First Occurrence of Coffee Leaf Rust Caused by Hemileia vastatrix on Coffee in Saudi Arabia

Round 1

Reviewer 1 Report (Previous Reviewer 2)

The authors have adequately addressed my comments. 

Author Response

Dear Professor,

We greatly appreciate all the critiques and comments raised from you. Those comments were extremely helpful for us to improving our paper, and they provided valuable guidance for our future study.

Yours sincerely,

Associ. Prof. Ahmed M. Ismail

Reviewer 2 Report (New Reviewer)

The entitled with the manuscript “First Report of Coffee Leaf Rust Caused by Hemileia vastatrix on Coffee in Saudi Arabia” was identified rust pathogen by morphologically and molecularly. This is a case study and the manuscript after the corrected is hugely improved. 

The changes made in the abstract section are sufficient and generally reflect the content of the article. On the other hand, keywords should be insufficient and rewritten as coffee, leaf rust, molecular analysis.

The introduction section is sufficient

Materials and Methods section is sufficient

Please re-arranged the figure 2 and the figures are also overlapped, they need to be renumbered. Please also correct it in the text.

Discussion section is sufficient

Line 136: "Koch's postulates" technique should be written in more detail and literature information should be given.

Line 137: “ ….. C. arabica seedlings…” It must be clearly stated which species and variety is used here. State clearly that this genotype is susceptible to the disease. If not, the article should be rejected.

Line 146: After how many days and according to which scale the disease evaluation was performed. Explain by referring to the literature.

Line 186-187: For the text, make a "citation for figures 1c and 1d".

Lİne 208: All "for figure 2 letters" must be cited in the text.

Line 228-272. Since the figure 3 is incomplete, no evaluation can be made.

Line 324: Please cite the literature to support this claim. Please cite the literature.

Line 325: Please cite the literature to support this claim. Please cite the literature.

Line 326: Please cite the literature to support this claim. Please cite the literature.

Line 327: Please cite the literature to support this claim. Please cite the literature.

Line 335-336: “….  migrated to Saudi Arabia from neighboring African countries  ….” Wheat rust diseases are known to follow such a migration path (Yr9 and Ug99 rust movement in wheat). You can support your idea with wheat rust.

Result part; Provide clear information about the results of the survey.

Discuss the "Survey results".

Discuss the results of "Phylogenetic Analysis".

Literature part; The first letters of the working name must be either always lower case or always upper case. Rewrite according to the journal rules.

Literature part; Journal names should always be shortened or spelled out. Rewrite according to the journal rules.

Literature part; Either always italicize the journal names or write them plain. Let's rewrite according to journal rules.

Line 11-12 and 273-277 and in the title section; high similarity has been identified and should be rewritten. Especially "Keith, L. M., Sugiyama, L. S., Brill, E., Adams, B. L., Fukada, M., Hoffman, K. M., ... & Melzer, M. J. (2022). First report of coffee leaf rust caused by Hemileia vastatrix on coffee (Coffea arabica) in Hawaii. Plant Disease, 106(2), 761." The similarity between the name and the article should be specifically minimised.

Comments for author File: Comments.pdf

Minor editing of English language required

Author Response

Response to Reviewer 2 Comments

Dear Professor,

We greatly appreciate all the critiques and comments from you. Those comments are extremely helpful for us to improving our paper, and they provide valuable guidance for our future study. According to these comments, we have carefully improved our manuscript.

We have made improvments in throughout the whole manuscript as recommended, and these revisions made to the manuscript were marked up using the “Track Changes” function to be easily viewed by the editors and reviewers.

Hereby please find the response for all the raised comments by the reviewer:

Comments and Suggestions for Authors:

The entitled with the manuscript “First Report of Coffee Leaf Rust Caused by Hemileia vastatrix on Coffee in Saudi Arabia” was identified rust pathogen by morphologically and molecularly. This is a case study and the manuscript after the corrected is hugely improved. 

The changes made in the abstract section are sufficient and generally reflect the content of the article. On the other hand, keywords should be insufficient and rewritten as coffee, leaf rust, molecular analysis.

The introduction section is sufficient

Materials and Methods section is sufficient

Please re-arranged the figure 2 and the figures are also overlapped, they need to be renumbered. Please also correct it in the text.

Discussion section is sufficient

Point 1:

Line 136: "Koch's postulates" technique should be written in more detail and literature information should be given.

Response 1:

Koch's postulates" technique is a method of which the potential pathogen associated with observed symptoms reinoculated to the same host to reproduce the same symptoms. The pathogenicity methodology is explained well in this section and the refereed literature is mentioned.  

Point 2:

Line 137: “ ….. C. arabica seedlings…” It must be clearly stated which species and variety is used here. State clearly that this genotype is susceptible to the disease. If not, the article should be rejected.

Response 2:

We have used the local variety “khawlani”, this variety is the main and widely cultivated across the production areas of Jazan region.

Point 3:

Line 146: After how many days and according to which scale the disease evaluation was performed. Explain by referring to the literature.

Response 3:

The incidence of H. vastatrix was calculated after 30 days using the scale established by Julca-Otiniano et al. (2019).

Point 4: Line 186-187: For the text, make a "citation for figures 1c and 1d"

Response 4: citation has done

Point 5: Lİne 208: All "for figure 2 letters" must be cited in the text.

Response 5: citation has done

Point 6: Line 228-272. Since the figure 3 is incomplete, no evaluation can be made.

Response 6: the figure was moved overlapped between pages

Point 7: Line 324: Please cite the literature to support this claim. Please cite the literature.

Line 325: Please cite the literature to support this claim. Please cite the literature.

Line 326: Please cite the literature to support this claim. Please cite the literature.

Line 327: Please cite the literature to support this claim. Please cite the literature.

Response 7: literature cited in the mentioned in the highlighted lines.

Point 8: Line 335-336: “….  migrated to Saudi Arabia from neighboring African countries  ….” Wheat rust diseases are known to follow such a migration path (Yr9 and Ug99 rust movement in wheat). You can support your idea with wheat rust.

Response 8: We supported our idea with the suggested subject.

Point 9: Result part; Provide clear information about the results of the survey.

Response 9: The survey results have been explained in details.

Point 10:: Discuss the "Survey results".

Response 10: Survey results have discussed in the first part of discussion

Point 11: Discuss the results of "Phylogenetic Analysis".

Response 11: Phylogenetic Analysis results have discussed

Point 12: Literature part; The first letters of the working name must be either always lower case or always upper case. Rewrite according to the journal rules.

Response 12: The literature section have improved according to the journal rules

Point 13: Literature part; Journal names should always be shortened or spelled out. Rewrite according to the journal rules.

Response 13: The journal names have improved according to the journal rules

Point 14: Literature part; Either always italicize the journal names or write them plain. Let's rewrite according to journal rules.

Response 14: The journal names have improved according to the journal rules

Point 15: Line 11-12 and 273-277 and in the title section; high similarity has been identified and should be rewritten. Especially "Keith, L. M., Sugiyama, L. S., Brill, E., Adams, B. L., Fukada, M., Hoffman, K. M., ... & Melzer, M. J. (2022). First report of coffee leaf rust caused by Hemileia vastatrix on coffee (Coffea arabica) in Hawaii. Plant Disease, 106(2), 761." The similarity between the name and the article should be specifically minimised.

Response 15: This comment has been taken in consideration and the title has modified to minimize the similarity.

Point 16: Comments on the Quality of English Language

Minor editing of English language required

Response 16: English language editing has been don throughout the whole manuscript.

Point 17: Comment in line 49 ;

Response 17: The old cited reference is removed as there is no updated information available regarded this statement.

Point 18: Comment in line 53, farmers often combine organic goat manure with stone mulching in coffee production, Is this statement true?

Response 18: Yes, this statement true based on the personal interviews with many farmers as many farms are applying the organic cultural practices in terms of fertilization.

Point 19: Comment in line 93, Its is appropriate to state which specialist made the diagnosis.

Response 19: I have done the morphological examination and molecular characterization (Ahmed Mahmoud Ismail).

All the rest comments and corrections suggested in the whole manuscript have been taken in consideration and marked up using the “Track Changes” function to be easily viewed by the editors and reviewers.

Yours sincerely,

Associ. Prof. Ahmed M. Ismail

Round 2

Reviewer 2 Report (New Reviewer)

Acceptance of the article is favourable. However, some errors I have identified need to be corrected.

I wish you success in your work.

Comments for author File: Comments.pdf

Minor editing of English language required

Author Response

Dear Professor,

We again greatly appreciate all the critiques and comments from you. Those comments are extremely helpful for improving our paper, and they provide valuable guidance for our future study. According to raised minor comments, we have carefully improved our manuscript.

We have made improvments in throughout the whole manuscript as recommended, and these revisions made to the manuscript were marked up using the “Track Changes” function to be easily viewed by the editors and reviewers.

We would like to thank you for your critical revision of the whole manuscript and we really appreciate your efforts and time for improving the quality of our work. 

On behave of all authors 

My Regards

Prof. Ahmed M. Ismail 

Author Response File: Author Response.pdf

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

The reviewer has commented the suggestion for the authors in the PDF file uploaded. Other suggestion are listed below.

1. More genes and species can be analyzed in molecular phylogenetic research. Closely related species of Hemileia vastatrix are necessary. Are there any multi-gene phylogenetic analyses for Hemileia vastatrix or Hemileia?

2. Invasiveness, morphological similarities and differences for different sampling of Hemileia vastatrix from different areas can be compared and discussed.

3. The authors noted that distribution/occurrence of Hemileia vastatrix in Saudi Arabia may cause by a migration from Africans. But the Hemileia vastatrix report from adjacent African countries are absent in this manuscript. If there are no Hemileia vastatrix report in North Africa, it should be noted in the manuscript. The reviewer suggests the morphological and multi-gene phylogenetic analyses are beneficial to clarify this point.

Comments for author File: Comments.pdf

 English writing of this manuscript can be improved.

Author Response

Dear Professor,

We greatly appreciate all the critiques and comments raised from you. Those comments were extremely helpful for us to improving our paper, and they provided valuable guidance for our future study. According to these comments, we have carefully improved our manuscript,

We have taken in consideration all the raised comments by the reviewer and made improvments in throughout the whole manuscript as recommended, and these revisions made to the manuscript were marked up using the “Track Changes” function to be easily viewed by the editors and reviewers.

Yours sincerely,

Associ. Prof. Ahmed M. Ismail

Author Response File: Author Response.docx

Reviewer 2 Report

The authors provide a well-documented report of the presence of H. vastatrix in Saudi Arabia.  If the herbarium mentioned is not an herbarium indexed by Index herbariorum, I suggest they also deposit voucher specimens in an Index Hebariorum listed herbarium.  Below are several suggested edits, which should be compared carefully to the original document, as in many cases I have just removed or inserted one word to make the English flow better. 

Abstract:  (Suggested changes to wording): Coffee leaf rust (CLR) is a disease caused by the biotrophic fungus Hemileia vastatrix.  The Jazan region in Saudi Arabia has long been one of the last coffee-producing regions in the world free of this disease.  In August 2023, CLR was initially observed in coffee farms in Jazan and may have expanded beyond the initial observation.  The source of infection is unknown, however, CLR could have entered Jazan from the nearest coffee-producing countries that have the pathogen.  During a scheduled survey in August 2023, symptoms were observed including yellowish-orange lesions that frequently combined to form chlorotic lesions and exhibited the powdery appearance of yellow uredospores on the abaxial surface of leaves…..

Line 22: The pathogenicity of H. vastatrix specimens…

Line 24: Based on our findings this is the first documentation of H. vastatrix causing CLR in Saudi Arabia.

Line 29: One of the five largest families of flowering plants, the Rubiaceae comprises three subfamilies, 620 genera and 13000 species, including coffee plants. Arabica (Coffea arabica L.) and Robusta (Coffea canephora L. Linden) are the two main types of coffee grown in the world.

Line 32:  I’m not certain the line “It is crucial to understand that…” is strictly necessary in this paper.  Does this have bearing on the first report of a pathogen?

Line 43: The authors are understandably proud of the coffee produced in their country, but I don’t think the references cited support the subjective statement about the quality of Saudi Arabian coffee.  Suggested wording: “Coffee in Saudi Arabia is primarily grown using organic cultivation methods without the use of synthetic pesticides, herbicides or fertilizers. (4,5) Farmers frequently incorporate organic goat dung with stone mulching (6).  

Line 60: Despite recently published papers (12-14) which reported for the first time the association of certain fungi with diseases on coffee in Saudi Arabia, little….

Line 62: In the Jazan region, coffee farmers observed rust-like symptoms on several coffee trees with many trees heavily defoliated.  Upon the request of the farmers, a scheduled survey was conducted…

Line 66: This study used morphological and molecular approaches including phylogenetic analyses to identify the rust fungus causing disease on coffee trees in Jazan.

Line 75: KFUH?  Please list the herbarium code.

Line 80:  The 18S-ITS1-5.8S-ITS2-28S sequence generated by ITS1 and ITS4 is generally considered one gene region, not three.

Line 85: The PCR amplification conditions were as follows:

Line 94: Not certain what “multi sequence alignment” is referred to here as “concatenated”.  Usually if several gene regions are used (TUB, EF, ACT) then creating an alignment with all of the gene regions is “concatenated”.  Here, it looks like only the ITS region was aligned, which wouldn’t equal concatenation.

Line 95:  The taxonomic relationship of the Hemileia vastatrix isolates was investigated through phylogenetic analysis of the ITS gene region. 

Line 119: Plants were place in a dark humid chamber…

Line 125: ….lesions with varying degrees of severity, and on the abaxial surface exhibited the powdery appearance of yellow uredospores (Figure 1a,b). Uredospores were ovoid to reniform, convex on the dorsal side, with a smooth hyaline to pale yellow-orange wall on the ventral side, finely to coarsely echinulate, measuring ….

Line 155: …( c) close focus on upper lesions with tissue necrosis

Line 170: Uredospores of H. vastatrix. 

Line 174: The identity of H. vastatrix specimens collected in this study was further confirmed based on ITS sequencing, with generated sequences sharing 99-100% identity with publicly available sequences identified as H. vastatrix. The results of a phylogenetic analysis based on the ITS region for our six specimens and sequences retrieved from GenBank (Table 1) is illustrated in Figure 2.  

Line 178:  (The location of the sequences on the tree within the clade that is full of nearly identical sequences is probably not particularly significant and may be a result of the alignment.)

Line 243: The pathogenicity of the…

Line 254: Coffee farmers across the different regions of southwest Saudi Arabia face several challenges to their agricultural output. 

Line 257: The results presented here provide new information about the identification and documentation of the presence of H. vastatrix on coffee in Saudi Arabia.

Line 260: ….Jazan region and surrounding areas. 

Line 263: In this study, the ITS sequence data was sufficient to support the identification of six specimens of H. vastatrix.  (Remove the rest beginning “approving….”  In this case, the ITS region was sufficient, but that’s not true for many major groups of fungi and this is too broad a statement to make based solely on this paper). 

Line 279: Coffee has been cultivated in the south-western region of Saudi Arabia for several centuries, mostly through seed propagation. 

Line 283: …distribution of the resistant coffee cultivars…

Line 284: The latter is due to the coffee plantation replacement plan implemented by the Saudi government to fight and replace invasive Khat (qat) trees (Catha edulis) with coffee trees.  The population structure of H. vastatrix in Saudi Arabia has not yet been investigated and this study is the first to report the occurrence of this pathogen on coffee in Saudi Arabia.  

Adequate, specific suggestions provided. 

Author Response

Dear Professor,

We greatly appreciate all the critiques and comments raised from you. Those comments were extremely helpful for us to improving our paper, and they provided valuable guidance for our future study. According to these comments, we have carefully improved our manuscript,

We have taken in consideration all the raised comments by the reviewer and made improvments in throughout the whole manuscript as recommended, and these revisions made to the manuscript were marked up using the “Track Changes” function to be easily viewed by the editors and reviewers.

Yours sincerely,

Associ. Prof. Ahmed M. Ismail

Author Response File: Author Response.docx