Dry Rot Caused by the Complex Colletotrichum falcatum and Thielaviopsis paradoxa Emerges as a Key Stalk Disorder in Newly Expanded Sugarcane Plantations from Northwestern São Paulo, Brazil

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The MS presented the occurrence of the three stem-related disorders in commercial sugarcane files, and constructed multiple regression model to see which disorders affect the total productivity of sugarcane most. Furthermore, the pathogens that causes the red rot and stem dry rot were identified by the morphology of the fugal hyphae and their spore. The investigation has good practical guiding significance for farmers in front-line production of sugarcane in Brazil, especially under background that Brazil is the leading player, exporter and the World’s largest sugarcane. However, the MS should be improved from several aspects, such as writing, need further language polish; Additional experiments: to do 18 rDNA sequencing of the identified fungi, and give a direct and more accurate identification of these pathogens that cause red rot and stem dry rot. I recommend the MS be accepted after a major revision.

 

1.     Language should be polished by a professional expert in the field. Such as the first sentence of abstract, “This study presents results from research on the incidence of three stem-related disorders 16 in commercial sugarcane fields in six municipalities in northwest São Paulo and the associated 17 productivity losses”. This sentence is very long, and appeared three “in” to restrict the stem-related disorders.

 

2.     In the line 23 of the abstract, it said that “…the three stem-related disorders…”, however, the reader did not know the third one besides the mentioned stem dry rot, and the red rot. Hence, I think it most to detailed descript it, and figure out which is the third disorders here. Because this is the abstract, more clearly and summarized of the content, the reader will understand it more clearly.

 

3.     Additional experiment should be included in identification of the pathogens. Using the 18S rDNA sequencing of the identified fungi, and to construct the phylogenetic trees according to the sequences.

 

4.     In results part, especially the part of pathogens’ identification (Figure 3 and Figure 4), just two sentences’ description, and directly give the results. However, I think there may have two pathogens could cause the dry rot. Because there are two different type spores from the bottom picture of Fig. 4, one is ellipsoid, and another one is cylindrical. How to explain it? 

Comments on the Quality of English Language

Extensive editing of English language required

Author Response

Our response to Reviewer 1:

We appreciate your encouragement and suggestions for improving the manuscript submitted to Agronomy. We considered all our suggestions by incorporating the changes needed, which can be detected in the version of the manuscript with track-changes. 

Now we approach each of our individual considerations:

1.     Language should be polished by a professional expert in the field. Such as the first sentence of the abstract, “This study presents results from research on the incidence of three stem-related disorders  in commercial sugarcane fields in six municipalities in northwest São Paulo and the associated productivity losses”. This sentence is very long, and appeared three “in” to restrict the stem-related disorders.

  Please, apologize for not presenting our original manuscript in a more polished scientific English fashion. Though I am not a native speaker, I thoroughly reviewed the English writing of the entire text to ensure that presentation and readability in formal English is restored to the manuscript.

2.     In the line 23 of the abstract, it said that “…the three stem-related disorders…”, however, the reader did not know the third one besides the mentioned stem dry rot, and the red rot. Hence, I think it is best to describe all of them in detail, so we can figure out which are the three disorders here. Because this is the abstract, more clearly and summarized of the content, the reader will understand it more clearly.

 The abstract has been fixed to include the description of all three stalk disorders in detail, and the missing contents added so the manuscript is now complete. The manuscript now has an introduction, a description of the pathosystem, a phrase describing the importance of the study, the hypothesis testing, the main results and a brief discussion phrase. Guided by the new abstract content, we also described all three disorders in the main text, using photographs and relevant literature.

3.     Additional experiments should be included in identification of the pathogens. Using the 18S rDNA sequencing of the identified fungi, and to construct the phylogenetic trees according to the sequences.

 Unfortunately we were unable to provide this information, since the 3,400 samples surveyed in this study have been destroyed years ago. Though the study did not include phylogenetics to answer our hypothesis since we were focused on determining the prevalence of the three stalk related disorders based on phenotypic characters, we still included an important topic on isolation and identification of the pathogens associated with the internal dry rot disease complex. This identification was based on micro-morphological characters, including optical and electron microscopy, as presented in the text. A more thorough study that would include phylogenetic analyses would be helpful to test hypotheses on species diversity. But this should be a topic for another study.

4.     In the results part, especially the part of pathogens’ identification (Figure 3 and Figure 4), just two sentences’ description, and directly give the results. However, I think there may be two pathogens that could cause dry rot. Because there are two different types of spores from the bottom picture of Fig. 4, one is ellipsoid, and another one is cylindrical. How to explain it?

This is an excellent consideration. First of all, we apologize for the poor presentation of the information contained in those figures. Please check the newly constructed Figure 5 and the more detailed description of the two pathogens identified: Colletotrichum falcatum and Thielaviopsis paradoxa. About you concern of the two different spores detected in T. paradoxa, these two types of spores are commonly found in this fungal species. We also added the following paragraph describing the fungus species: 

"Concerning the micro-morphological characterization of T. paradoxa isolated from dry rot symptomatic stalks, the fungal strains produced long and unbranched phialides, measuring 73.0–180.5 × 6.0–9.0 μm (Figure 5 D). Two distinct types of conidia were identified: the primary conidia, hyaline, produced in chains, cylindrical, and unicellular, measuring 2.0–4.0 × 6.0–10.0 μm (Figure 5 D, F); and the aleurioconidia, which turn brown upon maturation, are produced holoblastically, either singly or in short chains, ovoid to subglobose in shape, measuring 3.0-4.0 × 8.0-10 μm (Figures 5D, 4E, and 4F). Perithecia were not observed." 

 We conclude the description with the following paragraph:

The morphological characteristics observed for C. falcatum strains aligned with those formerly described by Costa et al. [1] and Hossain et al. [2]. Similarly, the specific characteristics of T. paradoxa strains resembled those in the description of the pathogen in the CABI Compendium [3]. 

Literature: 

1. Costa, M.M.; Silva, B.A.A.S.; Moreira, G.M.; Pfenning, L.H. Colletotrichum Falcatum and Fusarium Species Induce Symptoms of Red Rot in Sugarcane in Brazil. Plant Pathol. 2021, 70, 1807–1818, doi:10.1111/ppa.13423.
2. Hossain, M.I.; Ahmad, K.; Vadamalai, G.; Siddiqui, Y.; Saad, N.; Ahmed, O.H.; Hata, E.M.; Adzmi, F.; Rashed, O.; Rahman, M.Z.; et al. Phylogenetic Analysis and Genetic Diversity of Colletotrichum Falcatum Isolates Causing Sugarcane Red Rot Disease in Bangladesh. Biology 2021, 10, 862, doi:10.3390/biology10090862.
3. Center for Agriculture and Bioscience International (CABI) Ceratocystis Paradoxa (Black Rot of Pineapple). 2022, 12157, doi:10.1079/cabicompendium.12157.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript of Tatiane Carla Silva and co-authors is devoted to study of the incidence of three stem-related disorders in commercial sugarcane fields in northwest São Paulo. In my opinion, this work is not a scientific study, but an analysis of a specific situation in commercial sugarcane fields, an analysis of sugarcane plant diseases and crop loss. It is unclear from the manuscript what new the authors established and what is the scientific significance of the results obtained? In addition, the manuscript is carelessly formatted and contains a number of errors, inconsistencies, etc.

 

The main comments

Title

Article title is too long.

Abstract

The abstract does not contain the main conclusion to understand the significance of the main results obtained.

Introduction

The manuscript hypothesis (lines 112-11) does not contain a scientific problem requiring research.

Materials and methods

- 2.1. Figure 5 contains different information than indicated in the text (lines 130-133).

- 2.3. This section begins with a strange phrase “In data shared by the sugar mill, dry rot of sugarcane stalk was considered…”.

The methodology for preparing plant material for analysis for possible phytopathogens (fungi, bacteria, viruses) is unclear (treatment with soap, alcohol, sodium hypochlorite, plating on agar with antibiotic). Did the authors try to find only a specific phytopathogen, without taking into account the possibility of other diseases?

There is no description of scanning microscopy, which is shown in Figures 3 and 4.

Figure 4 legend describes the BDA medium, which is not included in the materials and methods.

How many plants were analyzed to determine phytopathogens.

Results

-The results mainly describe the yield loss analysis. Description of the definition of phytopathogens that cause diseases is 2 lines (329-330).

- There is no way to understand what is in Fig. 2. The caption on the Figure and figure legend (grey) does not match.

-Figures 3 and 4 legends are not accurately described. Figure 3 also contains scanning microscopy.

- There is no figure from plants damaged by the pest. How did the authors establish that this pest was the cause?

- However, what about other plant pathogens? Were they not discovered at all?

Discussion

-This section as a whole does not contain much discussion of its own results.

- Lines 388-392. This is not shown in Figure 3C

- Lines 397-399. It is unclear how these species and the diseases they cause differ.

- Lines 412-414. It's not written in English.

Conclusion

 

This section should contain the main conclusions from your own results.

Author Response

Our response to Reviewer 2:

We appreciate your encouragement and suggestions for improving the manuscript submitted to Agronomy. We considered all our suggestions by incorporating the changes needed, which can be detected in the version of the manuscript with track-changes. 

Now we approach each of our individual considerations:

The manuscript of Tatiane Carla Silva and co-authors is devoted to study of the incidence of three stem-related disorders in commercial sugarcane fields in northwest São Paulo. In my opinion, this work is not a scientific study, but an analysis of a specific situation in commercial sugarcane fields, an analysis of sugarcane plant diseases and crop loss. It is unclear from the manuscript what new the authors established and what is the scientific significance of the results obtained? In addition, the manuscript is carelessly formatted and contains a number of errors, inconsistencies: We apologize for allowing this manuscript submission with the careless formatting and inconsistencies pointed out by the reviewer. A major revision has now been made in the hopes of attending the reviewers expectations. We have approached the reviewer's concern about the relevance of the scientific problem tackled in our study in the item Introduction, below.

The main comments

Title

Article title is too long. : The title was shortened to: "Dry rot caused by the complex Colletotrichum falcatum and Thielaviopsis paradoxa emerges amongst the key stalk disorders in newly expanded sugarcane plantations from northwestern São Paulo, Brazil

Abstract

The abstract does not contain the main conclusion to understand the significance of the main results obtained: We fixed the entire manuscript following suggestions from Reviewer 1. To attend the suggestion from Reviewer 2 we added the following paragraph: "We concluded that the stalk internal dry rot, as a disease complex associated with both C. falcatum and T. paradoxa, was the most important disorder in sugarcane fields from the northeastern São Paulo region. A sustainable pest management program is needed to reduce the impact of all three stalk-associated disorders on the regional sugarcane production."

Introduction

The manuscript hypothesis (lines 112-11) does not contain a scientific problem requiring research: Our hypothesis was described in the following paragraph: "We hypothesized that the stalk red rot disease is the major stalk related disorder for the recently established sugarcane commercial fields in northeastern São Paulo, due to its higher incidence, widely distribution and the high yield losses caused." We respect the point of view of the Reviewer 2, but have to disagree with him/her. Since the stalk related disorders are considered an important problem for the local sugarcane industries in northeastern São Paulo, surveying their regional field distribution, prevalence and yield impact across 34 fields in 6 distinct counties, was the way of addressing the relevant scientific hypothesis. Distinctly from applying an experimental design with a limited number of factors, we applied extensive field sampling of healthy and diseased / injured plants for crop loss assessment to guide decision making in pest management (using ecological principles discussed by L. V. Madden and G. Hughes: https://apsjournals.apsnet.org/doi/pdf/10.1094/PHYTO.1999.89.11.1088, and E. A. Ampt and collaborators: https://link.springer.com/article/10.1007/s10658-018-1573-x )

Materials and methods

- 2.1. Figure 5 contains different information than indicated in the text (lines 130-133). We checked lines  130-133 and Figure 3 for consistency. Thanks for your observation.

- 2.3. This section begins with a strange phrase “In data shared by the sugar mill, dry rot of sugarcane stalk was considered…”. Thanks for your observation on this item. This item is now 2.4, and it begins with the following sentence:

Based on information  shared by the regional sugar mills, the stalk internal dry rot disorder has been associated with the simultaneous infection by C. falcatum and T. paradoxa (Usina Santa Adélia Sugarcane Mills, personal information). To confirm this a priori assumption,, we aimed to assert  the etiology  of the stalk internal dry rot disorder by field surveying symptomatic stalks. 

The methodology for preparing plant material for analysis for possible phytopathogens (fungi, bacteria, viruses) is unclear (treatment with soap, alcohol, sodium hypochlorite, plating on agar with antibiotic). Following your consideration, we specified in more detail the methodology for preparing the plant material for pathogens isolations. Please refer to item 2.4.

Did the authors try to find only a specific phytopathogen, without taking into account the possibility of other diseases? No, in our isolation, by adopting PDA as a non-selective culture medium, our goal was to allow other pathogens to grow, not only C. falcatum or T. paradoxa. However, these two pathogens were the only one isolated from the infected sugarcane stalks.

There is no description of scanning microscopy, which is shown in Figures 3 and 4. Following your consideration and suggestion we included the description of electron microscopy in the M&M of the revised manuscript, which reads as follows:

For SEM, 5 mm diameter samples of plug colonies with fungal sporulation were collected and fixed in 70% formalin acetic alcohol (FAA) [48] and stored under refrigeration. The fixed samples were dehydrated in ethanol series treatment (at 70, 80, 90 and 99.5%), dried at critical points and metallized with gold [49]. The images were acquired using a Zeiss EVO/LS15 Scanning Electron Microscope at the Chemistry–Physics Department (UNESP, Ilha Solteira Campus).  

Figure 4 legend describes the BDA medium, which is not included in the materials and methods: The medium was, in fact, PDA, which is described in the M&M. BDA is the Portuguese form of the same medium. We apologize for that mistake too.

How many plants were analyzed to determine phytopathogens: From the total number of infected stalks, we analyzed 100 plants and conducted proper fungal isolations.

Results

-The results mainly describe the yield loss analysis. Description of the definition of phytopathogens that cause diseases is 2 lines (329-330): We corrected this mistake of oversimplifying the description of the pathogens, including the additional item 3.4 with a more thorough description. Now this item reads as this:

"By the indirect isolation technique, we detected the simultaneous infection of the pathogens C. falcatum and T. paradoxa in 99% of sugarcane stalks with dry rot symptoms, which confirmed the a priori assumption based on information shared by the Usina Santa Adélia Sugarcane Mills."

Regarding the micro-morphological characterization of C. falcatum isolated from the infected stalks (Figure 5 A-C), the fungal strains had setae that were septate, dark brown in color, and rounded at the apex, measuring between 75.5 and 110 μm (Figure 5 B). The conidia were either sickle-shaped or spindle-shaped, hyaline, and aseptate, with 15.5-25 × 5.0 μm (Figures 5 A and C). Appressoria and perithecia were not observed. 

Concerning the micro-morphological characterization of T. paradoxa isolated from dry rot symptomatic stalks, the fungal strains produced long and unbranched phialides, measuring 73.0–180.5 × 6.0–9.0 μm (Figure 5 D). Two distinct types of conidia were identified: the primary conidia, hyaline, produced in chains, cylindrical, and unicellular, measuring 2.0–4.0 × 6.0–10.0 μm (Figure 5 D, F); and the aleurioconidia, which turn brown upon maturation, are produced holoblastically, either singly or in short chains, ovoid to subglobose in shape, measuring 3.0-4.0 × 8.0-10 μm (Figures 5D, 4E, and 4F). Perithecia were not observed.

The morphological characteristics observed for C. falcatum strains aligned with those formerly described by Costa et al. [22] and Hossain et al. [50]. Similarly, the specific characteristics of T. paradoxa strains resembled those in the description of the pathogen in the CABI Compendium [28]."

The new Figure 5 describing the pathogen's micromorphology was also built to attend the Reviewers' recommendations.

- There is no way to understand what is in Fig. 2. The caption on the Figure and figure legend (grey) does not match: Figure 2 has been checked and its legend and capture match. We added the following missing capture: Means followed by the same letter are not significantly different by the Scott-Knott test at p ≤ 0.05 

-Figures 3 and 4 legends are not accurately described. Figure 3 also contains scanning microscopy:

Following your suggestion, we rebuilt these figures, merging them into a single one (Figure 5), containing only the optical and the electron photomicrographs depicting the fungal micromorphology.

- There is no figure from plants damaged by the pest. How did the authors establish that this pest was the cause?: We constructed and incorporated a new Figure 1 depicting the stalk red rot, the pineapple sett rot, the internal dry rot (from A to E) and, following your suggestion, we included details of the pest Mahanarva fimbriolata, with the following description, from F to J: "Pre-sprouted sugarcane seedlings infested with the spittlebug M. fimbriolata, with a soap foam- like exudation on the roots, which is typically produced by the insect pest nymphs (F). Damaged sugarcane stalks,  become thinner, presenting cracks and deterioration due to the spittlebug attack (G). The straw left in the field, as a result of mechanized harvesting of green sugarcane, harbors M. fimbriolata and keeps the populations of the spittlebug high (H). Spittlebug adult stage close to  a massive foam exudation (I) and on a sugarcane leaf (J)." The presence of the foam-like exudation or the presence of nymphs and adults of M. fimbriolata were used as criteria to determine the association of the spittlebug with the respective disorder. 

- However, what about other plant pathogens? Were they not discovered at all? Yes, we were not able to recover other pathogens other than C. falcatum and T. paradoxa in our sampling.

Discussion

-This section as a whole does not contain much discussion of its own results.

Following your suggestions, we have completely restructured the discussion section of the manuscript to get it more focused on our own results and to propose a sustainable pest management program as good practices [54] to reduce the impact of all three stalk-associated disorders on the regional sugarcane production. We apologize for the presentation of the former version of the discussion, which was indeed not very focused at all and even contained information written in Portuguese. 

- Lines 388-392. This is not shown in Figure 3C. This phrase was removed from the discussion.

- Lines 397-399. It is unclear how these species and the diseases they cause differ. Following your suggestions, we restructured this paragraph, which is now presented at the end of the discussion section and reads as follows:

Finally, for a more effective management of sugarcane red rot, we should highlight the need for additional studies to reveal a more complex etiology and species diversity of the pathogens associated with both the red rot and the pineapple rot in a broader survey across traditional and newly expanded sugarcane cropping areas from São Paulo State. Three species of Colletotrichum (C. falcatum, C. plurivorum and C. siamense) [20,22] and also three species of Fusarium (F. sacchari, F. proliferatum, and F. madaense) [22] have already been associated with the sugarcane red rot disease in plantations from Alagoas (Northeastern Brazil), Minas Gerais (Southeastern Brazil), or Paraná state (Southern Brazil). Though C. falcatum has been considered the major species and the most aggressive on sugarcane stalks and leaves  [20,22], Fusarium can also cause the typical red rot symptoms, either alone or in co-infection with Colletotrichum [22]. In contrast, the Fusarium species also induced symptoms of pokkah boeng, which was not caused by any of the Colletotrichum species [22]. In addition, C. plurivorum, whose sexual stage was observed on the surface of sugarcane stems, did not induce stalk rot or leaf symptoms  [22]. 

- Lines 412-414. It's not written in English. Please accept our sincere apologies for that mistake. 

Conclusion

This section should contain the main conclusions from your own results: The Conclusion section was rewritten and reads as follows now:

The red rot disease caused by C. falcatum,, the internal dry rot disease complex associated with both C. falcatum and T. paradoxa, and the spittlebug-induced budding were major stalk disorders with widespread distribution in sugarcane fields from northwestern São Paulo, occurring in 88 to 92% of the 34 fields surveyed, and causing significant yield losses. On average, the yield losses associated with these three stalk disorders were 14.2 (± 3.8) tons.ha-1, which represented 20.1 (± 5.2) % of the total estimated sugarcane yield.  The disorder with the highest level of incidence was the stalk internal dry rot, with an average of 19.5% (95% C.I. = 3.7%), followed by the spittlebug-induced budding [7.4% (95% C.I. = 2.4%)] and the red rot disease [5.8% (95% C.I. = 2.4%)], with similar incidences. A sustainable pest management program  is needed to reduce the impact of all three stalk-associated disorders on the regional sugarcane production.

Reviewer 3 Report

Comments and Suggestions for Authors

1.      First of all, the title is too complicated and wordy. It should be seriously shortened.

2.      The current abstract is unacceptable. At first glance, the abstract lacked many important details. For instance, the background and methods are missing, and the objectives are not well declared. Because of these lacks, the results are not well interpreted and what the study contributed or what can be studied based on the current research are missing.

3.      The introduction should be well prepared. In this current version, the introduction took too much space for the sugarcane in Brazil, which should be summarized in a single paragraph. However, some important details are missing, such as the symptoms of the diseases, or their mechanisms, etc. Last but not least, the citations in the introduction are not acceptable. Citations should be at the end of a sentence not a paragraph, and there are many arguments that should have been cited. Please redesigned with 3 paragraphs including sugarcane, C. falcatum, T. paradoxa

4.      In the materials and methods, the segmentation of this section is not clear. Therefore, the whole process and the ways to conduct experiment of the study are difficult to understand. The identification should be referred at the molecular level, so in this case, “detection” is more preferable. If there was a DNA sequencing or PCR technique applied, it should have been described here.

5.      Because of the lack of coherence in the materials and methods, the evaluation of the results is not possible at the current version. Moreover, without molecular techniques, how could the pathogens be identified at species level? The results should divide into subheadings.

6.      The discussion encounters the same issue as the introduction. The section is too fragmented, and contains many uncited arguments. At some points, it is similar to a review rather than a regular research study.

7.      Additionally, there are too many run-on sentences along with poor grammar, spelling and vocabulary which make the manuscript lowly readable. A sentence in Portuguese can be found in lines 412-414, I assume that the manuscript has not been well prepared and reviewed before submission. Furthermore, in my opinion, a 2017-2018 survey is not up to date enough.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Our response to Reviewer 3:

We appreciate your encouragement and suggestions for improving the manuscript submitted to Agronomy. We considered all our suggestions by incorporating the changes requested which can be detected in the version of the manuscript with track-changes. 

Now we approach each of our individual considerations:

1. First of all, the title is too complicated and wordy. It should be seriously shortened: 

We shortened the title as suggested. The title was shortened to: "Dry rot caused by the complex Colletotrichum falcatum and Thielaviopsis paradoxa emerges amongst the key stalk disorders in newly expanded sugarcane plantations from northwestern São Paulo, Brazil

2.  The current abstract is unacceptable. At first glance, the abstract lacked many important details. For instance, the background and methods are missing, and the objectives are not well declared. Because of these lacks, the results are not well interpreted and what the study contributed or what can be studied based on the current research are missing: 

Following yours and Reviewer 1 suggestions, the abstract has been fixed to include the description of all three stalk disorders in detail, and the missing contents added so the manuscript is now complete. The manuscript now has an introduction, a description of the pathosystem, a phrase describing the importance of the study, the hypothesis testing, the main results and a brief discussion phrase. Guided by the new abstract content, we also described all three disorders in the main text, using photographs and relevant literature.

3. The introduction should be well prepared. In this current version, the introduction took too much space for the sugarcane in Brazil, which should be summarized in a single paragraph. However, some important details are missing, such as the symptoms of the diseases, or their mechanisms, etc. Last but not least, the citations in the introduction are not acceptable. Citations should be at the end of a sentence not a paragraph, and there are many arguments that should have been cited. Please redesign with three paragraphs including sugarcane, C. falcatum, T. paradoxa.

Following your suggestion, we completely restructured the Introduction section, included the symptoms of the diseases and insect pest disorder, a newly constructed Figure 1 to illustrate all three stalk disorders focused on the study, and most importantly, we thoroughly checked every assertion at the sentence level and included the corresponding citation. We apologize for the inconveniently formatted citation in the original version of the manuscript. They are now professionally cited using a Zotero library. In general, the Introduction section is now fully redesigned following a more clear line of thinking.

4. In the materials and methods, the segmentation of this section is not clear. Therefore, the whole process and the ways to conduct experiments of the study are difficult to understand. The identification should be referred to at the molecular level, so in this case, “detection” is more preferable. If there was a DNA sequencing of PCR technique applied, it should have been described here. 

Following your suggestion, we better segmented the M&M section and used the same sequence of items in the results section for clarity. We have not applied DNA sequencing or PCR techniques because of the extensive sampling conducted (a total of 3,400 sugarcane stalks examined). 

5. Because of the lack of coherence in the materials and methods, the evaluation of the results is not possible at the current version. Moreover, without molecular techniques, how could the pathogens be identified at species level? The results should be divided into subheadings.

Following your suggestion, we better segmented the results section into subheadings  using the same structure from M&M items, for clarity. We used phenotypic characterization, based on micromorphological characteristics (using both optical and electron microscopy) to identify the pathogens.

6. The discussion encounters the same issue as the introduction. The section is too fragmented, and contains many uncited arguments. At some points, it is similar to a review rather than a regular research study.

Following yours and Reviewer 2' suggestions, we have completely restructured the discussion section of the manuscript to get it more focused on our own results and to propose a sustainable pest management program to reduce the impact of all three stalk-associated disorders on the regional sugarcane production. We apologize for the presentation of the former version of the discussion, which was indeed not very focused at all and even contained information written in Portuguese. 

7. Additionally, there are too many run-on sentences along with poor grammar, spelling and vocabulary which make the manuscript lowly readable. A sentence in Portuguese can be found in lines 412-414, I assume that the manuscript has not been well prepared and reviewed before submission. Furthermore, in my opinion, a 2017-2018 survey is not up to date enough.

We apologize for the poor presentation of the former version of the manuscript, which was indeed not very focused at all and even contained information written in Portuguese. The current version has been scrutinized for accuracy, readability and flow of thoughts. We hope that we have now attended your expectation.

About your last comment: Although the 2017/18 sampling might not sound up to date, it represents a complete cycle of staggering sugarcane planting that began in 2011, that probably culminated in the highest incidence of all three stalk disorders. For the particular reason of revealing the relative importance of stalk associated pathogens and insect pests that survive or overwinter in crop residues accumulated over the year, this sampling year was very important. After 2018, all the 34 fields have been completely  renewed, after one year crop rotation with soybeans or  snap beans cropping.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Minor editing of English language required

Comments on the Quality of English Language

Minor editing of English language required

Author Response

Thank you for the prompt assessment of the revised version of our manuscript. We understood that there was no concern about the content of the former version of the manuscript and that the minor English editing suggested will be assessed by MDPI Agronomy upon acceptance. 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors certainly did a great job on the manuscript, tried to take into account and correct all the comments made. But for me personally, the scientific significance of the work remained unclear. The fact that the observed fungi and insects most often cause these stalked-related disorders is described in the abstract and introduction section. Plus, the authors received preliminary information from the regional sugar mills, as they themselves described. Is there scientific significance in the frequency of plant infestation by each of the three pathogens studied in commercial fields?

Some comments

-Figure 1 is included in the Introduction section. Therefore, it is difficult to conclude whether it is part of previously published data or the result of the research carried out in this work.

-There are no control healthy plants in Figure 1.

-The authors conclude that “C. falcatum,, the internal dry rot disease complex associated with both C. falcatum and T. paradoxa, and the spittlebug-induced budding were major stalk disorders”. But as they themselves claim, they did not find any other pathogens at all. During plant analysis they used 70% alcohol and 2% sodium hypochlorite, and the sowing was done on a medium with an antibiotic tetracycline intended for the growth of fungi. So it was at least: Identification of fungal pathogens associated with sugarcane stalk internal dry rot

 

-What is it “weak-PDA”?

Author Response

          We appreciate your suggestions, in this second round of review,  for improving the manuscript submitted to Agronomy. They have been incorporated in the final version of the manuscript, and can be easily checked since they are shaded in yellow along the text.

           Now we approach each of our individual considerations:

The authors certainly did a great job on the manuscript, tried to take into account and correct all the comments made. But for me personally, the scientific significance of the work remained unclear. [Because of] the fact that the observed fungi and insects most often cause these stalked-related disorders is described in the abstract and introduction section. Plus, the authors received preliminary information from the regional sugar mills, as they themselves described [their occurrence]. Is there scientific significance in the frequency of plant infestation by each of the three pathogens studied in commercial fields? 

         We understood the point of view of the Reviewer 2, which was expressed in the first round of review and properly answered. Now, this point of view was presented as a question about the scientific significance of the study. Since the stalk related disorders were considered/hypothesized as important crop health problem for the local sugarcane industries from northeastern São Paulo, the scientific significance of the study resided in the extensive surveying of their regional field distribution, their prevalence and the corresponding yield impact across 34 fields in 6 distinct counties in this new frontier for sugarcane expansion.  Distinctly from applying an experimental design with a limited number of factors, we applied extensive field sampling of healthy and diseased / injured plants for crop loss assessment. This way, our study is very significant as it revealed the extent of the damage caused by these stalk disorders and the negative impact on sugar yield, which can guide decision making in pest management both regionally and probably in other locations with similar conducive environments. 

Some [other] comments:

• Figure 1 is included in the Introduction section. Therefore, it is difficult to conclude whether it is part of previously published data or the result of the research carried out in this work.

         We included Figure 1 in the Introduction as an informative and helpful tool to describe, in detail, the three stalk disorders, using images captured during the execution of this current research. None of these images are from previous studies.

• There are no control healthy plants in Figure 1.

          Following your suggestion, we edited Figure 1 to include an image (F) containing both a healthy and a diseased / injured stalk. 

• The authors conclude that “C. falcatum, the internal dry rot disease complex associated with both C. falcatum and T. paradoxa, and the spittlebug-induced budding were major stalk disorders”. But as they themselves claim, they did not find any other pathogens at all. During plant analysis they used 70% alcohol and 2% sodium hypochlorite, and the sowing was done on a medium with an antibiotic tetracycline intended for the growth of fungi. So it was at least: Identification of fungal pathogens associated with sugarcane stalk internal dry rot.

          That is a correct interpretation from Reviewer 2.

• What is “weak-PDA”?

         That is a lower strength PDA medium, made with ⅕ of the potato dextrose content in the full PDA medium. We fixed the term in the manuscript replacing the term "weak-PDA" with low strength PDA.

Reviewer 3 Report

Comments and Suggestions for Authors

1.      After the first revision, the quality of the manuscript has been significantly improved. However, there are some small typos that need to be addressed before publication:

a.       Some scientific names are not italicized.

b.      A paragraph should not be separated by a figure and its caption. See lines 90-124.

c.       Some letters need to be superscripted.

d.      And some other typos.

2.      Moreover, sampling locations along with their general environmental conditions should be clearly indicated.

3.      In addition, for “identification”, a molecular method should be used. However, there was none. Therefore, I suggest correcting the term into “Detection”.

Comments on the Quality of English Language

Minor editing of English language required

Author Response

         Thank you for the prompt assessment of the revised version of our manuscript. Now we approached each of our individual considerations:

1. After the first revision, the quality of the manuscript has been significantly improved. However, there are some small typos that need to be addressed before publication.

         a. Some scientific names are not italicized: We fixed the scientific names that were not italicized, as indicated.

          b.  paragraph should not be separated by a figure and its caption. See lines 90-124: We also fixed this issue, by keeping this paragraph altogether and before the Figure 1 and its caption.

          c. Some letters need to be superscripted: We also searched and fixed the letters that were not superscripted.

           d. And some other typos: We tried to identify as many typos as possible and hope to have corrected them all.

      2. Moreover, sampling locations along with their general environmental conditions should be clearly indicated.

          Following your suggestion we added the following information: "The region’s existing vegetation is very fragmented and impoverished from an original interior Atlantic Forest (seasonal semideciduous forest) in transition to Cerrado (savanna) lowlands.  The average altitude is 347 m. Sugarcane plantation currently occupies 31% of the area, while natural vegetation represents only 5% (according to MapBiomas, https://brasil.mapbiomas.org/estatisticas/)."

3. In addition, for “identification”, a molecular method should be used. However, there was none. Therefore, I suggest correcting the term into “Detection”:

         Following your suggestion we replace the term identification with detection.