The Colonization of Grape Bunch Trash by Microorganisms for the Biocontrol of Botrytis cinerea as Influenced by Temperature and Humidity
Round 1
Reviewer 1 Report
Report on Agronomy-1008049
Title: The colonization of grape bunch trash by microorganisms for the biocontrol of Botrytis cinerea as influenced by temperature and humidity
- Brief summary of the content of the manuscript
The paper is motivated by investigating the effects of temperature and relative humidity on the colonization of grape bunch trash by six biocontrol agents of Botrytis cinerea. This fungus at the origin of Botrytis bunch rot (BBR), a disease that causes important economic damage to the grapevines. Traditionally, the BBR control has been based on the application of chemical fungicides, but a search for natural products that can replace or complement them is encouraged. Therefore, an experimental study of six commercial biocontrol agents (BCA) was performed. These BCA were applied to bunch trash collected in 2018 and 2019 with 5 different incubation temperatures and five relative humidity levels. For the data analysis, an ANOVA was carried in which the factors were the BCA, the temperature, the relative humidity and the colonization period. The variable of interest is the number of colony forming units (CFUs). The variability in the CFUs was also assessed by calculating coefficients of variation within each BCA as affected by the other factors. The numbers of CFUs were also rescaled and fit to equations that accounted for the combined effect or temperature and relative humidity.
The Akaike information criterion (AIC) was used for equation selection.
- Reasoning behind my recommendation
The paper deals with a very important problem of disease control on grapevines. The statistical analysis of BBR severity with respect to relevant factors is well conducted. However, an analysis of covariance (ANCOVA) may have been required because some variables may be considered as quantitative : the temperature, the relative humidity and the BCA colonization time” is quantitative. Figures 3 and 5 underscores this fact, since extrapolation is considered.
- Provide more lists of your minor for the improvement of the manuscript.
In lines 120, 154, 183, 185 and 189, there are confusion between “Number of CFUs” and “CFUs”. I suggest to add “number of” before CFUs
Author Response
Dear Editor, Dear reviewer,
We are submitting the revision of the manuscript agronomy- 1008049, entitled “The colonization of grape bunch trash by microorganisms for the biocontrol of Botrytis cinerea as influenced by temperature and humidity”.
We would like to thank you and the reviewers for the comments.
We have addressed all the editions and comments proposed by the reviewers. We also added a Figure to better explain the methodology used.
Best regards,
The authors
--------------------------------------------------------------------
- Brief summary of the content of the manuscript
The paper is motivated by investigating the effects of temperature and relative humidity on the colonization of grape bunch trash by six biocontrol agents of Botrytis cinerea. This fungus at the origin of Botrytis bunch rot (BBR), a disease that causes important economic damage to the grapevines. Traditionally, the BBR control has been based on the application of chemical fungicides, but a search for natural products that can replace or complement them is encouraged. Therefore, an experimental study of six commercial biocontrol agents (BCA) was performed. These BCA were applied to bunch trash collected in 2018 and 2019 with 5 different incubation temperatures and five relative humidity levels. For the data analysis, an ANOVA was carried in which the factors were the BCA, the temperature, the relative humidity and the colonization period. The variable of interest is the number of colony forming units (CFUs). The variability in the CFUs was also assessed by calculating coefficients of variation within each BCA as affected by the other factors. The numbers of CFUs were also rescaled and fit to equations that accounted for the combined effect or temperature and relative humidity.
The Akaike information criterion (AIC) was used for equation selection.
- Reasoning behind my recommendation
The paper deals with a very important problem of disease control on grapevines. The statistical analysis of BBR severity with respect to relevant factors is well conducted. However, an analysis of covariance (ANCOVA) may have been required because some variables may be considered as quantitative : the temperature, the relative humidity and the BCA colonization time” is quantitative. Figures 3 and 5 underscores this fact, since extrapolation is considered.
A: The type of statistical analysis should be functional to the type of data to be analyzed and to the aim of the study. In our case, we are interested in the response of BCAs to varying conditions of quantitative variables that are continuous by nature (i.e. time, temperature and relative humidity); therefore, the levels we used (1,3, .. days; 5, 10 .. °C, etc.) may be considered only samples of many possible values we could extract from the continuous variable. In such a situation, the linear regression is considered the most suitable analysis (Clewer and Scarisbrick 2001). In the present study, the ANOVA was used only for understanding the significant factors in the experiment and their relative weight (as explained variance) and not for analyzing the differences between levels. It is not interesting to know if 5°C is different from 10°C, because then the difference between 6/7°C and 10°C should be also verified (and this create almost unlimited possibilities). Instead, it is interesting to analyze the response of BCA to the different temperature by fitting equations to experimental data, as we did.
G. Clewer, D. H. Scarisbrick, Practical statistics and experimental design for plant and crop science. John Wiley & Sons LTD, Chichester, 2001.
- Provide more lists of your minor for the improvement of the manuscript.
In lines 120, 154, 183, 185 and 189, there are confusion between “Number of CFUs” and “CFUs”. I suggest to add “number of” before CFUs
A: we modified the manuscript as suggested.
Reviewer 2 Report
Lines 2-4 Title: change the title in a more appropriate form regard your research done, use words as: predict, modeling, statistical model, simulation study, model simulation,…..
Lines 11-13: use italic for all the latin names
Line 29: grapevine – without s
Line 78: B. cinerea in italic
Line 104: „After the suspension…“ - put „that“ and comma (,) after the word „After“
Line 105: use „without“ instead of „minus“
Line 125: put „the“ in the form „was highest“
Line 348-349: „effective and to then…“, delete „to“
Discussion: interpretations and some conclusions from your work are missing; which of the 6 BCAs would you reccomend to the farmers of your province, region or your country regard temperature and relative air humidity and why? Which microorganism resulted better for reccomendations - bacteria, yeast, fungus - which will you reccomend and why? Can you draw conclusions in a simple way to be more interesting for the readers?
It is very difficult to obtain the same results in optimal controlled and in non-optimal uncontrolled conditions as are usual in vineyard. Unfortunately, in the research the investigations on the efficacy of BCAs on B. cineraea in that T and RH controlled conditions are missing, so it is impossible to conclude something about the biocontrol efficacy. Hope that this questions will be answered in further researches.
Author Response
Dear Editor, Dear reviewers,
We are submitting the revision of the manuscript agronomy- 1008049, entitled “The colonization of grape bunch trash by microorganisms for the biocontrol of Botrytis cinerea as influenced by temperature and humidity”.
We would like to thank you and the reviewers for the comments.
We have addressed all the editions and comments proposed by the reviewers.
We submit two versions of the manuscript file: the first is the manuscript revised with track changes and the second is the manuscript revised.
Best regards,
The authors
---------------------------------------------------------------------------
Lines 2-4 Title: change the title in a more appropriate form regard your research done, use words as: predict, modeling, statistical model, simulation study, model simulation,…..
A: We studied the effect of temperature and humidity on BCA colonization and represented these data by means of equations. The work was not intended to simulate or predict the BCA colonization. Therefore, we find the current title appropriate.
Lines 11-13: use italic for all the latin names
A: Modified
Line 29: grapevine – without s
A: Modified
Line 78: B. cinerea in italic
A:Modified
Line 104: „After the suspension…“ - put „that“ and comma (,) after the word „After“
A:Modified
Line 105: use „without“ instead of „minus“
A:Modified
Line 125: put „the“ in the form „was highest“
A:Modified
Line 348-349: „effective and to then…“, delete „to“
A:Done
Discussion: interpretations and some conclusions from your work are missing; which of the 6 BCAs would you reccomend to the farmers of your province, region or your country regard temperature and relative air humidity and why? Which microorganism resulted better for reccomendations - bacteria, yeast, fungus - which will you reccomend and why?
A:Recommend the best BCA for the farmers is not our aim in this work; in addition, the paper shows that the best BCA also depends on weather conditions at the time of application and in the following days.
Can you draw conclusions in a simple way to be more interesting for the readers? It is very difficult to obtain the same results in optimal controlled and in non-optimal uncontrolled conditions as are usual in vineyard. Unfortunately, in the research the investigations on the efficacy of BCAs on B. cineraea in that T and RH controlled conditions are missing, so it is impossible to conclude something about the biocontrol efficacy. Hope that this questions will be answered in further researches.
A:Of course we will do.
Reviewer 3 Report
General comment
This study deals with the effect of environmental conditions on the growth of several microorganism showing a potential interest for the biocontrol of B. cinerea, the causal agent of Botrytis bunch rot. This article is interesting because it doesn’t restrict on testing the effect of T and RH on the growth of microorganisms, but it also aims to provide tangible tools to the farmers for choosing a microorganism according to weather conditions.
It is always difficult to analyze results when considering multiple factors together which is rather well done in this manuscript. However, some points have to be clarified and the ANOVA should be completed by a post hoc test through multiple pairwise comparisons. Moreover, the ANOVA demands to first consider the results of interactions before considering the effect of single factors when interactions are significant.
Please also consider to add a point in the discussion about the differences observed between bacteria, yeasts and molds.
Specific commentary
Abstract
L11-13: species in italic
L22: R2: exponent for 2
Materials and Methods
L74-76: It could be worth to explain how much these agricultural practices, i.e. grape variety, cutting and spacing, are representative of the location.
L77-78: Likewise, it could be worth to give an idea about the occurrence and the impact of B. cinerea in the region. Since the fungicide are ineffective in controlling the fungus, is it in general a yearly concern? Or some years?
L78: B. cinerea in italic
L86: How did you choose the BCAs: all commercially available products? All commercially available species? The most used products?
L92: Table 1. Maybe you could add the strategy(ies) used by each BCA against B. cinerea if you know it.
L102-103: As I understand the preceding paragraphs you placed 1 aliquot of 0.1 g of bunch trash per Petri dish and 3 petri dishes for each T/RH combination. I don’t understand how do you manage the sampling times. As it is written I understand that at each time you washed the bunches before re-incubating them until the next sampling time. If this technic was really employed, it poses a problem because you almost went back at beginning at each sampling time regarding the BCA colonization, at least in surface. Or did you have three replicates by T/RH couple and by sampling time?
You could provide a picture of your experiment.
L105: Dilutions from “no-dilution” to ??. Same dilution factor for molds, yeasts and bacteria?
L111: It is the first time you mention berries, before you only mentioned bunch trash.
L113-114: Ok for the homogeneity of variances. What about the normality of the distributions, as least regarding the residuals?
L109-104: What about a post-hoc test through multiple pairwise comparison associated to ANOVA? The ANOVA indicates that the factors have a significant effect or not, but does not indicates which modalities of each factor produce significant differences.
L163: N0 as defined by the manufacturer data or by the initial count on PDA?
Results
L181-188: You should summarize this results in a table.
Be careful: as the effect of the triple interaction is significant, you usually cannot consider the effects of individual factors, nor the effect of the double interactions. In details, it depends on the results that should be provided by the post hoc test.
L189-196: Calculating this CV is interesting. It allows to quickly understand that each BCA is affected by T/RH and/or by incubation period. However, I insist on the post hoc test associated to the ANOVA that will give you the same result with more details.
L197-208: Ok, you rescaled each BCA thanks to the highest CFU value obtained for this BCA. After reading your M&M, I was not sure if you rescaled each BCA thanks to the highest value obtained for the considered BCA or thanks to the highest value obtained regarding all BCAs together. You should clarify it in the M&M, i.e. “each value was then divided by the highest value found in the experiment” (L121-122).
L207-208: “and of different numbers of days after the BCA treatment in (B) and (C)”. You tested each T/RH combination, didn’t you? In (B) and (C) the bars are therefore mean of different number of days and different RH and means of different number of days and different T in (B) and (C) respectively.
L241: “RMSE<0.79”. In table 3, RMSE for TAE is 0.84.
L242: Why is the equation (5) only solved (or only worked?) at 100 % RH? Maybe you explained it in the M&M, but I cannot find the explanation easily. 100 % is not the optimal RH for all species, nor the main environmental condition in the field, I hope.
L247-248: I agree regarding AMY, but regarding TAE Figures 2 and 3 showed almost no growth at 35 °C: how is it now possible to confirm that only 5 days are necessary to reach the maximal colonization? Or else, it is relative to the colonization at 35 °C, but 5 days to reach a colonization almost inexistant is not a performance. Do you understand what I mean?
L259-260: Please develop the legend (products A to F) in this figure.
L275-278: Please remind the optimal temperature for each BCA in the figure.
Discussion
L325-340: Actually, I think this is a key point to explain your results, and it needs to be discussed in deep. The differences are not only explained by nutritional requirements, but also by the nature of each BCA. You compared bacteria, yeasts and molds: you have to link your results regarding T, RH and colonization time with the data available about these organisms.
Similarly, you should discuss the differences observed between AMY and TAE which are based on the same species, Bacillus amyloliquefaciens, but not on the same strain. This species being also more or less related to B. subtilis (SER).
L346-351: You’re right, this is another key data for the choice and the use of a BCA. The key factor is less the time and the conditions necessary to obtain the highest colonization than the time and the conditions necessary to obtain a colonization high enough to outcompete/inhibit B. cinerea. This level of colonization being probably dependent on the BCA considered and also dependent on its strategy against B. cinerea.
Author Response
Dear Editor, Dear reviewers,
We are submitting the revision of the manuscript agronomy- 1008049, entitled “The colonization of grape bunch trash by microorganisms for the biocontrol of Botrytis cinerea as influenced by temperature and humidity”.
We would like to thank you and the reviewers for the comments.
We have addressed all the editions and comments proposed by the reviewers.
We submit two versions of the manuscript file: the first is the manuscript revised with track changes and the second is the manuscript revised.
Best regards,
The authors
-------------------------------------------------------
Abstract
L11-13: species in italic
A:Modified
L22: R2: exponent for 2
A:Modified
Materials and Methods
L74-76: It could be worth to explain how much these agricultural practices, i.e. grape variety, cutting and spacing, are representative of the location.
A:We modified the sentence to explain it. However, the vineyard was only used to collect bunch trash samples and not for performing field experiments. Therefore, detailed description of the vineyard characteristics may not be required.
L77-78: Likewise, it could be worth to give an idea about the occurrence and the impact of B. cinerea in the region. Since the fungicide are ineffective in controlling the fungus, is it in general a yearly concern? Or some years?
A:Please, see the answer to the previous comment.
L78: B. cinerea in italic
A:Modified
L86: How did you choose the BCAs: all commercially available products? All commercially available species? The most used products?
A:We selected some commercially available products and others that are in an advanced phase of registration processes. Therefore, all the BCAs are formulations.
L92: Table 1. Maybe you could add the strategy(ies) used by each BCA against B. cinerea if you know it.
A:We explain why we did not consider the BCA mechanism of action in the discussion.
L102-103: As I understand the preceding paragraphs you placed 1 aliquot of 0.1 g of bunch trash per Petri dish and 3 petri dishes for each T/RH combination. I don’t understand how do you manage the sampling times. As it is written I understand that at each time you washed the bunches before re-incubating them until the next sampling time. If this technic was really employed, it poses a problem because you almost went back at beginning at each sampling time regarding the BCA colonization, at least in surface. Or did you have three replicates by T/RH couple and by sampling time?
A:We had 3 replicates for T/RH combinations, for each sampling time (e.g., at 20°C we had 3 replicates that were analyzed after 1 day, 3 (different) replicates analyzed after 3 days, etc.).
You could provide a picture of your experiment.
A:Sorry, but we cannot imagine a picture that could increase the value or the understanding of the manuscript.
L105: Dilutions from “no-dilution” to ??. Same dilution factor for molds, yeasts and bacteria?
A:We modified the sentence in order to make it clearer.
L111: It is the first time you mention berries, before you only mentioned bunch trash.
A:Yes, sorry it was a mistake. We worked on bunch trash.
L113-114: Ok for the homogeneity of variances. What about the normality of the distributions, as least regarding the residuals?
A:We added a sentence to specify the normal distribution of the data.
L109-104: What about a post-hoc test through multiple pairwise comparison associated to ANOVA? The ANOVA indicates that the factors have a significant effect or not, but does not indicates which modalities of each factor produce significant differences.
A:Generally, we agree with all the comment concerning the ANOVA analysis as basic statistical analysis. However, the use of the ANOVA should be functional to the type of data to be analyzed and to the aim of the study. In our case, we are interested in the response of BCAs to varying conditions of variables that are continuous by nature (i.e. time, temperature and relative humidity); therefore, the levels we used (1,3, .. days; 5, 10 .. °C, etc.) may be considered only samples of many possible values we could extract from the continuous variable. In such a situation, the post-hoc test is not usually used (and for some experts it is also incorrect). In the present study, the ANOVA was used only for understanding the significant factors in the experiment and their relative weight (as explained variance) and not for analyzing the differences between levels. It is not interesting to know if 5°C is different from 10°C, because then the difference between 6/7°C and 10°C should be also verified (and this create almost unlimited possibilities). Instead, it is interesting to analyze the response of BCA to the different temperature by fitting equations to experimental data, as we did.
L163: N0 as defined by the manufacturer data or by the initial count on PDA?
A:It is the CFU number assessed at time 0, when the treatment was performed. We better specified that in the text.
Results
L181-188: You should summarize this results in a table.
Be careful: as the effect of the triple interaction is significant, you usually cannot consider the effects of individual factors, nor the effect of the double interactions. In details, it depends on the results that should be provided by the post hoc test.
A:Please, see the answers to previous comments concerning the post-hoc test.
L189-196: Calculating this CV is interesting. It allows to quickly understand that each BCA is affected by T/RH and/or by incubation period. However, I insist on the post hoc test associated to the ANOVA that will give you the same result with more details.
A:Please, see the answers to previous comments concerning the post-hoc test.
L197-208: Ok, you rescaled each BCA thanks to the highest CFU value obtained for this BCA. After reading your M&M, I was not sure if you rescaled each BCA thanks to the highest value obtained for the considered BCA or thanks to the highest value obtained regarding all BCAs together. You should clarify it in the M&M, i.e. “each value was then divided by the highest value found in the experiment” (L121-122).
A:We better specified this in the M&M section.
L207-208: “and of different numbers of days after the BCA treatment in (B) and (C)”. You tested each T/RH combination, didn’t you? In (B) and (C) the bars are therefore mean of different number of days and different RH and means of different number of days and different T in (B) and (C) respectively.
A:The bars show for days (A), temperature (B) and relative humidity (C) the overall average of the different treatments, with their variability as whiskers.
L241: “RMSE<0.79”. In table 3, RMSE for TAE is 0.84.
A:Modified
L242: Why is the equation (5) only solved (or only worked?) at 100 % RH? Maybe you explained it in the M&M, but I cannot find the explanation easily. 100 % is not the optimal RH for all species, nor the main environmental condition in the field, I hope.
A:That is for simplicity. Describing all the responses at different RH would have been to long for the manuscript.
L247-248: I agree regarding AMY, but regarding TAE Figures 2 and 3 showed almost no growth at 35 °C: how is it now possible to confirm that only 5 days are necessary to reach the maximal colonization? Or else, it is relative to the colonization at 35 °C, but 5 days to reach a colonization almost inexistant is not a performance. Do you understand what I mean?
A:The relative colonization of bunch trash by TAE at 35°C was low (0 to 0.2 relative colonization), but in Figure 4 we represented the number of days required to attain the maximal colonization of bunch trash at each temperature. We observed that the maximal colonization (i.e., the highest CFUs) for TAE was reached in less than 5 days at 35°C.
L259-260: Please develop the legend (products A to F) in this figure.
A:Done
L275-278: Please remind the optimal temperature for each BCA in the figure.
A: Done
Discussion
L325-340: Actually, I think this is a key point to explain your results, and it needs to be discussed in deep. The differences are not only explained by nutritional requirements, but also by the nature of each BCA. You compared bacteria, yeasts and molds: you have to link your results regarding T, RH and colonization time with the data available about these organisms.
Similarly, you should discuss the differences observed between AMY and TAE which are based on the same species, Bacillus amyloliquefaciens, but not on the same strain. This species being also more or less related to B. subtilis (SER).
A:As suggested, we added this point to the discussion
L346-351: You’re right, this is another key data for the choice and the use of a BCA. The key factor is less the time and the conditions necessary to obtain the highest colonization than the time and the conditions necessary to obtain a colonization high enough to outcompete/inhibit B. cinerea. This level of colonization being probably dependent on the BCA considered and also dependent on its strategy against B. cinerea.
Round 2
Reviewer 3 Report
- L77-78: Likewise, it could be worth to give an idea about the occurrence and the impact of B. cinerea in the region. Since the fungicide are ineffective in controlling the fungus, is it in general a yearly concern? Or some years?
A: Please, see the answer to the previous comment.
Reviewer: You did not perform your study under field conditions, I agree. However, you have to contextualize your study. Explain how negative the impact of B. cinerea is in the area where the bunch trash was collected is therefore not unnecessary data in my opinion.
- L102-103: As I understand the preceding paragraphs you placed 1 aliquot of 0.1 g of bunch trash per Petri dish and 3 petri dishes for each T/RH combination. I don’t understand how do you manage the sampling times. As it is written I understand that at each time you washed the bunches before re-incubating them until the next sampling time. If this technic was really employed, it poses a problem because you almost went back at beginning at each sampling time regarding the BCA colonization, at least in surface. Or did you have three replicates by T/RH couple and by sampling time?
A: We had 3 replicates for T/RH combinations, for each sampling time (e.g., at 20°C we had 3 replicates that were analyzed after 1 day, 3 (different) replicates analyzed after 3 days, etc.).
Reviewer: Ok. Thus, clearly write it at line 101: “There were 3 Petri plates 100 (replicates) for each T/RH regime and for each sampling time”
- You could provide a picture of your experiment.
A: Sorry, but we cannot imagine a picture that could increase the value or the understanding of the manuscript.
Reviewer: In my opinion, a good picture always increases the value of a manuscript. First, it breaks the monotony of the reading. Then, a picture is always more explanatory than a paragraph. Maybe I had no question about sampling times if a judicious picture had been provided.
L109-104: What about a post-hoc test through multiple pairwise comparison associated to ANOVA? The ANOVA indicates that the factors have a significant effect or not, but does not indicates which modalities of each factor produce significant differences.
A: Generally, we agree with all the comment concerning the ANOVA analysis as basic statistical analysis. However, the use of the ANOVA should be functional to the type of data to be analyzed and to the aim of the study. In our case, we are interested in the response of BCAs to varying conditions of variables that are continuous by nature (i.e. time, temperature and relative humidity); therefore, the levels we used (1,3, .. days; 5, 10 .. °C, etc.) may be considered only samples of many possible values we could extract from the continuous variable. In such a situation, the post-hoc test is not usually used (and for some experts it is also incorrect). In the present study, the ANOVA was used only for understanding the significant factors in the experiment and their relative weight (as explained variance) and not for analyzing the differences between levels. It is not interesting to know if 5°C is different from 10°C, because then the difference between 6/7°C and 10°C should be also verified (and this create almost unlimited possibilities). Instead, it is interesting to analyze the response of BCA to the different temperature by fitting equations to experimental data, as we did.
Reviewer: Ok, I understand the answer and I agree with you.
L207-208: “and of different numbers of days after the BCA treatment in (B) and (C)”. You tested each T/RH combination, didn’t you? In (B) and (C) the bars are therefore mean of different number of days and different RH and means of different number of days and different T in (B) and (C) respectively.
A: The bars show for days (A), temperature (B) and relative humidity (C) the overall average of the different treatments, with their variability as whiskers.
Reviewer: I agree with your answer, but I still not agree with the way it is written in the manuscript: in (B) and (C), it is not only the means of numbers of days after the BCA treatment. Why do you not change the legend by what you wrote in you answer?
L242: Why is the equation (5) only solved (or only worked?) at 100 % RH? Maybe you explained it in the M&M, but I cannot find the explanation easily. 100 % is not the optimal RH for all species, nor the main environmental condition in the field, I hope.
A: That is for simplicity. Describing all the responses at different RH would have been to long for the manuscript.
Reviewer: Ok, I understand, but why did you choose 100 % as the most representative RH do describe? Certainly, because it corresponds to the best RH for 3 of 6 BCAS, but you have to provide an explanation for your choice.
Author Response
Dear Editor, Dear reviewer,
We are submitting the revision of the manuscript agronomy- 1008049, entitled “The colonization of grape bunch trash by microorganisms for the biocontrol of Botrytis cinerea as influenced by temperature and humidity”.
We would like to thank you and the reviewers for the comments.
We have addressed all the editions and comments proposed by the reviewers. We also added a Figure to better explain the methodology used.
Best regards,
The authors
------------------------------------------------------------------------
- L77-78: Likewise, it could be worth to give an idea about the occurrence and the impact of B. cinerea in the region. Since the fungicide are ineffective in controlling the fungus, is it in general a yearly concern? Or some years?
A: Please, see the answer to the previous comment.
Reviewer: You did not perform your study under field conditions, I agree. However, you have to contextualize your study. Explain how negative the impact of B. cinerea is in the area where the bunch trash was collected is therefore not unnecessary data in my opinion.
A: We added a sentence in the paragraph to explain the impact of B. cinerea in the area
- L102-103: As I understand the preceding paragraphs you placed 1 aliquot of 0.1 g of bunch trash per Petri dish and 3 petri dishes for each T/RH combination. I don’t understand how do you manage the sampling times. As it is written I understand that at each time you washed the bunches before re-incubating them until the next sampling time. If this technic was really employed, it poses a problem because you almost went back at beginning at each sampling time regarding the BCA colonization, at least in surface. Or did you have three replicates by T/RH couple and by sampling time?
A: We had 3 replicates for T/RH combinations, for each sampling time (e.g., at 20°C we had 3 replicates that were analyzed after 1 day, 3 (different) replicates analyzed after 3 days, etc.).
Reviewer: Ok. Thus, clearly write it at line 101: “There were 3 Petri plates 100 (replicates) for each T/RH regime and for each sampling time”
- You could provide a picture of your experiment.
A: Sorry, but we cannot imagine a picture that could increase the value or the understanding of the manuscript.
Reviewer: In my opinion, a good picture always increases the value of a manuscript. First, it breaks the monotony of the reading. Then, a picture is always more explanatory than a paragraph. Maybe I had no question about sampling times if a judicious picture had been provided.
A: We added a Figure to better explain the methodology
L109-104: What about a post-hoc test through multiple pairwise comparison associated to ANOVA? The ANOVA indicates that the factors have a significant effect or not, but does not indicates which modalities of each factor produce significant differences.
A: Generally, we agree with all the comment concerning the ANOVA analysis as basic statistical analysis. However, the use of the ANOVA should be functional to the type of data to be analyzed and to the aim of the study. In our case, we are interested in the response of BCAs to varying conditions of variables that are continuous by nature (i.e. time, temperature and relative humidity); therefore, the levels we used (1,3, .. days; 5, 10 .. °C, etc.) may be considered only samples of many possible values we could extract from the continuous variable. In such a situation, the post-hoc test is not usually used (and for some experts it is also incorrect). In the present study, the ANOVA was used only for understanding the significant factors in the experiment and their relative weight (as explained variance) and not for analyzing the differences between levels. It is not interesting to know if 5°C is different from 10°C, because then the difference between 6/7°C and 10°C should be also verified (and this create almost unlimited possibilities). Instead, it is interesting to analyze the response of BCA to the different temperature by fitting equations to experimental data, as we did.
Reviewer: Ok, I understand the answer and I agree with you.
L207-208: “and of different numbers of days after the BCA treatment in (B) and (C)”. You tested each T/RH combination, didn’t you? In (B) and (C) the bars are therefore mean of different number of days and different RH and means of different number of days and different T in (B) and (C) respectively.
A: The bars show for days (A), temperature (B) and relative humidity (C) the overall average of the different treatments, with their variability as whiskers.
Reviewer: I agree with your answer, but I still not agree with the way it is written in the manuscript: in (B) and (C), it is not only the means of numbers of days after the BCA treatment. Why do you not change the legend by what you wrote in you answer?
A: we modified the legend as suggest.
L242: Why is the equation (5) only solved (or only worked?) at 100 % RH? Maybe you explained it in the M&M, but I cannot find the explanation easily. 100 % is not the optimal RH for all species, nor the main environmental condition in the field, I hope.
A: That is for simplicity. Describing all the responses at different RH would have been to long for the manuscript.
Reviewer: Ok, I understand, but why did you choose 100 % as the most representative RH do describe? Certainly, because it corresponds to the best RH for 3 of 6 BCAS, but you have to provide an explanation for your choice.
A: we added a sentence in materials and methods to explain why we reported only the fitting for 100% RH.
