the sequences of SIX genes between Foa and Fol, as well as among different Foa isolates, could reveal important insights into host specificity and pathogenicity. We have added some clarification on this point in the Discussion section.

While our current study focused on the presence or absence of selected SIX genes, we acknowledge that further work involving gene sequencing and bioinformatic analysis is necessary to assess sequence diversity and potential functional implications. We have now added a paragraph in the Discussion emphasizing the value of such future work to improve our understanding of SIX gene evolution and virulence mechanisms in Foa.

Reviewer 3 Report

Comments and Suggestions for Authors

In this study, the authors have characterized the aggressiveness of 10 isolates of Fusarium oxysporum f. sp. albedinis (Foa) is from three main date palm production areas in Morocco by inoculation tests and reported the association between the aggressiveness of the isolates and the SIX gene homologs they possess. The objectives of this paper are clear and the results obtained are very interesting. However, the content of this paper is preliminary for a full paper and does not contain enough experimental data to persuade PLANTS readers. I recommend that the authors add some experimental data based on my comments below and submit it as a new paper.

 

1. Major comments

• In this paper, the authors describe a relationship between aggressiveness of Foa and the SIX gene, but it is not appropriate to determine that relationship with data from only 10 isolates of Foa. The authors should investigate using at least 30 or more isolates of Foa.
• In this paper, the authors have identified the presence or absence of SIX genes in Foa, but have not confirmed that each SIX gene is expressed in the host; if the authors postulate SIX genes are involved in the aggressiveness of Foa, the expression of each SIX gene in each isolate should be confirmed. That is, the authors should present data verifying the expression of each SIX gene within the host by RT-qPCR or immunoblotting or immunostaining.
• When clarifying the relationship between the aggressiveness of a plant pathogen and a candidate pathogenic gene (in this paper, the SIX gene), it is common for the authors to generate a disruptant strain of the candidate pathogenic gene and a genetic complement of the disruptant strain to verify that it is a pathogenic gene. Since this paper does not include such data, it is not possible to determine the relationship between the aggressiveness of the isolates and the SIX genes they possess from the data in this paper.

２．Minor comments

1)Title and Introduction: ”Fusarium oxysporum albedinis” should be “Fusarium oxysporum f. sp. albedinis”.

2) Introduction: “f. sp.” should be written by “normal letters”.

3) Page 2, 3^rd paragraph, “Based on previous work reported in other Fusarium species, they associated the SIX9 and SIX11 to pathogenicity.”: The authors should cite the reference (previous work). What does “format” mean?

4) Material and Methods: The authors should include “MCA method” and “the method to prepare the hierarchical tree” in the M&M section.

5) Page 4, 1^st paragraph, “the conidial suspension”: The authors should describe the concentration of conidial suspension (spores per mL).

6) Page 4, 1^st paragraph, “Disease severity was assessed one-, two- and three-months post-inoculation by recording the number of wilted leaves (disease incidence) and the number of completely collapsed plants per isolate (disease severity).”: The authors should indicate how many inoculation tests were performed.

7) Page 4, 2^nd paragraph: This paragraph appears incomplete. What experiments did the authors perform after the incubation? The authors should describe the method to identify Foa (Koch's postulates definition).

8) Page 4, 2.4. Amplification of SIX genes: “positive control”: The authors should describe the strain numbers (ID) of the positive control F. oxysporum f. sp. lycopersici races 1 and 2 in the M&M section.

9) Page 8, 1^st paragraph, “In addition, re-isolation of Foa from wilted leaves confirmed that it caused the observed symptoms.”: The authors should include images of diseased plants inoculated with re-isolated Foa.

10) Page 12, 1^st paragraph, “Sasaki et al. (2014)” should be “Sasaki et al. (2015)”.  “f.sp. couple” should be “f. sp. cepae”. “Lieven et al.” should be “Lievens et al.”.

Author Response

Reviewer 3

The reviewer commented ‘the authors should investigate using at least 30 or more isolates of Foa.

We thank the reviewer for raising this important point regarding sample size. We fully acknowledge that a larger number of isolates would strengthen the statistical power and generalizability of our findings. However, due to logistical constraints and the limited availability of well-characterized Fusarium oxysporum f. sp. albedinis (Foa) isolates, our current study was limited to 10 representative strains. Despite this limitation, the isolates covered a large geographical area and we observed consistent and biologically meaningful patterns linking the presence of specific SIX genes (e.g., SIX12 and SIX13) with higher levels of aggressiveness. When more well-characterized isolates are available, it will be worth it to perform such larger analysis.

 

2. The reviewer asked that ‘the authors have identified the presence or absence of SIX genes in Foa but have not confirmed that each SIX gene is expressed in the host; if the authors postulate SIX genes are involved in the aggressiveness of Foa, the expression of each SIX gene in each isolate should be confirmed. That is, the authors should present data verifying the expression of each SIX gene within the host by RT-qPCR or immunoblotting or immunostaining.’’

We thank the reviewer for this insightful comment. We agree that the presence of a SIX gene does not necessarily indicate its functional expression during host infection. While our study focused on establishing a preliminary association between the presence of selected SIX genes and the aggressiveness of Fusarium oxysporum f. sp. albedinis (Foa) isolates, we fully acknowledge that confirming gene expression would provide stronger evidence for their correlation and involvement in pathogenicity.

The expression analysis by RT-qPCR or protein-level assays (e.g., immunoblotting or immunostaining) was beyond the scope of the current work. However, we have now added a clear statement in the Discussion section recognizing this as a limitation and highlighting the need for future transcriptomic or proteomic studies to confirm in planta expression of individual SIX genes.

 

3. The reviewer commented that ‘When clarifying the relationship between the aggressiveness of a plant pathogen and a candidate pathogenic gene (in this paper, the SIX gene), it is common for the authors to generate a disruptant strain of the candidate pathogenic gene and a genetic complement of the disruptant strain to verify that it is a pathogenic gene. Since this paper does not include such data, it is not possible to determine the relationship between the aggressiveness of the isolates and the SIX genes they possess from the data in this paper.’’

We thank the reviewer for this excellent observation. We agree that gene disruption and complementation experiments are considered the gold standard for confirming the functional role of candidate pathogenicity genes, such as the SIX genes studied here. These approaches would indeed provide direct evidence of a gene’s contribution to the aggressiveness of Fusarium oxysporum f. sp. albedinis (Foa).

However, our current study was first focused on association evidence based on gene presence/absence patterns and observed aggressiveness in planta. We have now clarified in the Conclusion that while our findings suggest a potential association between certain SIX genes (notably SIX12 and SIX13) and higher aggressiveness, we recognize that functional validation through gene knockout and complementation is essential for establishing causality. This is work we are definitely going to do in the future, as we have completed it with many other fungal pathogens in the past. This said, we consider our study to be a foundational step that highlights candidate virulence-associated genes, and we strongly support future research that incorporates reverse genetics approaches to confirm their functional roles in Foa pathogenicity.

 

4. The reviewer noted Title and Introduction: “Fusarium oxysporum albedinis” should be “Fusarium oxysporum sp. albedinis”.

We thank the reviewer for pointing out this taxonomic correction. We have revised the manuscript to consistently use the correct nomenclature, “Fusarium oxysporum f. sp. albedinis,” in the title, Introduction, and throughout the text where applicable.

 

5. The reviewer asked that ‘Page 2, 3rd paragraph, “Based on previous work reported in other Fusarium species, they associated the SIX9 and SIX11 to pathogenicity.”: The authors should cite the reference (previous work).’’

We have now added a reference to support the statement regarding the association of SIX9 and SIX11 with pathogenicity in other Fusarium species.

 

6. The reviewer commented on Material and Methods: The authors should include “MCA method” and “the method to prepare the hierarchical tree” in the M&M section.

We thank the reviewer for highlighting this omission. We have now added a detailed description of both the Multiple Correspondence Analysis (MCA) and the hierarchical clustering method in the Materials and Methods section.

 

7. The reviewer commented Page 4, 1st paragraph, “the conidial suspension”: The authors should describe the concentration of conidial suspension (spores per mL).

We thank the reviewer for pointing out the need for more detailed methodological information. We have now revised the text in the Materials and Methods section to specify the concentration of the conidial suspension, which was 1 × 10⁶ spores/mL, used for inoculating the date palm seedlings.

 

8. Page 4, 1st paragraph, “Disease severity was assessed one-, two- and three-months post-inoculation by recording the number of wilted leaves (disease incidence) and the number of completely collapsed plants per isolate (disease severity).”: The authors should indicate how many inoculation tests were performed.

We thank the reviewer for this important clarification request. We have now updated the Materials and Methods section to indicate that two independent inoculation tests were performed, each using ten date palm seedlings per Foa isolate. This information has been added to the methodology.

 

9. Page 4, 2nd paragraph: This paragraph appears incomplete. What experiments did the authors perform after the incubation? The authors should describe the method to identify Foa (Koch's postulates definition).

We thank the reviewer for pointing out the incomplete description of the re-isolation and identification procedures. We have revised the paragraph to clearly describe the steps performed after incubation and to ensure that the identification of Fusarium oxysporum f. sp. albedinis (Foa) followed Koch’s postulates.

 

10. Page 4, 2.4. Amplification of SIX genes: “positive control”: The authors should describe the strain numbers (ID) of the positive control F. oxysporum f. sp. lycopersici races 1 and 2 in the M&M section.

As suggested by the reviewer, we added the strain numbers in method section (race 1: CBS646.78; race 2: CBS645.78) (Lievens et al., 2009). The strain numbers (ID) were provided by Prof. Martijn Rep (University of Amsterdam, Netherlands)

 

11. Page 8, 1st paragraph, “In addition, re-isolation of Foa from wilted leaves confirmed that it caused the observed symptoms.”: The authors should include images of diseased plants inoculated with re-isolated Foa.

As suggested by the reviewer, we included the figure 5 which shows the symptoms observed on date palm seedling after inoculation with the re-isolated Foa.

 

12. Page 12, 1st paragraph, “Sasaki et al. (2014)” should be “Sasaki et al. (2015)”. “f.sp. couple” should be “f. sp. cepae”. “Lieven et al.” should be “Lievens et al.”.

Sasaki et al. (2014)” has been corrected to “Sasaki et al. (2015)”.

“f.sp. couple” has been corrected to “f. sp. cepae”.

“Lieven et al.” has been corrected to “Lievens et al.”

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The author has basically answered my question, and there are no other suggestions.

Author Response

>>The author has basically answered my question, and there are no other suggestions.

Response:

We would like o thank the reviewer for their time and effort.

Reviewer 3 Report

Comments and Suggestions for Authors

I thank the authors for their responses to my comments. I understand what the authors are thinking, but considering the standard of papers that can be published in PLANTS, you should at least do RT-qPCR experiments to confirm that the genes are expressed. As the authors responded, this study is ”a foundational step that highlights candidate virulence-associated genes”. I would judge it to be lacking in content for an acceptable paper in plants.

Author Response

>>>I thank the authors for their responses to my comments. I understand what the authors are thinking, but considering the standard of papers that can be published in PLANTS, you should at least do RT-qPCR experiments to confirm that the genes are expressed. As the authors responded, this study is ”a foundational step that highlights candidate virulence-associated genes”. I would judge it to be lacking in content for an acceptable paper in plants.

Response:

First, we thank the reviewer for their time and effort reviewing this manuscript.

Second, as explained in the last revision and body of the text, the main objective of this study was to explore/determine the presence/absence of these genes across various Foa isolates, not to assess the expression levels of those genes at this point. Doing so, going forward, is our plan, but not as part of this manuscript.

We hope this clarifies our studies' scope and objectives for the Editor to make an informed decision.

Thank you so much for understanding.

Fouad Daayf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

I understand the authors' views.
I consider this paper acceptable for publication in PLANTS.