Ozone in Droplets and Mist in Inhibition of Phytopathogenic Microbiota
Round 1
Reviewer 1 Report
The manuscript (Ozone in droplets and mist in inhibition of phytopathogenic microbiota) evaluates the potential of ozonated water against phytopathogenic bacteria and fungi.
L10 I prefer to use Microgram/liter instead of Part per million. Convert, please.
L19 You wrote (underlying the power of ozone in treating fungal contamination). I do think it is too general. I prefer to be more specific because you only tested B. cinerea.
L40 You wrote (Overall, there are over 150 bacterial, and over 8000 fungal species that damage the plant). Please carefully check the numbers of bacterial and fungal species.
L135 Please write the composition and preparation of brain heart infusion agar.
The results in Figures 3 and 4 are not compatible and consistent. I can see from the picture that 4 ppm mist is better than 4 ppm droplet while in Figure 4 the droplet application is a little more effective. Can you explain?
L456 You wrote (the growth of plant pathogens that could replace the conventional biocidal agents that are considered highly toxic). I do not agree with this as you only tested the potential of ozone on two phytopathogenic bacteria and only one fungus. Several studies should be handled against several plant pathogens (including viruses) to conclude the potential of ozone against plant pathogens.
I think the manuscript should be checked carefully for the English language. I recommend you use an English editing service.
You can find below some suggestions to improve the writing.
L27 Please change to (, resulting in over 220 billion USD).
L53 Grammatical error change to (which have proven effective in inhibiting).
L56 Please change to (with a maximum concentration not exceeding 0.001%).
L102 Please change to (Tap water … ).
L162 Please change to (until cell turbidity… ).
L305 Change to the development and application
L332 Change to achieved by an ozone concentration of
L348 Change to none of the E. amylovora have been published so far
L356 Change to E. carotovora were achieved, respectively.
L388 Change to sprayer in the form of droplets is more efficient in inhibiting bacterial growth.
L397 Change to aqueous ozone applied in the form of the droplets.
L327 Change to not much data on the effect of ozone
L435 Change to young microbial cultures that make them
L449 Change to applying ozone in the form of droplets
Author Response
Dear reviewer 1,
Please see the attachment.
With kind regards,
authors
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This paper presents tests on the impacts of aqueous ozone on microbial colony formation based on concentration and method of delivery. Although the preliminary data appears sound, there are major deficiencies in the explanation of the methods, especially as relating to how the data were generated. I outline my major concerns below. Although all errors are not noted in their entirety, there are many typos and language errors throughout that also require editing.
Additional information is needed on how colonies were counted – per the methods there were no dilutions made, just direct mixing of treated cells with BHI/PDA agar (concentration not specified) – which would result in bacterial growth throughout the medium, not only on the surface. You are reporting 106 CFU/mL with 1mL of bacteria per plate. How can you count 106 colonies per plate? Or 104 colonies per plate for that matter? And how would you differentiate between the two quantitatively – both of these CFU numbers would yield a complete lawn on a single plate. If the colonies were not actually counted, they should not be presented (or analyzed) as quantitative data, they should be treated as qualitative. These must be addressed in order to evaluate the remainder of the study, because the quality of the data and interpretations thereof depend on the methods and quality of colony counts.
It is also a major problem that the preliminary experiment was conducted with more replicates (3) than the “main” experiment (2 replicates).
Figures showing percent reduction (Figures 1b, 4b, 4d, 4f, and 5b) are not very helpful, especially since the data are redundant with the other data being presented (Figures 1a, 4a, 4c, 4e, and 5a) and are not visibly helpful, being on a linear instead of log scale. If it is desired to keep the percent reduction figures, I recommend they be included as supplemental. The viable cell count graphs should remain in the main text and should be the basis of statistical comparison.
Figure 3 would greatly benefit if higher resolution images are available. The cell concentration needs specified for the images also, not just the treatment group. And if different dilutions are presented in each panel, these need to be specified – otherwise, the visible numbers of colonies distinguishable do not visibly appear to match any dataset presented in the graphs in Figure 4.
Figure 3d and 3e are the same image – this error needs corrected.
Viable cell counts should include control values – these are included in Figure 2, but need added to Figures 4 & 5.
The introductory and conclusion text suggests field application of ozone, but only (barely) considers phytotoxicity in a post-harvest application setting. It would be nice to include discussion of published data regarding phytotoxicity of aqueous ozone. Similarly, it would be nice to include discussion of how ozone – as a non-specific antimicrobial – may impact beneficial microbes, or the microbiome at large if used in field settings.
Line 15 (and throughout – see also lines 144-145, 166-167, 219, etc.) – please use exponential notation [104 CFU/ml] instead of 4 log, unless this format is specifically requested by the journal.
Line 263-264 needs a qualifier that 2ppm mist was not bactericidal to the tested bacteria at high or medium concentrations (because did show inhibitory effects for low concentrations of bacteria). In fact,
Author Response
Dear reviewer 2,
please see the attachment.
With kind regards,
authors
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors investigated Ozone in droplets and mist in inhibition of pathogenic microorganism. It is important to explore new agents for controlling pathogenic microorganism. Therefore, the topic deserves to be investigated. Manuscript is clearly written, however, some points should be improved. My recommendations are as follows:
Point 1: the title must be redesigned.
Point 2: Abstract- the abstract must be redesigned and focus on the effect of different aqueous Ozonated water in form of droplets and mist, on the survival of phytopathogenic bacteria Erwinia amylovora, Pseudomonas syringae, and Agrobacterium tumefaciens and fungus Botrytis cinerea; ozone concentrations and exposure time; significant finding.
Point 3: Even though the microbial removal of E. coli was enhanced with the increasing aqueous ozone concentration and time of exposure the differences were not significant. Effects of different ozone water concentration and exposure time on survival rate of selected plant pathogens?
Point 4: Also, the authors should significantly improve the discussion and try to explain the mechanism of antifungal activity of ozone.
Author Response
Dear reviewer 3,
please see the attachment.
With kind regards,
authors
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The comments were answered by authors.
I still believe you should write the composition of brain heart infusion agar.
Thank you
Author Response
Thank you very much for your efforts and time. We have used commercially available brain heart infusion agar, and stated the exact manufacturer and country of origin, as is customary in writing a research paper. Hence, we must respectfully decline your suggestion.
With kind regards.
Reviewer 2 Report
In the revised manuscript, the authors have satisfactorily addressed several concerns, however, there remain concerns that have not been addressed in a satisfactory manner.
I maintain that additional replicates should be included. The experiments in question are simple and inexpensive, and there appears to be no valid justification for not adding additional replicates other than the authors personal preference. Comparing several treatments with each other, with only 2 replicates each, is poor scientific practice and leaves large room for doubts regarding the validity of the data – especially as it is unclear whether the first 2 replicates were conducted at the same time.
There remain glaring language errors that have not been addressed.
Although the methods section has been amended, section 2.3.2 still does not specify that dilutions were conducted after treatment with ozone.
Figure 3: Because the plates are inconsistent in the dilution used, the graphical utility of the images is lost and becomes visually misleading in regard to quantitative comparison. For this reason, I recommend this figure be removed or moved to supplemental.
Author Response
Please see the attachment
With kind regards.
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors have improved the manuscript. It is now in much better shape.
Author Response
Thank you for your efforts and time.
With kind regards.
