Integrated Pathogen Management in Stevia Using Anaerobic Soil Disinfestation Combined with Different Fungicide Programs in USA, Mexico, and Paraguay
, Guillermo A. Enciso-Maldonado 2, Marco Maidana-Ojeda 2, Jose F. Diaz-Najera 3, Sergio Ayvar-Serna 3, Lindsey D. Thiessen 1 and H. David Shew 1Round 1
Reviewer 1 Report
Laboratory and field studies were conducted on the efficacy of ASD with cornmeal or wheat carbon sources or fungicide applications to control stem rot and Septoria leaf spot of stevia and evaluate cost-effectiveness. Overall, all objectives were met in the study, appropriate statistical analyses were conducted, and presented with an excellent discussion.
Introduction
Ln 83: Water field capacity should be corrected as field capacity
Ln 172: into de soilà into the soil
Ln 186: Please provide more information (material and manufacturer) about the black plastic mulch. Is it an O2 impermeable film?
Figures
Figures 3 and 4: Legend 2019-22020 should be corrected as 2019-2020.
References
Please make sure to scientific names are correct in the reference list. E.g. reference 71 should be corrected as follows.
71. Rogers, L.W.; Koehler, A.M. Nondestructive Sampling to Monitor Macrophomina phaseolina Root Colonization in 796 Overwintering Stevia . Plant Heal. Prog. 2021, XX, 1–3, doi:10.1094/php-10-20-0092-br.
Author Response
Response to Reviewer 1 Comments
Comments and Suggestions for Authors
Laboratory and field studies were conducted on the efficacy of ASD with cornmeal or wheat carbon sources or fungicide applications to control stem rot and Septoria leaf spot of stevia and evaluate cost-effectiveness. Overall, all objectives were met in the study, appropriate statistical analyses were conducted, and presented with an excellent discussion.
Response: We wanted to take a moment to express our sincere appreciation for your excellent review of our work. Your insights and comments were truly invaluable in helping us to improve the quality and clarity of this manuscript. We included a track changes document so you can verify the changes made ( agronomy-2320012_Track _changes) and a clean version with all the changes accepted (agronomy-2320012_All_changes_acepted). Please use the second for comments and suggestions.
Introduction
Ln 83: Water field capacity should be corrected as field capacity
Response: We changed to field capacity through the document. Changes were highlighted in the document.
Ln 172: into de soilà into the soil
Response: We changed as suggested. Changes were highlighted in the document.
2.4. Effect of carbon sources incorporated into the soil on stevia stem rot incidence
Ln 186: Please provide more information (material and manufacturer) about the black plastic mulch. Is it an O2 impermeable film?
Response: We changed as suggested. Changes were highlighted in the document.
Immediately after the incorporation of carbon sources, microplots were flooded with water to field capacity and covered with black plastic mulch (polyethylene selective reflecting mulch 1.0 mm. thick; Berry Global©, Charlotte, USA) ensuring that the borders were completely sealed while the rest of the plots were left uncovered.
Figures
Figures 3 and 4: Legend 2019-22020 should be corrected as 2019-2020.
Response: We changed as suggested. Changes were highlighted in the document.
References
Please make sure to scientific names are correct in the reference list. E.g. reference 71 should be corrected as follows.
- Rogers, L.W.; Koehler, A.M. Nondestructive Sampling to Monitor Macrophomina phaseolina Root Colonization in 796 Overwintering Stevia . Plant Heal. Prog. 2021, XX, 1–3, doi:10.1094/php-10-20-0092-br.
Response: We changed as suggested. Changes were highlighted in the document.
Author Response File: 
Author Response.docx
Reviewer 2 Report
General Comment
A large number of lab and field experiments were conducted in three different countries. This study will provide useful information for further understanding of the response of ASDs to different soil climates. However, the author's description of the experimental data in the results section is too verbose and unreadable. In addition, a large number of abbreviations are used in the abstract as well as in the other sections, which makes it difficult for readers to read quickly.
Special Comment
1. L2 For the first time IPM is mentioned in the title, the full name should be written.
2. A conclusion (take home message) should be added at the end of Abstract section.
3. L137-139 Describe how many grams of carbon sources are added to 65 grams of soil? Description “2 kg of carbon source m-2 of soil” is not accuracy, because it depends on thickness and bulk density of soil.
4. L121 and L126 delete the unnecessary segment number “2.2.1.:” and “2.2.2.”
5. L141 write the full name of PAD as it appears first time.
6. L149 descripte how to measure the area.
7. L157 Table 1, Add the necessary information on soil properties such like the content of clay, silt and sand, soil bulk density, SOC, pH etc.
8. L381 What does “with in a rate 10.1 Mg ha-1 and” mean?
9. L394 Add “on” between two words “research and carbon”.
10. In the Discussion section, only Table 3 (L510), Table 2 (L589), Figs 3 and 4 (L573) are included. Please reference to other results.
11. To make it easier for readers, add 2-3 subheadings to the discussion section
Author Response
Response to Reviewer 2
Comments and Suggestions for Authors
A large number of lab and field experiments were conducted in three different countries. This study will provide useful information for further understanding of the response of ASDs to different soil climates. However, the author's description of the experimental data in the results section is too verbose and unreadable. In addition, a large number of abbreviations are used in the abstract as well as in the other sections, which makes it difficult for readers to read quickly.
Response: We would like to express our gratitude for the time and effort you dedicated to reviewing our work. Your thoughtful and constructive feedback has been incredibly valuable in improving the quality and clarity of our manuscript. Thanks to your insightful comments and suggestions, we were able to correct the writing and make it more precise and comprehensible. Additionally, we included a table of abbreviations, which we believe will enhance the readability of the document and make it easier for readers to follow along. Thank you once again for your excellent review. We included a track changes document so you can verify the changes made ( agronomy-2320012_Track _changes) and a clean version with all the changes accepted (agronomy-2320012_All_changes_acepted). Please use the second for comments and suggestions.
- L2 For the first time IPM is mentioned in the title, the full name should be written.
Response: We changed the title as suggested.
Integrated pathogen management in stevia using anaerobic soil disinfestation combined with different fungicide programs in USA, Mexico, and Paraguay
- A conclusion (take home message) should be added at the end of Abstract section.
Response: We added a conclusion at the end of Abstract section as suggested. Changes were highlighted in the document.
- L137-139 Describe how many grams of carbon sources are added to 65 grams of soil? Description “2 kg of carbon source m-2 of soil” is not accuracy, because it depends on thickness and bulk density of soil.
Response: The calculation description was added to Figure S1 in the supplemental material section.
- L121 and L126 delete the unnecessary segment number “2.2.1.:” and “2.2.2.”
Response: We changed as suggested. Changes were highlighted in the document.
- L141 write the full name of PAD as it appears first time.
Response: PDA was was spelled out in Ln 117
- L149 descripte how to measure the area.
Response: We changed as suggested and added a new reference.
The mycelial growth of S. rolfsii was measured as the area of the colony in cm2 using the ImageJ program [30].
Schneider, C.A.; Rasband, W.S.; Eliceiri, K.W. NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 2012, 9, 671–675.
- L157 Table 1, Add the necessary information on soil properties such like the content of clay, silt and sand, soil bulk density, SOC, pH etc.
Response: The decription of soil properties was added to Table S1 in the supplemental material section.
- L381 What does “with in a rate 10.1 Mg ha-1 and” mean?
Response: The typo was corrected and changes were highlighted in the document.
ASD = plastic covered saturated amended at 10.1 Mg ha-1 and cornmeal at 20.2 Mg ha-1.
- L394 Add “on” between two words “research and carbon”.
Response: The typo was corrected and changes were highlighted in the document.
“but it was effective in small areas suggesting that there is potential for its application for soilborne pathogen management but more research on carbon source type and mechanisms of action are needed”.
- In the Discussion section, only Table 3 (L510), Table 2 (L589), Figs 3 and 4 (L573) are included. Please reference other results.
Response: We changed as suggested and added a new paragraphs to the Discussion as following.
Ln 500 -516 discusses results from table 2, a new discusion was added.
Ln 519- to 574 discusses results from table 3
Ln 576- to 583 discusses results from figure 1, a new discusion was added.
Ln 584- to 589 discusses results from table 4 , a new discusion was added
Ln 590- to 620 discusses results from Figure 2 , 3 and 4
Ln 621- to 635 discusses results for supplementary tables.
- To make it easier for readers, add 2-3 subheadings to the discussion section.
Response: We changed as suggested and added new subheadings to the discussion section.
4.1. Inhibitory effect of fungistatic volatile compounds produced during ASD.
4.2. Impact of ASD on sclerotia, disease reduction, and weed control in microplots.
4.3. Effectiveness of integrating ASD and fungicide programs under field conditions.
Author Response File: Author Response.docx
Reviewer 3 Report
General comments:
The manuscript by Sanabria-Velazquez reports on the evaluation of ASD, and different fungicides, for the control of Septoria leaf spot and stem rot in the production of stevia. In general, the work describes a well-conceived study and its novelty lies in the fact that the field component was conducted at multiple sites representing multiple nations. One element of the manuscript that requires considerable attention is the unsuitable sentence structure encountered throughout the document. As an example (lines 487-488) the sentence reads: "...it was explored alternative pesticides for their use in stevia disease managment..."
This should read: "alternative pesticides were examined for their use in stevia disease managment..."
This is just one example and there are a vast number of instances in the text where this needs to be addressed. Thus, significant editing of the manuscript should be required prior to its acceptance.
Specific comments:
Lines 81-82: This is an incomplete statement. There are additional non-volatile metabolites that contribute to pathogen suppression.
Lines 85-87: ASD is associated with dramatic sequential changes in the soil microbiome that require coordinated activity of specific components of the altered microbiome. In addition, both bacterial and fungal elements of the ASD generated microbiome are not only to be required to yield the disease-suppressive active metabolome but themselves are capable of direct pathogen suppression. Therefore, integrating ASD with specific fungicides needs to assess what impacts such a treatment could have on this functional microbiome.
For instanace ASD is know to increase abundance of Trichoderma spp. (e.g T. asperellum and T. harzianum which parasitize the sclerotia of S. rolfsii) and various yeasts which can produce hydrocarbons that are antifungal in action. (the authors actually note this in the discussion)
The authors should recognize the potential negative influence of their fungicide treatments on the capacity of the ASD generated microbiome to function in disease suppression.
Line 97: This statement needs to be substantiated by the use of published citations of examples where such has been documented. The utility of botanical fungicides and soil amendments for the control of plant diseases has been evaluated over multiple decades. In large part, the efficacy of such methods has proven insufficient to warrant their use. By no means are these methods "new sustainable and efficacious" and the authors should edit this statement accordingly.
Line 115: How was inoculum prepared?
Line 121: How was the PA prepared? Provide some detail or specifically identify a citation that documents how it was formulated and its preparation.
Lines 126-130: This should be stated that 'fungicides containing azoxystrobin as the active ingredient were used in these trials'...you can then subsequently state the specific commercial formulations that were utilized.
Line 133: delete the first “was” in this sentence.
Line 138: State that a “Petri” plate was used.
Line 143: State that electrical tape was use “to minimize gaseous exchange with the external atmosphere”. The electrical tape will not completely exclude this process.
Line 151: How long were these monitored for regrowth and under what conditions were the fungi grown?
Line 155: Table 1: This table needs to be moved to a position in the text adjacent to the description of the study sites. What were the attributes of these soils? pH? Nutrient contents? Organic matter content? Etc.???
Lines 180-182: This description is confusing. The sentence appears to state that soils were infested with 350 g stevia debris colonized by S. rolfsii prior to carbon amendment. How was this inoculum source prepared? Just randomly obtained material from the field? Was this 350 g of material applied to each plot/block? How big were the plots and how was the material added to the soil? It is then stated that packets containing 10 sclerotia of the pathogen were added to each 'microplot'. There is no description of these microplots. What were their size? How many per treatment? A statement needs to be made at the beginning of this section that pathogen infestation utilized two methods of introduction into the soils.
Line 202: Weed biomass is the standard metric used in such assessments. The "number" of weeds is not appropriate as plant size among species is so dramatically variable.
Line 209: What was the organic matter employed for fertility? What were the NPK contents of this material?
Line 222: In the table 2 footnotes it is stated that molasses was applied at 20.2 Mg/ha...which value is correct. In addition, based upon the data in table 2 it would appear that the total carbon inputs for the combined C treatments (molasses + cornmeal or wheat bran) each treatment would have been 30 or 40 Mg/ha...is this correct? This value is greater than that the rate commonly utilized in such ASD trials. In addition, this would result in a significantly greater carbon input than when any of these carbon inputs were used independently. There is no mention of the potential impact of this greater C input when contrasting the single C input ASD treatments and the combined C input treatments. The authors need to clearly address this difference and possible impact on the findings both in the results and discussion.
Line 233: replace the word “After” with “Beginning at…”
Lines 245-246: How many plants were evaluated? At what intervals were these evaluations conducted? Was the same person conducting the evaluation at all time points? Visual assessments of this type can be less than accurate when multiple individuals with differing views of "percent disease" are employed in these evaluations.
Line 270: Indicate that mycelial growth was monitored in PDA media.
Line 274: These data are listed in the adjoining table and there is no need to repeat these values in the text...simply make the statement that there were no differences among these treatments. This needs to be repeated at many other points in the results section…some but not all of which I will point out.
Table 2: What were the rates for the combined C inputs? Was the cornmeal or wheat + molasses a combined rate of 30 or 40 Mg/ha? Please clarify.
Lines 286-300: Again, much of these data are directly provided in the table, there is no need to repeat these values in the text, simply state there were no differences among the ASD carbon treatments.
Table 3: What is % sclerotia? Is this % germination? If so, this needs to be stated in the table footnotes.
Lines 312-314: Again, why are you repeating the data in the text when they are clearly stated in table 3?
Lines 319-327: Continually identifying treatments as "plots amendeded with carbon source, flooded and covered with plastic mulch" is extremely tedious and is not necessary. Simply state "ASD treated C source amended plots"...the process of ASD was noted above and the attributes are well known.
The controls can be defined in the M&M as Control = no ASD, no mulch...and you can include a Control + mulch and define. Much easier for the reader.
Lines 362-363: Not significantly different? State one way or the other.
Lines 369-375: As above, reduce or remove much of this from the text as data are presented in table 4.
Line 387: delete …”the reduction of the”
Line 395: Based upon the statistics, there were no differences among these treatments; therefore it cannot be stated that the combination of ASD + azoxystrobin was the "most effective" because the other treatments including fungicide alone were just as effective.
Lines 400-406: this entire section can be replaced by one simple sentence: “Azoxystrobin application was the only treatment that significantly reduced SLS during 2020 and 2021 trials conducted in Mexico.”
Figure 2: The x-axis labels need to be defined in the figure heading...or the figures need to be edited to clearly state the treatments. What is organic? What is 'chemical'? The figures need to be able to stand alone without reference to the manuscript text.
Figure 3: Again, as in figure 2 address x-axis labels
Figure 4: Move this figure to follow the text describing these results. Clarify x-axis labels .
Line 504: Is this the correct citation? There is no note in the cited work regarding the fungistatic nature of ASD relative to resumption of sclerotia viability. Perhaps a different reference was intended to be cited?
Lines 589-591: But in other systems, ASD conducted using the cover crop as the carbon input was at times as effective as soil fumigation. See Dupont et al. 2021. https://0-doi-org.brum.beds.ac.uk/10.1016/j.apsoil.2021.104076
Author Response
Response to Reviewer 3
Comments and Suggestions for Authors
The manuscript by Sanabria-Velazquez reports on the evaluation of ASD, and different fungicides, for the control of Septoria leaf spot and stem rot in the production of stevia. In general, the work describes a well-conceived study and its novelty lies in the fact that the field component was conducted at multiple sites representing multiple nations. One element of the manuscript that requires considerable attention is the unsuitable sentence structure encountered throughout the document. As an example (lines 487-488) the sentence reads: "...it was explored alternative pesticides for their use in stevia disease managment..."
This should read: "alternative pesticides were examined for their use in stevia disease managment..."
This is just one example and there are a vast number of instances in the text where this needs to be addressed. Thus, significant editing of the manuscript should be required prior to its acceptance.
Response: Thank you very much for taking the time to review this manuscript. We appreciate your valuable feedback and suggestions. We are incredibly grateful for your suggestion to review the sentence grammar and fluency. Your comments helped refine the writing and ensure our message was conveyed more effectively. We are pleased to report that your suggestions were implemented and highlighted in the paper. Thank you once again for your time and effort in reviewing our paper. We included a track changes document so you can verify the changes made ( agronomy-2320012_Track _changes) and a clean version with all the changes accepted (agronomy-2320012_All_changes_acepted). Please use the second for comments and suggestions.
Lines 81-82: This is an incomplete statement. There are additional non-volatile metabolites that contribute to pathogen suppression.
Response: We changed the sentence to include other compounds and added a reference for readers to consult about recent work on this topic.
During the process, compounds unfavorable to pathogen survival a released into the soil [17,18]
Hewavitharana, S.S.; Klarer, E.; Reed, A.J.; Leisso, R.; Poirier, B.; Honaas, L.; Rudell, D.R.; Mazzola, M. Temporal Dynamics of the Soil Metabolome and Microbiome During Simulated Anaerobic Soil Disinfestation. Front. Microbiol. 2019, 10, 1–17, doi:10.3389/fmicb.2019.02365.
Lines 85-87: ASD is associated with dramatic sequential changes in the soil microbiome that require coordinated activity of specific components of the altered microbiome. In addition, both bacterial and fungal elements of the ASD generated microbiome are not only to be required to yield the disease-suppressive active metabolome but themselves are capable of direct pathogen suppression. Therefore, integrating ASD with specific fungicides needs to assess what impacts such a treatment could have on this functional microbiome.
Response: It is important to note that the authors intended to convey that the fungicides are applied to the plants after the completion of ASD and not simultaneously with it. We changed the sentence to make this statement more clear.
In the context of integrated management, after conducting ASD, it is possible to apply organic fungicide programs targeted at both the base and leaves of the stevia plants for further disease management [17]. One of these fungicides, used in Japanese traditional agriculture, is pyroligneous acid (PA), a byproduct of the pyrolysis process of plant material [18].
Line 97: This statement needs to be substantiated by the use of published citations of examples where such has been documented. The utility of botanical fungicides and soil amendments for the control of plant diseases has been evaluated over multiple decades. In large part, the efficacy of such methods has proven insufficient to warrant their use. By no means are these methods "new sustainable and efficacious" and the authors should edit this statement accordingly.
Response: We edited the sentence as suggested. Changes were highlighted in the document.
Using organic amendments and botanical fungicides to manage plant pathogens could be a promising alternative for enhancing the productivity of stevia smallholder farmers when the use of chemical fungicides is limited.
Line 115: How was inoculum prepared? (review notes)
Response: We added the sentence explaining how sclerotia was prepared as suggested.
To obtain sclerotia of S. rolfsii, 4-week-old stevia plants were inoculated with the mycelia of S. rolfsii in the greenhouse. After three weeks, the sclerotia were collected from the dead plants and utilized in the field experiments. Additionally, the plant debris colonized by S. rolfsii was utilized to infest microplots.
Line 121: How was the PA prepared? Provide some detail or specifically identify a citation that documents how it was formulated and its preparation.
Response: We added a sentence and reference detailing the methodology for the preparation of PA.
Campos, A.D. Processo de Coleta e Produção Do Extrato Pirolenhoso Para Uso Agrícola. Circ. Técnica 178 - Embrapa 2018, 1–4.
Lines 126-130: This should be stated that 'fungicides containing azoxystrobin as the active ingredient were used in these trials'...you can then subsequently state the specific commercial formulations that were utilized.
Response: We edited the sentence as suggested. Changes were highlighted in the document.
Fungicides containing azoxystrobin as the active ingredient were used with a rate of 230 a.i. g ha−1 in the trials [6,8,26,27]. In Paraguay, Quadris™ (22.9% a.i., Syngenta, Greensboro, NC, USA) and in Mexico, the fungicide Amistar™ (25% a.i., Syngenta, Greensboro, NC, USA).
Line 133: delete the first “was” in this sentence.
Response: We edited the sentence as suggested. Changes were highlighted in the document.
Line 138: State that a “Petri” plate was used.
Response: We edited the sentence as suggested. Changes were highlighted in the document.
Line 143: State that electrical tape was use “to minimize gaseous exchange with the external atmosphere”. The electrical tape will not completely exclude this process.
Response: We edited the sentence as suggested. Changes were highlighted in the document.
Line 151: How long were these monitored for regrowth and under what conditions were the fungi grown?
Response: We added the sentence explaining the monitoring of growth and incubation. Changes were highlighted in the document.
Mycelia from all the colonies were transferred to a fresh PDA medium to check regrowth, incubating in darkness for four weeks at room temperature.
Line 155: Table 1: This table needs to be moved to a position in the text adjacent to the description of the study sites. What were the attributes of these soils? pH? Nutrient contents? Organic matter content? Etc.???
Response: We added supplementary Table S1.
Lines 180-182: This description is confusing. The sentence appears to state that soils were infested with 350 g stevia debris colonized by S. rolfsii prior to carbon amendment. How was this inoculum source prepared? Just randomly obtained material from the field? Was this 350 g of material applied to each plot/block? How big were the plots and how was the material added to the soil? It is then stated that packets containing 10 sclerotia of the pathogen were added to each 'microplot'. There is no description of these microplots. What were their size? How many per treatment? A statement needs to be made at the beginning of this section that pathogen infestation utilized two methods of introduction into the soils.
Response: The size of the microplots was stated at the beginning (1 m2). We clarified that the Experimental unit was each microplot (5 rep). We also described that sclerotia in the packets were used to measure the effectiveness of the treatments on sclerotia germination and the debris to evaluate disease progression after ASD was completed.
To obtain sclerotia of S. rolfsii, 4-week-old stevia plants were inoculated with the mycelia of S. rolfsii in the greenhouse. After three weeks, the sclerotia were collected from the dead plants and utilized in the field experiments. Additionally, the plant debris colonized by S. rolfsii was utilized to infest microplots.
Line 202: Weed biomass is the standard metric used in such assessments. The "number" of weeds is not appropriate as plant size among species is so dramatically variable.
Response: We agree with this comment and will incorporate this variable to complement our weed count in future works. However, we counted the number of weeds in 1 m2 as previously described in other research published research and added these citations for the present work.
- Woźniak, A. Effect of Crop Rotation and Cereal Monoculture on the Yield and Quality of Winter Wheat Grain and on Crop Infestation with Weeds and Soil Properties. Int. J. Plant Prod. 2019, 13, 177–182, doi:10.1007/s42106-019-00044-w.
- Schappert, A.; Schumacher, M.; Gerhards, R. Weed Control Ability of Single Sown Cover Crops Compared to Species Mixtures. Agronomy 2019, 9.
Line 209: What was the organic matter employed for fertility? What were the NPK contents of this material?
Response: We added the NPK content and the corresponding citation for the common practices.
For all trials, fertility with 0.5 kg m-2 of compost (2N-2P-2K), plastic mulch, drip irrigation, and management practices were employed as commonly performed by smallholder farmers [2]. No other chemical treatments were added to the test plots during the experiments.
Line 222: In the table 2 footnotes it is stated that molasses was applied at 20.2 Mg/ha...which value is correct. In addition, based upon the data in table 2 it would appear that the total carbon inputs for the combined C treatments (molasses + cornmeal or wheat bran) each treatment would have been 30 or 40 Mg/ha...is this correct? This value is greater than that the rate commonly utilized in such ASD trials. In addition, this would result in a significantly greater carbon input than when any of these carbon inputs were used independently. There is no mention of the potential impact of this greater C input when contrasting the single C input ASD treatments and the combined C input treatments. The authors need to clearly address this difference and possible impact on the findings both in the results and discussion.
Response: We addressed this in the discussion as suggested. Changes were highlighted in the document.
In this work, a higher rate (20 to 40 Mg ha-1) of carbon sources for ASD was incorporated into the soil compared to previous works [33,74,75]. The addition of these high amounts of carbon to soil may have a significant impact on soil health and fertility. Greater carbon input can provide more food for microbes, which may lead to increased microbial activity and production of CO2 [29]. In addition, excessive carbon input can lead to imbalances in soil nutrients and can negatively impact soil health, such as nitrogen immobilization [76], soil acidity [77], and higher N2O emissions [73,78]. To avoid these adverse effects, it is important to carefully manage the amount, C:N ratio, and type of carbon added to the soil [50]. Further research is needed to understand the impact of these higher rates of carbon when used for ASD in different edaphoclimatic conditions.
Line 233: replace the word “After” with “Beginning at…”
Response: We edited the sentence as suggested. Changes were highlighted in the document.
Lines 245-246: How many plants were evaluated? At what intervals were these evaluations conducted? Was the same person conducting the evaluation at all time points? Visual assessments of this type can be less than accurate when multiple individuals with differing views of "percent disease" are employed in these evaluations.
Response: Disease was recorded weakly, starting 15 DAP, only plants from the center row were evaluated. Different raters in each country evaluated disease. A diagram was developed and published in Plant Disease to improve interrater reliability. The reference to this work was cited in the manuscript:
Only for the field trials, SLS severity progression and plant yield were evaluated. Severity progression was evaluated through visual assessment of the percentage of ne-crotic leaf area of stevia with symptoms only from the center rows of each plot [32].
- Sanabria-Velazquez, A.D.; Enciso-Maldonado, G.A.; Maidana-Ojeda, M.; Diaz-Najera, J.F.; Thiessen, L.D.; Shew, H.D. Validation of Standard Area Diagrams to Estimate the Severity of Septoria Leaf Spot on Stevia in Paraguay, Mexico, and the USA. Plant Dis. 2022, In press, doi:https://0-doi-org.brum.beds.ac.uk/10.1094/PDIS-07-22-1609-RE.
Line 270: Indicate that mycelial growth was monitored in PDA media.
Response: This was already mentioned in the methods section.
Sclerotia of S. rolfsii in the mesh packets were recovered after soil disinfestation, cleaned, surface-disinfested, and plated on Bromophenol-blue PDA medium to evaluate their viability. Sclerotia viability was evaluated in percentage, considering 100% viable in case all sclerotia germinated.
Line 274: These data are listed in the adjoining table and there is no need to repeat these values in the text...simply make the statement that there were no differences among these treatments. This needs to be repeated at many other points in the results section…some but not all of which I will point out.
Response: We edited the section as suggested. Changes were highlighted in the document.
Table 2: What were the rates for the combined C inputs? Was the cornmeal or wheat + molasses a combined rate of 30 or 40 Mg/ha? Please clarify.
Response: This was already mentioned in the methods section. Each carbon source was 20 Mg/ha and when combined 40 Mg/ha for micrplot experiments. In field trials, molasses was incorporated at 10.1 Mg/ha, cornmeal at 20.2 Mg/ha, and when combined 30 Mg/ha.
Lines 286-300: Again, much of these data are directly provided in the table, there is no need to repeat these values in the text, simply state there were no differences among the ASD carbon treatments.
Response: We edited the section as suggested. Changes were highlighted in the document.
Table 3: What is % sclerotia? Is this % germination? If so, this needs to be stated in the table footnotes.
Response: We edited the section as suggested. Changes were highlighted in the document.
Lines 312-314: Again, why are you repeating the data in the text when they are clearly stated in table 3?
Response: We edited the section as suggested. Changes were highlighted in the document.
Lines 319-327: Continually identifying treatments as "plots amendeded with carbon source, flooded and covered with plastic mulch" is extremely tedious and is not necessary. Simply state "ASD treated C source amended plots"...the process of ASD was noted above and the attributes are well known.
The controls can be defined in the M&M as Control = no ASD, no mulch...and you can include a Control + mulch and define. Much easier for the reader.
Response: We edited the section as suggested. Changes were highlighted in the document.
Lines 362-363: Not significantly different? State one way or the other.
Response: We edited the section as suggested. Changes were highlighted in the document.
Lines 369-375: As above, reduce or remove much of this from the text as data are presented in table 4.
Response: We edited the section as suggested. Changes were highlighted in the document.
Line 387: delete …”the reduction of the”
Response: We edited the section as suggested. Changes were highlighted in the document.
Line 395: Based upon the statistics, there were no differences among these treatments; therefore it cannot be stated that the combination of ASD + azoxystrobin was the "most effective" because the other treatments including fungicide alone were just as effective.
Response: We edited the section as suggested. Changes were highlighted in the document.
Lines 400-406: this entire section can be replaced by one simple sentence: “Azoxystrobin application was the only treatment that significantly reduced SLS during 2020 and 2021 trials conducted in Mexico.”
Response: We edited the section as suggested. Changes were highlighted in the document.
Figure 2: The x-axis labels need to be defined in the figure heading...or the figures need to be edited to clearly state the treatments. What is organic? What is 'chemical'? The figures need to be able to stand alone without reference to the manuscript text.
Response: We edited the section as suggested. Changes were highlighted in the document.
Figure 3: Again, as in figure 2 address x-axis labels
Response: We edited the section as suggested. Changes were highlighted in the document.
Figure 4: Move this figure to follow the text describing these results. Clarify x-axis labels .
Response: We edited the section as suggested. Changes were highlighted in the document.
Line 504: Is this the correct citation? There is no note in the cited work regarding the fungistatic nature of ASD relative to resumption of sclerotia viability. Perhaps a different reference was intended to be cited?
Response: We appreciate the comment. We corrected the mistake, and a new reference was added.
Hewavitharana, S.S.; Mazzola, M. Effect of Different Gramineae Carbon Inputs on Efficacy of ASD for Control of Macrophomina phaseolina in Strawberry. In Proceedings of the Phytopathology; Amer Phytopathological Soc 3340 Pilot Knob Road, St Paul, Mn 55121 USA, 2017; Vol. 107, p. 46.
Lines 589-591: But in other systems, ASD conducted using the cover crop as the carbon input was at times as effective as soil fumigation. See Dupont et al. 2021. https://0-doi-org.brum.beds.ac.uk/10.1016/j.apsoil.2021.104076
Response: We clarified that and added a new reference
The use of cover crops such as cowpea, annual ryegrass, oat, rye, and mustard, among others, may reduce the costs of ASD [38] and has shown to be as effective as soil fumigation in other perennial systems such as apple [80].
DuPont, S.T.; Hewavitharana, S.S.; Mazzola, M. Field Scale Application of Brassica Seed Meal and Anaerobic Soil Disinfestation for the Control of Apple Replant Disease. Appl. Soil Ecol. 2021, 166, 104076, doi:https://0-doi-org.brum.beds.ac.uk/10.1016/j.apsoil.2021.104076.
Author Response File: Author Response.docx
