Plant Protection Products to Control Alternaria Brown Spot Caused by Alternaria alternata in Citrus: A Systematic Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is well structured, identifies and presents the clear questions, has comprehensive literature, and follows the basic rules of systematic review. The study focused on Alternaria Brown Spot (ABS) in citrus. For ABS control, authors concentrate on summarizing the (1) Substances and their effectiveness, (2) Methods to test the substances, and (3) Reasons for failure or hindrance in horizontal resistance of ABS.

However, no disease occurrence and control mechanism at the molecular level is presented. It would be better to add the mechanism of disease establishment, including the subcellular and the genome level. How the resistance in resistant genotypes work? How many genes are reported and their pathway to control the ABS in resistant genotypes?   

In the methodology section:

L93: The criteria to select GRP3 and GRP4 should be discussed. The advantage of studying these groups over others may be clear.

L100: In Figure 1, “reading for overview” may be rephrased as “to take an overview” or else

L103: Group 3: bio-control may need deeper insight. There may be a deeper subclassification of Bio-control.   

In the Results section:

L 170: If the selected research didn’t relate to the topic, then how did they search, and what were the criteria to relate / reject?

L172: “One of the most numerous reports was group 8”. Nonetheless, it seems neglected in the manuscript. However, it should be elaborated and discussed in detail.

L184: Heading 3.3 is still methodology, not results

In the Discussion section:

L227: 4.1: The heading “natural substances to control ABS” is not representative of the text under it.

L236: “The higher the concentration, the greater the effect.”, This statement may be acceptable to a specific limit, after which it may be considered a toxic level or over-use of the substance. Hence, an optimum range should be discussed.

L237: 4.1.1: MGA was frequently used, and VCA was rarely reported. Hence, here should discuss the advantages of VCA. Why the author of the VCA-based study preferred it in comparison to well-reputed techniques.

The answer regarding “Reasons of failure or hindrance in horizontal resistance of ABS” is hard to find against various methods and chemicals. It would be better to elaborate clearly and suggest the possible solutions.

L575: It seems the “summary” rather than the “conclusion”.  In conclusion, instead of repeating the results, it’s suggested to report the best control of ABS, the best method to control ABS, and the major causes of failure to control ABS at the field level.

 

Author Response

Reviewer #1:

Comments and Suggestions for Authors

Reviewer: The manuscript is well structured, identifies and presents the clear questions, has comprehensive literature, and follows the basic rules of systematic review. The study focused on Alternaria Brown Spot (ABS) in citrus. For ABS control, authors concentrate on summarizing the (1) Substances and their effectiveness, (2) Methods to test the substances, and (3) Reasons for failure or hindrance in horizontal resistance of ABS.

Authors: We truthfully thank the reviewer’s 1comments as these suggestions have clearly improved the manuscript. We have very carefully reviewed it after considering each comment. A point-by-point response to every requested change is provided below. We have followed all the suggestions to correct the manuscript.

Comments 1: However, no disease occurrence and control mechanism at the molecular level is presented. It would be better to add the mechanism of disease establishment, including the subcellular and the genome level. How the resistance in resistant genotypes work? How many genes are reported and their pathway to control the ABS in resistant genotypes?  

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have included the information suggested by the reviewer. However, we do not want to distract from the main objective of this review. Perhaps the title of the review was too broad. This systematic review focuses on substances used in the laboratory and field against ABS. A more specific title may help avoid confusion. We have changed: Substances to control Alternaria Brown Spot caused by Alternaria alternata in Citrus: A systematic review

It should be noted that the bibliography referring to “mechanism of disease establishment” and “resistant genotypes” is very extensive and would require a review itself. For example, as detailed in the present review (Figure 1), group 6: Metabolic pathways (53 reports) and group 8: Molecular characterization and diversity (63 reports), have a total of 116 reports, which would imply a systematic review only to address these topics.  These two groups have not been full text reviewed at this point, because it would have meant an excessively extensive and diverse study. However, they are clearly very interesting topics. Therefore, we have included some information but not a large amount of information regarding these topics, since this review is focused on natural substances and fungicides that have shown effectiveness against A. alternata.

In the introduction: (page 2, line 54)

Alternaria spp. have specific cells known as appressoria, which play an important role in recognizing the host through certain hydrophobic materials released from the host surface [9,10]. The pathogenicity of Alternaria alternata in citrus primarily depends on the biosynthesis of the host-selective ACT toxin, which is released during conidial germination [11]. This toxin compromises the integrity of host cell plasma membranes, ultimately leading to cell death, and subsequently spreads through the vascular system [12,13]. In addition to toxin production, the fungus secretes cell wall-degrading enzymes (CWDEs), which are also critical for successful infection of citrus tissues (Ma et al., 2019). Within necrotic citrus tissue, A. alternata must detoxify reactive oxygen species (ROS) to survive in the oxidative environment and establish successful colonization (Wu, 2020).

In the results section: (page 17, line 604)

4.3. Breeding for disease-resistant

Beyond chemical and technical management strategies, breeding for disease-resistant citrus varieties offers also a promising approach to control Alternaria brown spot (ABS). This strategy requires a systematic review by itself due to the large number of reports, which was not the focus of this review. The main information during the classification phase was that proteomic analyses comparing susceptible and resistant citrus genotypes have revealed differential protein expression following infection. Resistant varieties exhibit elevated levels of proteins involved in ROS metabolism and immune responses, suggesting these factors play a key role in resistance mechanisms (Santos Dória et al., 2019). Furthermore, susceptibility to A. alternata has been associated with mitochondrial RNA processing. Disruptions in this process can sensitize citrus plants to the ACT toxin, exacerbating disease symptoms (Ohtani et al., 2002).

Comments 2: L93: The criteria to select GRP3 and GRP4 should be discussed. The advantage of studying these groups over others may be clear.

Response 2: Thank you for your suggestion. The criteria are explained in section 2.3. Step 3: Eligibility criteria for full-text review. Information has been added to this section to better clarify the selection criteria. As we have mentioned before, groups 6 and 8 are very broad and would require a systematic review for each. (page 4, line 116)

 

Comments 3: L100: In Figure 1, “reading for overview” may be rephrased as “to take an overview” or else

Response 3: Done. We've changed Figure 1. Thanks for the correction (page 4, line 110).

 

Comments 4: L103: Group 3: bio-control may need deeper insight. There may be a deeper subclassification of Bio-control.  

Response 4: Thank you for your suggestions. We have changed the title of the group to: Microorganisms and Natural Substances. When we analyzed this group of reports, we realized that they studied natural substances and the effect of some microorganisms against the pathogen. In these studies, we could compare the effectiveness of substances and microorganisms in controlling the growth of Alternaria alternata. The title "biocontrol" is excessively broad and includes techniques for which we did not find information. Furthermore, in the results, we have separated "microorganisms" from "natural substances." (page 6, line 203)

(In the Results section)

Comments 5: L 170: If the selected research didn’t relate to the topic, then how did they search, and what were the criteria to relate / reject?

Response 5: Dear reviewer, we understand your concerns and hope to be able to clarify them. The reports were initially included using a broad literature search with the search strategy: “Alternaria alternata” AND “Citrus”, detailed in the section “2.1. Step 1: Information sources and search strategy”. Systematic reviews often begin with a broad search (so as not to leave out potential studies), and then the studies containing the evidence to answer the previously posed questions are classified.  Therefore, some articles corresponded to different crops but sometimes included the word "citrus" or were not related to the main topic of the review (conferences with no outcome of interest; reports focused on post-harvest disease and not on ABS, wrong species, …). This is the usual process for systematic reviews.

These details are provided in the Materials and Methods section:

2.1. Step 1: Information sources and search strategy

2.3. Step 3: Eligibility criteria for full-text review

We have improved section 2.3 to make it clearer.

 

Comments 6: L172: “One of the most numerous reports was group 8”. Nonetheless, it seems neglected in the manuscript. However, it should be elaborated and discussed in detail.

Response 6: It is impossible to address all groups, as the review would be excessively long and inconsistent for comparing results (much of the work involves comparing the effectiveness of different substances against A. alternata. Combining this with information on genetic breeding can tricky). To better clarify this point, the title has been modified, and more details on genetic breeding have been added to the introduction, discussion and conclusions.

 

Comments 7: L184: Heading 3.3 is still methodology, not results

Response 7: You're right. Thanks for the suggestion. We've removed the section as it's fully described in Materials and Methods section 2.3. (page 4, line 196).

 

In the Discussion section:

Comments 8: L227: 4.1: The heading “natural substances to control ABS” is not representative of the text under it.

Response 8: Thank you for your suggestion. Therefore, we have changed the heading to “Microorganisms and natural substances to control ABS” (page 8, line 239).

 

Comments 9: L236: “The higher the concentration, the greater the effect.”, This statement may be acceptable to a specific limit, after which it may be considered a toxic level or over-use of the substance. Hence, an optimum range should be discussed.

Response 9: You're right. We've cut the sentence to avoid confusion. When talking about each substance, the ranges are specified and compared (page 8, line 248).

 

Comments 10: L237: 4.1.1: MGA was frequently used, and VCA was rarely reported. Hence, here should discuss the advantages of VCA. Why the author of the VCA-based study preferred it in comparison to well-reputed techniques.

Response 10: Thank you for pointing this out. We agree with this comment. Therefore, we have added a sentence to the paragraph to clarify this (page 8, line 262).

 

Comments 11: The answer regarding “Reasons of failure or hindrance in horizontal resistance of ABS” is hard to find against various methods and chemicals. It would be better to elaborate clearly and suggest the possible solutions. L575: It seems the “summary” rather than the “conclusion”.  In conclusion, instead of repeating the results, it’s suggested to report the best control of ABS, the best method to control ABS, and the major causes of failure to control ABS at the field level.

 

Response 11: Thank you for your suggestions. We greatly appreciate the suggestions for improving the conclusions. Therefore, the conclusions have been completely rewritten for greater clarity. “Reasons of failure at field level” have been specified in the conclusions. (page 17, line 616).

 

Greetings
Hugo Merle

Reviewer 2 Report

Comments and Suggestions for Authors

Detail comments:

 

1. The title is need to rewrite as "Control of Alternaria Brown Spot (Alternaria alternata (Fr.) Keissler) in Citrus: to "Control of Alternaria Brown Spot caused by Alternaria alternata in Citrus:

 

2. Rewrite the aim of study in briefly

 

3. Line 20:

What is this meant "ABS"? must be explain it

 

4. Rewrite the keywords includes Alternaria alternata; Citrus; systematic review; natural substances; Alternaria Brown Spot; Nova; Leanri;

 

5. What is the difference between  section 4.2 and 4.2.2? the authors must change the title of section.

 

6. The discussion is unclear and ambiguously, as well as, the authors wrote about "Microorganisms to control ABS" very briefly and mentioned to "microorganisms can be dangerous" but about fungicides are very widely without mentioned to their dangerous  

 

7. "4.1.2. Biocontrol and natural substances to control ABS":

This section is need to separate among subjects

 

8. The conclusion is a very long and poorly, and must rewrite in one paragraph, in briefly, and what are you concluded from your study?

 

 

Author Response

Reviewer #2:

Comments and Suggestions for Authors

Authors: We sincerely appreciate the comments and suggestions from reviewer # 2. We have modified the manuscript based on these suggestions and we believe they have served to improve the manuscript.

 

Comments 1: The title is need to rewrite as "Control of Alternaria Brown Spot (Alternaria alternata (Fr.) Keissler) in Citrus: to "Control of Alternaria Brown Spot caused by Alternaria alternata in Citrus:

Response 1: Thank you for pointing this out. We agree with this comment. Therefore, we have changed the title according to the reviewer's suggestion (page 1, lines 2 and 3).

 

Comments 2: Rewrite the aim of study in briefly

Response 2: Thanks for the suggestion. We've shortened the wording of the aim of study as suggested (page 1, lines 19-23; and page 2, lines 80-84).

 

Comments 3: Line 20: What is this meant "ABS"? must be explain it

Response 3: Agree. We have, accordingly, define the acronym clearly the first time it appears in the abstract: Alternaria Brown Spot (ABS) is one of the most critical … (page 1, Line 15).

 

Comments 4: Rewrite the keywords includes Alternaria alternata; Citrus; systematic review; natural substances; Alternaria Brown Spot; Nova; Leanri;

Response 4: Done. We've changed the keywords based on the reviewer's suggestion. Thanks for the correction (page 1, line 34-35).

 

Comments 5: What is the difference between  section 4.2 and 4.2.2? the authors must change the title of section. 

Response 5: Thank you for pointing this out. We have changed the title of section according to the reviewer's suggestion. The new title is: Detail of fungicide groups to control ABS (page 12, line 428).

 

Comments 6: The discussion is unclear and ambiguously, as well as, the authors wrote about "Microorganisms to control ABS" very briefly and mentioned to "microorganisms can be dangerous" but about fungicides are very widely without mentioned to their dangerous 

Response 6: Agree. We have, accordingly, improved the manuscript. We have removed the word "dangerous" because as the reviewer comments, any new treatment (fungicide, natural or microorganisms) can be dangerous, and all its effects must be evaluated. (page 9, lines 305-307). The length of this section is in accordance with the number of articles found on this topic. Of the 98 reports analyzed, only 9 focused on the use of microorganisms against A. alternata, and none of them included field experiments. We have also added a consideration of the possible harmful effects of fungicides on the ecosystem (page 16, line 567-569).

Comments 7: "4.1.2. Biocontrol and natural substances to control ABS": This section is need to separate among subjects.

Response 7: Thank you for your comment. We've separated the sections based on your suggestions. Microorganisms now have their own section. We've modified the rest of the section to reflect on this new structure. 4.1.2. Microorganisms to control ABS; 4.1.3. Natural substances to control ABS (page 9 and 10).

 

Comments 8: The conclusion is a very long and poorly, and must rewrite in one paragraph, in briefly, and what are you concluded from your study?

Response 8: Thank you for your suggestions. You are correct. We have rewritten the conclusions following your instructions.

Greetings
Hugo Merle

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Rewrite all keywords

 

Author Response

Thank you for your suggestions.

Rewrite all keywords

We've modified the keywords based on your corrections.

We've removed the repetitive words in the title and added a few more.

We've kept the names of some varieties we consider important.