Integrated Assays and Microscopy to Study the Botrytis cinerea–Strawberry Interaction Reveal Tissue-Specific Stomatal Penetration

Round 1

Reviewer 1 Report

Article "Integrated assays and microscopy to study the Botrytis cinerea-strawberry interaction reveal tissue-specific stomatal penetration" by authors Lorena Rodriguez Coy et al presents the results of a qualitative analysis of damage to vegetative and generative organs of strawberries by gray mold.
The study is distinguished by a versatile original methodic approach.
The manuscript is formatted with some violations, the necessary fonts and numbering are missing.
In the last paragraph of the Introduction section, instead of formulating the hypothesis being tested, as well as the goals and objectives, the results are briefly stated. They should be removed or moved to the appropriate section, perhaps to the Conclusion section
The methodology mentions water agar without explaining what the concentration is, what quantity, how many repetitions. There is also an issue with PDA agar. Correct this.
It is necessary to indicate the company, city and country of the manufacturer.
The authors demonstrate that the achenes (nuts) have stomata, but do not show what this entails. It should be shown and discussed that these achenes do not germinate, or that they die during germination. Otherwise, it is not clear what this means. Is this important for breeding? It is unlikely that anyone will eat spoiled berries or process them...
After correction, the article can be published.

Author Response

We thank the reviewer for helping us improve our manuscript. Answers to each issue raised are below.

Article "Integrated assays and microscopy to study the Botrytis cinerea-strawberry interaction reveal tissue-specific stomatal penetration" by authors Lorena Rodriguez Coy et al presents the results of a qualitative analysis of damage to vegetative and generative organs of strawberries by gray mold.

The study is distinguished by a versatile original methodic approach. The manuscript is formatted with some violations, the necessary fonts and numbering are missing.

In the last paragraph of the Introduction section, instead of formulating the hypothesis being tested, as well as the goals and objectives, the results are briefly stated. They should be removed or moved to the appropriate section, perhaps to the Conclusion section

We thank the reviewer for the suggestion. The last paragraph has been incorporated at the conclusion of the manuscript.

The methodology mentions water agar without explaining what the concentration is, what quantity, how many repetitions. There is also an issue with PDA agar. Correct this.

It is necessary to indicate the company, city and country of the manufacturer.

Missing concentrations have been added, and the manufacturer of all reagents has been added throughout the methods section.

The authors demonstrate that the achenes (nuts) have stomata, but do not show what this entails. It should be shown and discussed that these achenes do not germinate, or that they die during germination. Otherwise, it is not clear what this means. Is this important for breeding? It is unlikely that anyone will eat spoiled berries or process them...

A line highlighting that achenes might a point of entry for fungal pathogens has been added to lines 613-623. This means that studying the distribution and function of achene stomata can provide tools to understand fungal penetration and may lead to the development of novel control strategies.

After correction, the article can be published.

Reviewer 2 Report

The core question addressed by this research is the infection mechanism of Botrytis cinerea, a highly important and devastating crop pathogen. This topic is undoubtedly relevant to the field of developing more effective plant protection technologies against this plant pathogen based on understanding the fundamental biology of B. cinerea. While not entirely novel in its broad subject matter, the research distinguishes itself by description the fungus's infection mechanism on different parts of strawberry, thereby addressing a clear gap in the understanding of infection mechanism.

This study significantly enhances the subject area by describing a straightforward method to facilitate the comparison of pathogenicity potential and fungicide sensitivity among different isolates. For methodological improvements, the authors might consider using chlorophyll fluorescence analysis and confocal microscopy for the quantification of damage (lesion) caused by B. cinerea. Scanning electron and confocal microscopy demonstrated a new infection route of the pathogen fungus B. cinerea, demonstrating that it penetrates through stomata of strawberry achenes. The methodology, repetition number and statistical analysis are appropriate. High-quality images effectively showcase the B. cinerea infection mechanisms on strawberry, proving to be highly informative. The figures, though complex, skillfully visualize the most important results in an accessible and intuitive manner.

The conclusions are strongly supported by the results data and microscopic pictures and clearly summarize the most important novelties of the study.

The references are appropriate and comprehensive, providing a solid foundation for the research citing the most important previous results connected to the studied question.  

Minor remarks:

Row 50: Although Wheat and corn was reported as host of B. cinerea, this is not considered as their mayor pathogens. Please delete them.

Rows 205-206: add colons (:) and correct to ’followed by 1:1 ethanol:hexamethyldisilazane’

Row 836: change “olletotrichum” to Colletotrichum”

Refs No. 63 and 65 are the same. Delete one of them.

Author Response

We thank the reviewer for helping us improve our manuscript. Answers to each issue raised are below.

The core question addressed by this research is the infection mechanism of Botrytis cinerea, a highly important and devastating crop pathogen. This topic is undoubtedly relevant to the field of developing more effective plant protection technologies against this plant pathogen based on understanding the fundamental biology of B. cinerea. While not entirely novel in its broad subject matter, the research distinguishes itself by description the fungus's infection mechanism on different parts of strawberry, thereby addressing a clear gap in the understanding of infection mechanism.

This study significantly enhances the subject area by describing a straightforward method to facilitate the comparison of pathogenicity potential and fungicide sensitivity among different isolates. For methodological improvements, the authors might consider using chlorophyll fluorescence analysis and confocal microscopy for the quantification of damage (lesion) caused by B. cinerea. Scanning electron and confocal microscopy demonstrated a new infection route of the pathogen fungus B. cinerea, demonstrating that it penetrates through stomata of strawberry achenes. The methodology, repetition number and statistical analysis are appropriate. High-quality images effectively showcase the B. cinerea infection mechanisms on strawberry, proving to be highly informative. The figures, though complex, skillfully visualize the most important results in an accessible and intuitive manner.

The conclusions are strongly supported by the results data and microscopic pictures and clearly summarize the most important novelties of the study.

The references are appropriate and comprehensive, providing a solid foundation for the research citing the most important previous results connected to the studied question. 

Minor remarks:

Row 50: Although Wheat and corn was reported as host of B. cinerea, this is not considered as their mayor pathogens. Please delete them.

Non-major hosts of B. cinerea have been removed.

Rows 205-206: add colons (:) and correct to ’followed by 1:1 ethanol:hexamethyldisilazane’

Thanks for highlighting. The colon has been added.

Row 836: change “olletotrichum” to Colletotrichum”

The mistake has been corrected.

Refs No. 63 and 65 are the same. Delete one of them.

We thank the reviewer for finding the duplication. We have corrected the issue.