Non-Thermal Plasma Can Be Used in Disinfection of Scots Pine (Pinus sylvestris L.) Seeds Infected with Fusarium oxysporum

Round 1

Reviewer 1 Report

All comments for Authors could find on attachment.

Comments for author File: Comments.pdf

Author Response

The authors thank very much for the constructive comments and suggestions that improved the content of the manuscript.

Responses to suggestions

Lines 56-59: Please add this work in which authors worked with effect of plasma on wheat seed and Fusarium fungus:

Answer: Reference „Kordas L., Pusz W., Czapka T., Kacprzyk R. 2015. The effect of low-temperature plasma on fungus colonization of winter wheat grain and on seed quality. Polish Journal of Environmental Studies 24 (1): 433-438” has been added to text and reference list. (now in lines: 59-63).

Lines 92-94: from which regions of Poland? Only from Mazowieckie voivodship?

Answer: The pine seeds were collected from Forest District in Chojnów, Mazowieckie voivodeship and provided by Institute of Forest Research in Sękocin Stary near Warsaw, Poland. It has been clarified in text (now lines: 100-102).

Lines 97-98: Write please from which plants and organs of plants was isolated Fusarium oxysporum?

Answer: Fusarium oxysporum, isolate 342, was Pinus sylvestris pathogen isolated from pine root and obtained from Bank of Plant Pathogens, Institute of Plant Protection – National Research Institute, Poznań, Poland.It has been clarified in text (now lines: 106-108).

 Lines 109-111: Have you re-isolated F. oxysporum from infected pines according Koch's rules?

Answer: The Fusarium oxysporum has not been re-isolated from seeds or germinated seeds during this study. Density of fungal spores was set using hemocytometer chamber. In addition, inoculation solution with spores were spread over PDA medium to confirm spore viability. Moreover, Control 2 (surface sterilized seeds inoculated with spores of F. oxysporum) developed the monoculture of F. oxysporum after incubation time on YPD medium.

Lines 162-163 I know that main aim of investigation was check effect of plasma on pine's seedlings but have you got any results on effect plasma on F. oxysporum colonies, growth etc.?

Answer: We are thankful for this valuable comment. Unfortunately, we did not check effect of plasma on F. oxysporum colonies, growth etc., but we will consider this suggestion in the future work.

 Lines 223-225: Please add this work:

Kordas L., Pusz W., Czapka T., Kacprzyk R. 2015. The effect of low-temperature plasma on fungus colonization of winter wheat grain and on seed quality. Polish Journal of Environmental Studies 24 (1): 433-438.

Answer: this reference has been discussed in the text. (now lines 244-248).

Reviewer 2 Report

The authors of this manuscript tested a non-thermal plasma to disinfect the pine seeds surface from fungal spores. This manuscript is well-written and has a high quality of illustrations especially graphical abstract and figure 1 are well prepared however all bar charts can be edited to improve the quality of presentation. Statistical analysis of the experimental data correctly performed. I believe that the article can be accepted for publication in the Forests journal after a moderate revision. To enhance the quality of the paper I have the following questions, comments, and suggestions:

Line 2-4: Please use the upper case for highlighted words (according to the journal requirements).

Line 6: Please add "and" before the name of the last author (according to the format of this journal).

Line 32: All % should be next to the value with no blank space.

Line 36: if you mean the results of this study my suggestion is to rewrite the last statement of the abstract as follow or a similar statement (I believe it will improve the scientific soundness of the text):
"According to the results of this study, non-thermal plasma can serve as a seed surface disinfectant in the regeneration system of different pine species."

Line 41: A very clear graphical abstract.
Please double-check if the graphical abstract needs a caption?

Line 50-51: Fungal attacks are often fatal and can sharply reduce the seed germination or even eliminate the seedling establishment.

Line 52: For more clarification please provide at least one reference to support this statement about seeds and fruits being a fungal spores carrier

Line 75: Please be more specific and add if these studies were performed on seeds and mention which crop in particular?

Line 85: or "being desired,"

Line 85: "has the goal to evaluate"

Line 122: Please define the AC if it is the first time that is used in line 122.

Line 147: Do you have a reference for this formula? Technically "germination rate is the speed of germination and has the time unit such as hours or days and the definition of Germination Rate is Time to reach 50% of total germination.
The GR that you mentioned here is more close to the term "germination Energy" which the unit is %. Please make it clear.

Line 157: Observation

Line 163: Please add a subheading 3.1.

Line 173, 194, 198: in Materials and Methods.

Line 203: You may edit the subheadings and add 3.1. to the upper section and then 3.2. for this as 3.2. Morphological Observation.

Line 255: that will help to establish a successful strategy to regenerate trees through seeds.

Author Response

The authors thank very much for the constructive comments and suggestions that improved the content of the manuscript.

Responses to suggestions

Line 2-4: Please use the upper case for highlighted words (according to the journal requirements).

Answer: Title has been corrected accordingly.

Line 6: Please add "and" before the name of the last author (according to the format of this journal).

Answer: it has been added.

Line 32: All % should be next to the value with no blank space.

Answer: it has been corrected accordingly along the text.

Line 36: if you mean the results of this study my suggestion is to rewrite the last statement of the abstract as follow or a similar statement (I believe it will improve the scientific soundness of the text):
"According to the results of this study, non-thermal plasma can serve as a seed surface disinfectant in the regeneration system of different pine species."

Answer: sentence has been modified according to reviewer’s suggestion.

Line 41: A very clear graphical abstract.

Please double-check if the graphical abstract needs a caption?

Answer: we have not found in the instructions for authors that graphical abstract needs any caption.

Line 50-51: Fungal attacks are often fatal and can sharply reduce the seed germination or even eliminate the seedling establishment.

Answer: this sentence has been modified accordingly.

Line 52: For more clarification please provide at least one reference to support this statement about seeds and fruits being a fungal spores carrier.

Answer: appropriate references have been provided to support above sentence (Mancini, V,; Romanazzi, 2014; Zhang et al. 2019).

Line 75: Please be more specific and add if these studies were performed on seeds and mention which crop in particular?

Answer: these studies were performed on polymer (PLA) and seeds of wheat which were contaminated with different microorganisms. It has been specified in the text (now lines 79-83)

Line 85: or "being desired,"

Answer: “being sought” has been modified to “being desired”.

Line 85: "has the goal to evaluate"

Answer: “is the goal..” has been modified to “has the goal..”

Line 122: Please define the AC if it is the first time that is used in line 122.

Answer: AC (alternating-current) has been defined in the text (now line 134).

Line 147: Do you have a reference for this formula? Technically "germination rate is the speed of germination and has the time unit such as hours or days and the definition of Germination Rate is Time to reach 50% of total germination.

The GR that you mentioned here is more close to the term "germination Energy" which the unit is %. Please make it clear.

Answer: Not all seed germination parameters are well used, there are different interpretations in literature. These include the germination rate GR (e.g. see discussion on Reseach Gate: https://www.researchgate.net/post/What_is_germination_rate). There we used the article Šerý et al. 2020 to determine GR, where we used the formula, the number of pine seeds, and the design of the experiment in our manuscript.

Line 157: Observation

Answer: It has been changed from “observation” to “Observation”

Line 163: Please add a subheading 3.1.

Answer: the subheading “3.1. Effect of Plasma Treatment on Seed Disinfection and Germination” has been added.

Line 173, 194, 198: in Materials and Methods.

Answer: As above, the common subheading was provided.

Line 203: You may edit the subheadings and add 3.1. to the upper section and then 3.2. for this as 3.2. Morphological Observation.

Answer: It has been modified according to reviewer’s suggestion.

Line 255: that will help to establish a successful strategy to regenerate trees through seeds.

Answer: This sentence has been modified accordingly.