Impact of Fusarium Species Composition and Incidence on Onion Basal Rot in Northeastern Israel

Round 1

Reviewer 1 Report

Please include the hypotheses that you were testing with this research.

In the first set of plant materials, you evaluated inbred lines that were flowering. Since onion is an outcrossing species, it suffers some inbreeding depression when inbreeding is conducted. The A, B, and C lines that are used in onion hybrid cultivar development are often inbred to a certain extent to ensure uniformity within the hybrid cultivar. Inbred lines are often weaker in their growth as compared to hybrid or open-pollinated cultivars. Please address how this level of inbreeding may have influenced the results that you observed with respect to disease incidence and severity. Also you were sampling plants that had been vernalized and were flowering. Please address how the disease incidence and severity might have been different if you had sampled dormant, mature bulbs. It would have been beneficial if you could have compared the hybrid cultivar produced from these inbred lines with the inbred lines in terms of disease incidence and severity.

Please include the names of the cultivars screened in section 2.2 of the M&M.

Do you have any past crop history for the fields mentioned in the paper? Do you know many onion crops have been grown previously in these fields?

Please check the attached file for more comments and proposed changes

Comments for author File: Comments.pdf

The quality of the English was fine. There were a few typos throughout the manuscript.

Author Response

Responses to Reviewer 1's comments

We thank the reviewer for investing substantial effort, which undoubtedly contributed to this manuscript. The remarks and suggestions undoubtedly improved this paper's scientific soundness and accuracy. Your contribution is greatly appreciated.

Please include the hypotheses that you were testing with this research.

Reply: This is good advice. We added the hypothesis at the end of the introduction: "According to the cumulating scientific data [9,20-22], our research hypothesis is that the Fusarium species variety involved in FBR is much larger than what we have so far discovered and that they thrive in complex compositions depending on the host plant and the environment. We also hypothesize that the impact of the disease in some commercial fields is more significant than previously thought." (lines 108-112).

In the first set of plant materials, you evaluated inbred lines that were flowering. Since onion is an outcrossing species, it suffers some inbreeding depression when inbreeding is conducted. The A, B, and C lines that are used in onion hybrid cultivar development are often inbred to a certain extent to ensure uniformity within the hybrid cultivar. Inbred lines are often weaker in their growth as compared to hybrid or open-pollinated cultivars. Please address how this level of inbreeding may have influenced the results that you observed with respect to disease incidence and severity.

Reply: This is indeed an important aspect that should be addressed. Still, there are significant knowledge gaps here, and we believe that our initial data provided here will encourage follow-up studies that will deepen our understanding of the subject. The following explanation was added to the Discussion (lines 595-605):

"It was shown here for the first time that the level of contamination in some fields is significantly higher than assumed and reaches 8% in certain varieties. While such a higher incidence is alarming, it may be affected by the level of inbreeding. Since onion is an outcrossing species, it may suffer inbreeding depression [33]. The different lines used in onion hybrid cultivar development are often inbred to a certain extent to ensure uniformity within the hybrid cultivar. Inbred lines are frequently weaker in their growth than hybrid or open-pollinated cultivars. Thus, the survey presented here must be followed by a more comprehensive and dedicated study to evaluate the disease severity in different commercial onion cultivars in various geographic regions, considering the pollination method and other cultivation aspects."

Also, you were sampling plants that had been vernalized and were flowering. Please address how the disease incidence and severity might have been different if you had sampled dormant, mature bulbs.

Reply: This is an intriguing remark. Young seedlings and dormant mature onion bulbs are the two most valuable plant phenological stages of Fusarium basal rot. Yet, the disease can occur at any stage of the crop cycle. We modified the text to pronounce this better: " Young seedlings and dormant mature onion bulbs are the two most valuable plant phenological stages of FBR. Yet, the disease can occur throughout the entire crop cycle [3-6]." (lines 45-47).

It would have been beneficial if you could have compared the hybrid cultivar produced from these inbred lines with the inbred lines in terms of disease incidence and severity.

Reply: Indeed. We took advantage of our fieldwork when we sampled onion bulbs for the Fusarium species identification study and made the disease incidence survey (although this is not the main focus of the current work). The Givat Yoav field that was studied in the survey included inbred plants that had been vernalized and were flowering. This made it possible to easily identify the diseased dry inflorescences and conduct the survey without reducing the crop yield. In contrast, the second, Yiron's field, was seeded with hybrid yellow Riverside (Orlando) cv. and was cultivated for bulb production (so the flowering stage was denied). Consequently, identified diseased bulbs near the season-ending (before the harvest) in 1500 plants could reduce the yield, and we decided to avoid it. Moreover, the different cultivations (for seeds or bulbs) make these two fields noncomparable.

As explained above, we made this preliminary assessment knowing that this is only the first step to uncovering the full extent of the disease's impact on Israel's onion commercial fields. This survey will hopefully trigger new questions (such as those you raise here) and evoke a following comprehensive work on the subject.

The following explanation was added to the Materials and Methods (lines 131-141):

"We utilized our fieldwork to sample onion bulbs for the Fusarium species identification study and conducted a disease incidence survey, although this is not the primary focus of the current work. The Givat Yoav field, which was included in the survey, consisted of inbred plants that had undergone vernalization and were in the flowering stage. This enabled us to easily identify diseased dry inflorescences and conduct the survey without reducing crop yield. In contrast, the second field, Yiron's field, was seeded with a hybrid yellow Riverside (Orlando) cultivar and was cultivated for bulb production, thus denying the flowering stage. Consequently, identifying diseased bulbs near the end of the season (before harvest) in 1500 plants could have reduced the yield, and we decided to avoid this. Moreover, the different cultivation purposes (seeds or bulbs) render these two fields noncomparable."

Please include the names of the cultivars screened in section 2.2 of the M&M.

Reply: The following information was added to the text as suggested: "Yiron's field (33°09'19.4 "N 35°34'23.1" E) was sown with the yellow Riverside (Orlando) cv. on January 9, 2022, and sampled on day 228 of sowing (August 25, 2022). The Givat Yoav field includes three onion cultivars, red female (Ha3 cv.), red male (Ha2 cv.), and yellow female (Ha1 cv.)". (lines 160-163).

Do you have any past crop history for the fields mentioned in the paper?

Do you know many onion crops have been grown previously in these fields?

Reply: The fields are routinely used for onion cultivation, but we don't have their crop history.

Specific Comments in the attached file

1. Line 12: We deleted "Fusarium Basal Rot" (which already appeared on line 10).
2. Line 33: The word "cultivar" was replaced by "crop" as advice.
3. Lines 65-66: The word "ailment" was replaced by "disease" as advice.
4. Line 91: The word " prevalence" was added.
5. Line 126: The word "the" was removed from the title.
6. Line 169: The word "scabs" was replaced by "scales (fleshy leaves)."
7. line 193: Corrected to "according to the methods described previously."
8. Line 248: We corrected "In" to "in."
9. Line 265: The term "off" is correct.
10. Line 245: Section 2.6. Germination Pathogenicity Assay: Yes, the germination percentage was reported relative to the uninfected control group. This explanation was embedded in the text: "… the seeds were photographed and washed, and their germination percentages, biomass, and epicotyl emergence percentages were measured, and compared to the mock uninfected control group." (lines 281-283).
11. As already stated: " The bulbs were incubated in a temperature-controlled incubator in the dark at 22±1°C for two weeks." (lines 299-300).
12. Figure 2. There is a possibility that other diseases (such as pink root) affect the onion plants. We focused our study on the basal rot disease.
13. We don't have the past crop history of the fields.
14. Line 398: The font size of "Neocosmospora" was corrected here and throughout the text.
15. "Fusarium solani" was replaced with " Neocosmospora" in Figures 5, 8, 10, 11, 13 and 14.
16. An X-axis label was added in Figure 11: Neocosmospora isolates
17. Line 530: The word "inquiring" was replaced with "interesting" as advised.
18. Line 577: The word "an" was deleted from the text.
19. Line 579: We corrected "Metam" to "metam".
20. Line 624: We corrected "Integrated" to "integrated."
21. Line 629: The sentence was corrected as advised to: "Fusarium basal rot disease (FBR) in onion (Allium cepa) is common worldwide."
22. Line 630: We added the word "that" as advised.
23. Duplicate references were corrected as advised.

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript ID: horticulturae-2923177

The paper entitled “Fusarium species composition in agricultural fields in northeastern Israel and its effect on onion basal rot disease” was carefully reviewed. 

The manuscript offers insightful information regarding the Fusarium species impacting onion cultivation in Israel. To enhance the manuscript’s utility, I recommend refining the data presentation for greater clarity and providing a more comprehensive discussion of the study’s implications. These revisions could significantly strengthen the foundation for devising novel disease management strategies. I suggest addressing these aspects to fortify the manuscript for publication.

Detailed comments:

Please check a few notes you can use to improve this manuscript:

-          The title is too long and complex, and it could be simplified by removing some unnecessary details (e.g. composition, in agricultural fields). It could be improved as follows: “Impact of Fusarium species on the incidence of basal rot in onions across northeastern Israel”.

-          Line 12: Delete “Fusarium Basal Rot”, which already appeared on line 10.

-          Line 118: The decision to evaluate disease severity in only one field while having two experimental fields is not fully justified. It is crucial to explain why only one field was chosen for the evaluation and how this choice affects the representativeness and generalizability of the results.

-          Line 159: The authors should explain why the specific number of 228 plates was chosen for incubation. They need to justify this number to ensure that it provides sufficient replication for statistical analysis and to account for variability within the pathogen population.

-          Line 279: It is essential to verify whether the assumptions of ANOVA, including normality and homogeneity of variances, were tested. Additionally, please clarify if any corrective measures were implemented to adjust for multiple comparisons.

-          Lines 286-295: Delete the first paragraph of the Results section, as the objectives of the study are already clearly stated in the Introduction section (lines 103-116).

-          Line 316: Delete “(Delia antiqua)”, which already appeared on line 150.

-          Lines 329-334: There's no need to repeat the methodology in the Results section. Please delete the following paragraph to avoid redundancy in the manuscript: “Final verification was performed using a molecular test that included PCR amplification of an oligonucleotide unique to the Fusarium genus (the Fa/R8 or 7cF/11aR primers targeting the RPB1 or RPB2 – the RNA polymerase largest or 2nd largest subunit and E1/E2, targeting the Fusarium translation elongation factor-1 alpha gene). The PCR products were verified using gel electrophoresis and identified using sequencing and homology identification”.

-          Overall, the manuscript provides valuable information on the incidence and control of FBR. However, it would benefit from more detail on the implications for disease management practices and future research directions to fill identified knowledge gaps.

Author Response

Responses to Reviewer 2's comments

We would like to express our sincere appreciation to the reviewer for the essential and helpful advice. The time and effort invested are greatly appreciated and certainly contributed to and improved the manuscript. Thank you.

The paper entitled "Fusarium species composition in agricultural fields in northeastern Israel and its effect on onion basal rot disease" was carefully reviewed. The manuscript offers insightful information regarding the Fusarium species impacting onion cultivation in Israel.

Reply: Thank you for the positive evaluation of our manuscript.

To enhance the manuscript's utility, I recommend refining the data presentation for greater clarity and providing a more comprehensive discussion of the study's implications. These revisions could significantly strengthen the foundation for devising novel disease management strategies. I suggest addressing these aspects to fortify the manuscript for publication.

All your remarks and suggestions were addressed carefully and thoroughly, as detailed below.

Detailed comments:

The title is too long and complex, and it could be simplified by removing some unnecessary details (e.g., composition in agricultural fields). It could be improved as follows: "Impact of Fusarium species on the incidence of basal rot in onions across northeastern Israel."

Reply: The title was corrected as advised.

Line 12: Delete "Fusarium Basal Rot," which already appeared on line 10.

Reply: Corrected as per your suggestion.

Line 118: The decision to evaluate disease severity in only one field while having two experimental fields is not fully justified. It is crucial to explain why only one field was chosen for the evaluation and how this choice affects the representativeness and generalizability of the results.

Reply: This is indeed an important aspect that should be addressed. We took advantage of our fieldwork when we sampled onion bulbs for the Fusarium species identification study and made the disease incidence survey (although this is not the main focus of the current work). The Givat Yoav field that was studied in the survey included inbred plants that had been vernalized and were flowering. This made it possible to easily identify the diseased dry inflorescences and conduct the survey without reducing the crop yield. In contrast, the second, Yiron's field, was seeded with hybrid yellow Riverside (Orlando) cv. and was cultivated for bulb production (so the flowering stage was denied). Consequently, identified diseased bulbs at the near season-ending (before the harvest) in 1500 plants could reduce the yield, and we decided to avoid it. Moreover, the different cultivations (for seeds or bulbs) make these two fields noncomparable.

It is important to emphasize that since there are significant knowledge gaps here, we made this preliminary assessment, knowing that this is only the first step to uncover the full extent of the disease impact in Israel's onion commercial fields. Our initial data provided here in this survey will hopefully trigger new questions and evoke a follow-up comprehensive work that will deepen our understanding of the subject. Such future studies should be dedicated to evaluating the disease severity in different commercial onion cultivars in various geographic regions, considering the pollination method and other cultivation aspects.

The following explanation was added to the Materials and Methods (lines 131-141):

"We utilized our fieldwork to sample onion bulbs for the Fusarium species identification study and conducted a disease incidence survey, although this is not the primary focus of the current work. The Givat Yoav field, which was included in the survey, consisted of inbred plants that had undergone vernalization and were in the flowering stage. This enabled us to easily identify diseased dry inflorescences and conduct the survey without reducing crop yield. In contrast, the second field, Yiron's field, was seeded with a hybrid yellow Riverside (Orlando) cultivar and was cultivated for bulb production, thus denying the flowering stage. Consequently, identifying diseased bulbs near the end of the season (before harvest) in 1500 plants could have reduced the yield, and we decided to avoid this. Moreover, the different cultivation purposes (seeds or bulbs) render these two fields noncomparable."

The following explanation was added to the Discussion (lines 585-595):

"It was shown here for the first time that the level of contamination in some fields is significantly higher than assumed and reaches 8% in certain varieties. While such a higher incidence is alarming, it may be affected by the level of inbreeding. Since onion is an outcrossing species, it may suffer inbreeding depression [33]. The different lines used in onion hybrid cultivar development are often inbred to a certain extent to ensure uniformity within the hybrid cultivar. Inbred lines are frequently weaker in their growth than hybrid or open-pollinated cultivars. Thus, the survey presented here must be followed by a more comprehensive and dedicated study to evaluate the disease severity in different commercial onion cultivars in various geographic regions, considering the pollination method and other cultivation aspects."

Line 159: The authors should explain why the specific number of 228 plates was chosen for incubation. They need to justify this number to ensure that it provides sufficient replication for statistical analysis and to account for variability within the pathogen population.

Reply: We made a series of transformations for each isolate until a pure colony was obtained. After conducting this isolation, we conclude with 228 isolates.

The following paragraphs were rewritten to explain this better (lines 181-190):

" The series of transformations for each colony to a new plate, performed each time by taking a colony agar disk from the young margins of the old colony and carefully tracking the colony morphology. Additionally, molecular verification using the ISSR and sequencing was done to ensure that a single species was obtained.

The isolates were classified according to their colony characteristics. Of these, 31 were selected according to their morphological and spores' characteristics resembling the Fusarium genus and subjected to molecular identification (PCR, gel electrophoresis, sequence determination, and homology search against the GenBank and the Fusarioid-ID databases)."

Line 279: It is essential to verify whether the assumptions of ANOVA, including normality and homogeneity of variances, were tested. Additionally, please clarify if any corrective measures were implemented to adjust for multiple comparisons.

Reply: ANOVA is based on the assumption that the data are sampled from populations that all have the same standard deviations (Gaussian distributions). Prism automatically tests this assumption with the Brown-Forsythe test and always reports the results. In most cases in our study, the Brown-Forsythe test accepted the null hypothesis (p > 0.05, the populations' standard deviations are equal). Only in the Orlando cv. seeds' epicotyl emergence, there was a small p-value (p = 0.0014), but since all other parameters of those tests (biomass in both onion varieties and epicotyl emergence in the Noam cv.) had the same standard deviations, we assumed that this p-value is due to the populations' differences.

The same goes for the bulbs' pathogenicity assay analysis, where all parameters had the same Brown-Forsythe test null hypothesis (p > 0.05), except for the necrotic severity index in the Orlando cv., where a test p = 0.0072 was obtained. Here, we also assume that this p-value is due to the population differences.

We edited the Statistical Analysis section 2.8 to reflect this: The data were analyzed using a one-way analysis of variance (ANOVA), the Brown-Forsythe test, and posterior Dunnett's test (which restricted to comparing the experimental groups against a single control group) at a significance level of p < 0.05.

Lines 286-295: Delete the first paragraph of the Results section, as the objectives of the study are already clearly stated in the Introduction section (lines 103-116).

Reply: The paragraph was deleted as advised.

Line 316: Delete "(Delia antiqua)," which already appeared on line 150.

Reply: You are right. Deleted.

Lines 329-334: There's no need to repeat the methodology in the Results section. Please delete the following paragraph to avoid redundancy in the manuscript: "Final verification was performed using a molecular test that included PCR amplification of an oligonucleotide unique to the Fusarium genus (the Fa/R8 or 7cF/11aR primers targeting the RPB1 or RPB2 – the RNA polymerase largest or 2nd largest subunit and E1/E2, targeting the Fusarium translation elongation factor-1 alpha gene). The PCR products were verified using gel electrophoresis and identified using sequencing and homology identification".

Reply: The paragraph was deleted as advised.

Overall, the manuscript provides valuable information on the incidence and control of FBR. However, it would benefit from more detail on the implications for disease management practices and future research directions to fill identified knowledge gaps.

Reply: Thank you for the positive evaluation of our manuscript. The Discussion section discussed the implications for disease management practices and future research directions. Again, we are thankful for all your comments and corrections.

Reviewer 3 Report

Line12, Line 119, Line 286-287, change ‘Fusarium Basal Rot (FBR)’ to ‘FBR’

Line 18-19, change ‘generalist’ to ‘generally’

Line 22, change ‘F. oxysporum, f.’ to ‘F. oxysporum f.’

Line 41, delete ‘fungus’

Line 42, change ‘forme specialist’ to ‘formae speciales’

Line 69, delete ‘species’

Line 76, Line 554, change ‘Fusarium’ to ‘F.’

Line 119, change ‘disease severity’ to ‘Disease Severity’

Line 120, Line 537, ‘Fusarium’ should be italicized.

Line 138, change ‘pathogens from diseased onion plants’ to ‘Pathogens from Diseased Onion Plants’

Line 162, dose ‘clean colonies’ mean ‘pure colonies’? How to obtain?

Line 166, Line 200, change ‘the gene bank’ to ‘the GenBank’

Line 167, change ‘extraction and molecular identification’ to ‘Extraction and Molecular Identification’

Line 169, change ‘according to [21’ to ‘according to the methods described previously [21’

Line 197, change ‘species and phylogenetic relationships’ to ‘Species and Phylogenetic Relationships’

Line 220, change ‘morphology and identification of the Fusarium species’ to ‘Morphology and Identification of the Fusarium Species’

Line 228, change ‘pathogenicity assay’ to ‘Pathogenicity Assay’

Line 238-240, why don’t use the seeds not treated with fungicides?

Line 248, change ‘(isolate B5, [21]).’ to ‘isolate B5 [21]).’

Line 262-263, change ‘(isolate B5, [21]).’ to ‘isolate B5 [21]).’

Line 278, change ‘confirm’ to ‘fulfill’

Line 279, change ‘analysis’ to ‘Analysis’

Line 296, change ‘disease incidence’ to ‘Disease Incidence’

Line 312, change ‘identification of the Fusarium species from the collected onions’ to ‘Identification of the Fusarium Species from the Collected Onions’

Line 318, change ‘228 fungi’ to ‘228 fungal isolates’

Line 354, delete ‘spp.’

Line 381, change ‘relationships between the Fusarium species’ to ‘Relationships between the Fusarium Species’

Line 519, what does ‘the other more specialist Fusarium species.’ mean?

Line 566, change ‘Metam sodium’ to ‘metam sodium’

Line 581, change ‘Prochloraz’ to ‘prochloraz’

Line 602, change ‘Integrated’ to ‘integrated’

References. Names of some journals are full, but some are abbreviated. Please according to the guidelines of the journal

Line 692-693, Line 694-695, Line 731-732 please provide the full information of these three references

Line 697, change ‘Associated with basal rot of Allium spp. In’ to ‘associated with basal rot of Allium spp. in’

Line 708-709, change ‘In’ to ‘in’

Line 724, change ‘Lily’ to ‘lily’

Line 725, the words ‘Fusarium proliferatum disease outburst in white onions from-’ should not be italicized.

Line 728, Line 744, change ‘f. Sp. Cepae’ to ‘f. Sp. cepae’

Line 730, delete ‘(Basel)’

Line 742, change ‘biology and evolution’ to ‘Biology and Evolution’

The quality of English language for this manuscript is good.

Author Response

Responses to Reviewer 3's comments

We would like to express our sincere appreciation to the reviewer for the essential and helpful advice. The time and effort invested are greatly appreciated and certainly contributed to and improved the manuscript. Thank you.

Detailed comments:

Line12, Line 119, Line 286-287, change ‘Fusarium Basal Rot (FBR)’ to ‘FBR’

Reply: Corrected as advised.

Line 18-19, change 'generalist' to 'generally'

Reply: The term "generalist species" in biology indicates a species able to thrive in a wide variety of environmental conditions, and that can use various resources. That is why we use this word and not 'generally.'

Line 22, change ‘F. oxysporum, f.’ to ‘F. oxysporum f.’

Reply: Corrected as advised.

Line 41, delete 'fungus'

Reply: Deleted as suggested.

Line 42, change ‘forme specialist’ to ‘formae speciales’

Reply: Right. Corrected as per your advice.

Line 69, delete 'species'

Reply: Deleted as advised.

Line 76, Line 554, change 'Fusarium' to 'F.'

Reply: Corrected as advised.

Line 119, change' disease severity' to 'Disease Severity'

Reply: Corrected as advised.

Line 120, Line 537, 'Fusarium' should be italicized.

Reply: Corrected as advised.

Line 138, change 'pathogens from diseased onion plants' to 'Pathogens from Diseased Onion Plants'

Reply: Corrected as advised.

Line 162, does 'clean colonies' mean' pure colonies'? How do you obtain it?

Reply: Corrected to pure. As already partly stated, we performed a series of transformations for each colony to a new plate, each time taking a colony agar disk from the young margins of the old colony and carefully tracking the colony morphology. Additionally, molecular verification using the ISSR and sequencing was done to ensure that a single species was obtained.

The above information was added to the text (lines 181-185).

Line 166, Line 200, change 'the gene bank' to 'the GenBank'

Reply: Corrected as advised. At lines 227-231, we rephrase the paragraph to be more accurate: "Similarity percentages between sequences against other already recognized species were determined online by the identification database, Fusarioid-ID, accessible at www.fusarium.org and the NCBI GenBank BLASTN search (National Center for Biotechnology Information, MD, USA, at: http://www.ncbi.nlm.nih.gov)."

Line 167, change 'extraction and molecular identification' to 'Extraction and Molecular Identification'

Reply: Corrected as advised.

Line 169, change 'according to [21' to 'according to the methods described previously [21'

Reply: Corrected as advised.

Line 197, change' species and phylogenetic relationships' to 'Species and Phylogenetic Relationships'

Reply: Corrected as advised.

Line 220, change 'morphology and identification of the Fusarium species' to 'Morphology and Identification of the Fusarium Species'

Reply: Corrected as advised.

Line 228, change 'pathogenicity assay' to 'Pathogenicity Assay'

Reply: Corrected as advised.

Line 238-240, why don't use the seeds not treated with fungicides?

Reply: You are right. This is a more logical choice. Still, it is hard to obtain such seeds since, according to the Israel Ministry of Agriculture and Rural Development, Plant Protection and Inspection Services regulations, all commercial seeds must be treated with general pesticide treatment (Captan) to reduce diseases. Thus, untreated seeds were unavailable.

Line 248, change ‘(isolate B5, [21]).’ to ‘isolate B5 [21]).’

Reply: Corrected as advised.

Line 262-263, change '(isolate B5, [21]).' to 'isolate B5 [21]).'

Reply: Corrected as advised.

Line 278, change 'confirm' to 'fulfill'

Reply: Changed as per your suggestion.

Line 279, change 'analysis' to 'Analysis'

Reply: Corrected as advised.

Line 296, change' disease incidence' to 'Disease Incidence'

Reply: Corrected as advised.

Line 312, change 'identification of the Fusarium species from the collected onions' to 'Identification of the Fusarium Species from the Collected Onions'

Reply: Corrected as advised.

Line 318, change ‘228 fungi’ to ‘228 fungal isolates’

Reply: Corrected as advised.

Line 354, delete 'spp.'

Reply: Corrected to: "Final verification of the Fusarium species identity...". We think it's better pronounced this way.

Line 381, change' relationships between the Fusarium species' to 'Relationships between the Fusarium Species'

Reply: Corrected as advised.

Line 519, what does 'the other more specialist Fusarium species.' mean?

Reply: Specialist species in biology are those adapted to narrow habitats, limited food resources, or other specific environmental conditions. The paragraph was corrected to explain this better: "The study revealed an interesting pattern. Neocosmospora SC appeared to be a generalist pathogens group with a weak host specialization and lesser virulence capability than the other more aggressive specialist Fusarium species (adapted to narrow host range)." (lines 530-533).

Line 566, change 'Metam sodium' to 'metam sodium'

Reply: Corrected as advised.

Line 581, change ‘Prochloraz’ to ‘prochloraz’

Reply: Corrected as advised.

Line 602, change 'Integrated' to 'integrated'

Reply: Corrected as advised.

References. Names of some journals are full, but some are abbreviated. Please, according to the guidelines of the journal

Reply: All journals' names were corrected to the abbreviated form to match the journal guidelines.

Line 692-693, Line 694-695, Line 731-732 please provide the full information of these three references.

Reply: The full information of these three references was added.

Line 697, change 'Associated with basal rot of Allium spp. In' to 'associated with basal rot of Allium spp. in'

Reply: Corrected as advised.

Line 708-709, change 'In' to 'in'

Reply: Corrected as advised.

Line 725, the words' Fusarium proliferatum disease outburst in white onions from-' should not be italicized.

Reply: Corrected as advised.

Line 728, Line 744, change 'f. Sp. Cepae' to 'f. Sp. cepae'

Reply: Corrected as advised.

Line 730, delete ‘(Basel)’

Reply: Corrected as advised.

Line 742, change 'biology and evolution' to 'Biology and Evolution'

Reply: Corrected as advised.

Reviewer 4 Report

Dear authors,

I have found your manuscript interesting and well written and I think it is basically ready for publication. I would like to suggest to add more info about how you checked for DNA quality and quantity after extraction (this is not present in the M&M section) and I also would like to ask why you decided to use 60°C as annealing temperature for the EF primer pairs, since the recommended temperature in the literature reference is 53°C for this primer pair.

Best regards,

Author Response

Responses to Reviewer 4's comments

We thank the reviewer for investing substantial effort, which undoubtedly contributed to this manuscript. The remarks and suggestions improved this paper's scientific soundness and accuracy. Your contribution is greatly appreciated.

I have found your manuscript interesting and well written and I think it is basically ready for publication.

Reply: Thank you for the positive evaluation of our manuscript.

I would like to suggest adding more info about how you checked for DNA quality and quantity after extraction (this is not present in the M&M section)

Reply: The following information was added to the text as per your advice: "The DNA concentration of purity test after extraction was done using NanoDrop™ One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) Microvolume spectrophotometric UV–vis method. The test ensures a high DNA quality (median value of 1.8 or above of absorbance ratio at wavelength 260/280 nm) and at least 30 nanogram/µL DNA." (lines 194-198).

I also would like to ask why you decided to use 60°C as annealing temperature for the EF primer pairs, since the recommended temperature in the literature reference is 53°C for this primer pair.

Reply: for the PCR, we used the commercial reaction mixture containing the VeriFi™ Hot Start Mix Red (PCR Biosystems, London, UK). We followed the manufacturer's instructions to ensure optimal conditions for the DNA polymerase: denaturation at 95°C for 1 minute, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 5 minutes (see lines 211-213).