Green Alternatives for the Control of Fungal Diseases in Strawberry: In-Field Optimization of the Use of Elicitors, Botanical Extracts and Essential Oils

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments to the author (Horticulturae-3193667)

The manuscript entitled “Green alternatives for the control of fungal diseases in straw berry: in-field optimization of the use of elicitors, botanical extracts and essential oils” is an interesting manuscript and the author (s) has performed a nice study by assessing the effects of various treatments. The study by Soppelsaet al is mainly focusing on the evaluation of various treatments on suppressing fungal pathogen thereby promoting green agriculture. Overall, the manuscript is well structured; presenting novelty, merit and authenticity of work. The results are reliable and manuscript is in accordance with the Journal’s scope. However, some short comings in text are needed for the improvement of manuscript. Some important suggestions and key questions regarding the work are as:

Importantly: I would suggest revising manuscript from the experts of this field for the scientific soundness as I feel, manuscript needs grammatical improvements. For example, in abstract, the word should be challenge, our experiments, The experimental trials were conducted (as the authors have worked with multiple pathogens). Thus, I would request to revise it to improve the scientific soundness.

Abstract

I would suggest only adding general introductory lines (1-2) on emerging fungicide resistance challenges importance in these pathogens.

Line 13-21: Reduced and shorten the methodology and focus on the results, please add numerical data to express the results within abstract viz., B. cinerea was significantly reduced how much was reduced etc.   

Reduce the key words

At first, add the full for of Kt and then use abbreviations

In table 1: How these doses were finalized?

In experiment 1 and two why Not the same plants were used like in first experiment 66 while in second 84, while in third 34.

How P. aphanis and B. cinerea were identified? And how the autour(s) were assured about the pathogenicity caused by these fungi.

Line 196: Change “according to the methodology of” to “as previously reported”.

Powdery mildew conidia this is totally wrong. Powdery mildew itself is disease and conidia are different than this.

Did these all treatments (listed in table 1) were confirmed under in vitro inhibition of these pathogens? If not, then how these concentrations were finalized. If these are the recommended concentration (by company), how the authors were sure about their efficacy in these experiments, This is not convincing at all.

There is no information/proof that these all concentrations effectively inhibit these fungal pathogens in the present study. Have you standardized this dose and method of inoculum addition? If not, how you arrived at this concentration?

Further, in controlled conditions particularly, any treatment/oil/chemical can be influenced by environmental factors viz, light, humidity, moisture, temperature and on plants as well. 

Line 217-218: please provide appropriate reference.

There is no information about the sequence of reference gene/ FaGPDH2 (in table 2) gene expression analysis.

How the gene expression data were calculated?

Line 252: were

Figure 2: Please add Mean±SD error bars on graph and the colors used in the graphs should be avoided by using black and white bars.

Please add outside tick-marks in all figures

In results section Table 2 should be Table 3, as table 2 has already been mentioned previously and the yield is always mentioned in kg, please convert and in table 2, CON, (2.47¹) need to check this value.

Figure 5: Replace these figures into graph with differential patterns.
Why these all genes were quantified/calculated only in the four treatments (why not in others)?

Discussion needed to elaborate more by justifying the results of the present study particularly the importance of oil and extracts.

Comments on the Quality of English Language

I would suggest revising manuscript from the experts of this field for the scientific soundness as I feel, manuscript needs grammatical improvements. For example, in abstract, the word should be challenge, our experiments, The experimental trials were conducted (as the authors have worked with multiple pathogens). Thus, I would request to revise it to improve the scientific soundness.

Author Response

REVIEWER#1

I would suggest only adding general introductory lines (1-2) on emerging fungicide resistance challenges importance in these pathogens.

REPLY: we are gratefull for this suggestion, However, Abstract is already rather long and the emerging of fungicide resistance is cleraly mentioned in the infroduction (Line  71-73)

Line 13-21: Reduced and shorten the methodology and focus on the results, please add numerical data to express the results within abstract viz., B. cinerea was significantly reduced how much was reduced etc.   

REPLY: we modified the text according to the suggestion

Reduce the key words

REPLY: Keyword number has been reduced

At first, add the full for of Kt and then use abbreviations

REPLY: we followed the request

How P. aphanis and B. cinerea were identified? And how the autour(s) were assured about the pathogenicity caused by these fungi.

REPLY: P. apahnis was identified by morphological observation under the microscope and identification was performed by molecular identification. For this purpose, ITS genes were amplified and sequences according to Sangiorgio et al., 2022.

Line 196: Change “according to the methodology of” to “

REPLT: done

Powdery mildew conidia this is totally wrong. Powdery mildew itself is disease and conidia are different than this.

REPLY: We apologize for the mistake and we corrected it

Did these all treatments (listed in table 1) were confirmed under in vitro inhibition of these pathogens? If not, then how these concentrations were finalized. If these are the recommended concentration (by company), how the authors were sure about their efficacy in these experiments, This is not convincing at all. 

There is no information/proof that these all concentrations effectively inhibit these fungal pathogens in the present study. Have you standardized this dose and method of inoculum addition? If not, how you arrived at this concentration?

REPLY: the tested essential oils are active in vitro against the pathogens used in this work. The choice of concentrations were based on manufacturer suggestions and on preliminary experiments made to assess their phytotoxicity. However, due to the low number of repetition, those date were not included in the manuscript. Moreover, the efficacy of the tested products is evident on the in vivo experiments as shown by our results

Concerning the standardization of the inoculation, this was performed on P. aphanis, whereas, for B. cinerea, as explained in the materials and methods, no inoculation was performed

Further, in controlled conditions particularly, any treatment/oil/chemical can be influenced by environmental factors viz, light, humidity, moisture, temperature and on plants as well. 

REPLY: we thanks the reviewer for the comment and we fully agree with it. In fact, the experiments were performed in commercial conditions either in greenhouse or tunnel. For the same reason, we did not include the data on in vitro experiments which have a rather limited values

Line 252: were

REPLY:done

Figure 2: Please add Mean±SD error bars on graph and the colors used in the graphs should be avoided by using black and white bars.

REPLY: We are grateful for this comments. However, the number in the graphs are percentages and not natural numbers, thus error bars are not appropriate for those type of graphs

Please add outside tick-marks in all figures

REPLY: done

In results section Table 2 should be Table 3, as table 2 has already been mentioned previously and the yield is always mentioned in kg, please convert and in table 2, CON, (2.47¹) need to check this value.

REPLY: done

Figure 5: Replace these figures into graph with differential patterns.

REPLY: done
Why these all genes were quantified/calculated only in the four treatments (why not in others)?

Discussion needed to elaborate more by justifying the results of the present study particularly the importance of oil and extracts.

REPLY: we are grateful for the suggestion and consequently we modified the discussion

 

 

 

There is no information about the sequence of reference gene/ FaGPDH2 (in table 2) gene expression analysis. How the gene expression data were calculated?

REPLY: thank you for the suggestion. We corrected the text and complete it with the missing information

Please add outside tick-marks in all figures

REPLY: done

In results section Table 2 should be Table 3, as table 2 has already been mentioned previously and the yield is always mentioned in kg, please convert and in table 2, CON, (2.47¹) need to check this value.

REPLY: done

Figure 5: Replace these figures into graph with differential patterns.

REPLY: done

 

Discussion needed to elaborate more by justifying the results of the present study particularly the importance of oil and extracts.

REPLY: we thank the reviewer for the suggestions and we modified the discussion accordingly. We also modified the conclusion

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This work evaluates the control efficacy of six commercial products to control grey mold and powdery mildew in strawberry plants under greenhouse conditions. Moreover, the authors also investigate the defence mechanisms, fruit production and quality.

The MS is well structured and provides detailed information using good language, which will be beneficial to readers and researchers in related fields. The MS is well prepared and easy to follow. However, some issues need further improvement. I only got several minor suggestions to further improve the MS.

1. Line 149. The basis for using dosage of commercial products needs to be appropriately explained.

2. Line 190. Which strain or isolate of P. aphanis was used?

3. Line 225. How many plants for each replication? How many replications? Are “CON” samples used as control for gene expression?

4. Suggest reflecting standard errors of each Mean in Figure 2, 3, and 4.

5. Line 270. It is better to display the preventive effects of powdery mildew using figures of strawberry leaves.

6. The results of Figure 5 are confusing. Zero folds is incorrect at 0 days after treatment. Please confirm the data of gene expression. Perhaps using column charts would be better. The significance of differences also needs to be annotated.

7. Table 2. Which samples do the significance represented by asterisks? Suggest removing the last line of Table 2 and adding letters after all the Means ± SE.

8. Experiment 3 has better control effects against powdery mildew Compared to Experiment 2. Those results should be discussed in the section of Discussion.

Author Response

Line 149. The basis for using dosage of commercial products needs to be appropriately explained.

REPLY: corrected

Line 190. Which strain or isolate of P. aphanis was used?

REPLY: wild isolates were collected for the same area were experiments were performed. The identity was confirmed by amplifying and sequencing ITS region.

Line 225. How many plants for each replication? How many replications? Are “CON” samples used as control for gene expression?

REPLY: we added the missing details(section 2.2)

Suggest reflecting standard errors of each Mean in Figure 2, 3, and 4.

REPLY: we are grateful for this suggestion. However, error bars are used correctly on natural number, whereas they should not be used in percentual values

Line 270. It is better to display the preventive effects of powdery mildew using figures of strawberry leaves.

REPLY: thanks for the suggestion. In strawberry, B. cinerea infects primarily flowers and consequently fruits. Furthermore, damages on flowers are the most important since they cause a direct loss of yield. Therefore, we believe that data on flowers, more than leaves, are relevant for practical application

The results of Figure 5 are confusing. Zero folds is incorrect at 0 days after treatment. Please confirm the data of gene expression. Perhaps using column charts would be better. The significance of differences also needs to be annotated.

REPLY: we are grateful for this comment and we agree that the figure needed to be improved. We included more information both in the material and methods and in the caption. Furthermore the Y axis label has been corrected

Table 2. Which samples do the significance represented by asterisks? Suggest removing the last line of Table 2 and adding letters after all the Means ± SE.

REPLY: done

Experiment 3 has better control effects against powdery mildew Compared to Experiment 2. Those results should be discussed in the section of Discussion.

REPLY: thank you for the suggestion. Concerning incidence (panel A of figures 3 and 4) there is no significant differences among experiments 2 and 3. We modified discussion by mentioning that, as far is cocenrned severity, the curative treatment (experiment 3) has a higher efficacy than the preventive one, despite not significantly different from the preventive treatment

 

 

 

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Although the reviewers have addressed most of the revision suggestions, I insist that the author should improve the charts, especially the lack of standard deviation. This is the most basic requirement for data presentation.

Author Response

Dear Editor, Dear Reviewer#2,

thanks for the suggestions and the time you dedicated to our manuscript. As requested we added the error bars to the graphs. As I previoulsy explained, error bars shold not be used on percentual value. Nonthless, the percentages have been arcsin transformed prior analysis of variance (see line 264) and thus, error bars can be used. We apologise for not having intially fullfilled your request

I'm now ulpoading the same recised version of the previsous round of revision. The only difference is that in it the new figures have been included

Author Response File: Author Response.pdf