Uptake, Distribution, and Activity of Pluronic F68 Adjuvant in Wheat and Its Endophytic Bacillus Isolate

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. “Key Contribution:”:

Delete it

 

2. Line 73-85:

Rewrite the aim of study in briefly and merge to end paragraph

 

3. In section “2.2” : the authors mentioned to “(seeds)”:

Seeds for who plant or plants and resource?

 

4. This term "probiotic" is not used in plant field but used endophyte. Therefore, must change in fulltext of manuscript

 

5. Line 374-377: From “These observations “ to end:

Delete "Further studies" from conclusions

 

Author Response

We thank the reviewer for their detailed comments. Please see the attachment for our detailed responses to all reviews. Thank you, sincerely, the authors.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review of Manuscript entitled: «Uptake, Distribution, and Activity of Pluronic F68 Adjuvant in Wheat and its Endophytic Bacillus isolate»
My comments:
1) What types of micelles are formed? Spherical, oval, mixed, etc.
2) The introduction is too small. I do not understand the self-use of pluronic as a reagent. As a delivery system, yes, I understand. Or is this a preliminary study, and then will the active components be loaded into micelles? I would like more specifics
3) More characteristics of the starting materials before and after the experiments are needed. Especially Fluorescent F68. Did the studied systems degrade during the experiments?
4) It is assumed that the Adjuvant will be administered with what substance. Do the authors intend to use the adjuvant individually?
5) I will clarify my questions with the author. In this article, F68 is used for visualization or as an adjuvant? If the first, then the work should be reworked for this concept. If the second, then the work should also be reworked. Now there is both visualization and constant references to its use as an adjuvant. It is not clear.
I recommend the publication of this work after major revision in Agrochemicals.

Author Response

We thank the reviewer for their detailed comments. Please see the attachment for our detailed responses to all reviews. Thank you, sincerely, the authors.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is a well-written manuscript with a clear goal: to examine a positive effect of Pluronic F68 on drought-stressed weed and its compatibility with the endophytic and rhizosphere microbiome. My questions and comments are:

Line 111: Seedlings were grown in quartz sand? Please specify.

Line 114: Please describe how PcO6 inoculum was grown.

Paragraph 2.3: In your experiment you studied the effect of F68, GB and F68+GB on wheat drought stress response. Why did you include PcO6 in this experiment? Why did you not test the effect of F68 and GB alone on wheat seeds without bacteria? Please explain the rationale for using PcO6. And give more background on this strain: is it growth-promoting? The way this experiment was organized, you cannot rule out that the effects you observed are attributed to some kind of interaction between F68 and the strain or GB and the strain. Also, you could test the effect of F68 and GB in wheat seedlings planted in natural soil. This would allow natural colonization of the rhizosphere. The use of a single strain for rhizosphere colonization is useful, but not close to realistic conditions.

Paragraph 2.4.2.: It is useful to study the effect of F68 to a second strain, an endophytic gram-positive Bacillus. It would be a lot more informative to conduct a metagenomics study on the endophytic and rhizosphere microbiome with and without addition of F68. 

Figure 5B: The V/Y shaped pairs of bacterial cells seem to be coryneforms.

Lines 311-318 in Discussion, 372 in conclusion: with two strain tested, the statement about the effect of F68 should not be broadened to the "bacterial microbiome". I would like to see a statement that more work is needed towards evaluating the effect of F68 and GB on the endophytic and rhizosphere microbiome, including metagenomics. 

Author Response

We thank the reviewer for their detailed comments. Please see the attachment for our detailed responses to all reviews. Thank you, sincerely, the authors.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

1. Line 75-83: from first line to "Protective compound.":
Rewrite the aim of study in briefly and merge to end paragraph

2. Line 83-85: "These experiments" to end:
Delete it 

3. In section “2.2” : the authors mentioned to “( wheat seeds)”:
The authors must mention the resource and cultivar of wheat seeds.

4. This term "probiotic" is not used in plant field but used endophyte. Therefore, must change in text of figure 1 and 2 of manuscript

5. The conclusions is poorly and need to rewrite without repeating the results and discussion but what are you concluded from your study.

 

Author Response

1. Line 75-83: from first line to "Protective compound.":
Rewrite the aim of study in briefly and merge to end paragraph

> We have further condensed the aim of the study provided as an overview in the Introduction to now read:

"In this paper the interactions of F68 were examined in wheat and a subset of its microbiome, consisting of a Gram-negative PcO6 epiphyte and a Gram-positive JunSE1L endophyte. The fate and transport of fluorescently labeled F68 in the rhizoplane were monitored to assess F68 membrane affinity, microbiome interactions, and mobility in plant cells. F68 biological activity in watered and droughted plants was compared to untreated controls or plants receiving the osmolyte glycine betaine (GB), utilized here as a known plant protective compound. These studies support suitability of F68 as an agricultural adjuvant."

2. Line 83-85: "These experiments" to end: Delete it 

> We have deleted the last sentence from the Introduction:

"These experiments aim to establish suitability of Pluronic F68 as an agricultural adjuvant for delivery of active ingredients as well as unique roles in improving nutrient utilization and repairing plant cell membranes damaged during abiotic stress."

3. In section “2.2” : the authors mentioned to “( wheat seeds)”:
The authors must mention the resource and cultivar of wheat seeds.

> Thank you for clarifying that we had omitted this information. We have begun section 2.2 with the following sentence providing the details of the wheat utilized in these studies: 

"Wheat seeds (Tritium aestivum, var. Juniper, Reg. No. CV-1021, PI 639951) is a hard red winter wheat developed by the Idaho Agriculture Experiment Station."

4. This term "probiotic" is not used in plant field but used endophyte. Therefore, must change in text of figure 1 and 2 of manuscript

> We have deleted "probiotic" from the Figure 1 and 2 captions. However, PcO6, is not an endophyte, it is a soil microbe the establishes an epiphytic life style on the wheat root surfaces (and does benefit the host plant against both biotic and abiotic stresses). The endophytic bacteria, JunSE1L, may also be released from roots to colonize the root surface, but PcO6 is a much more aggressive root-colonizer than JunSE1L and therefore dominates the root surface under our experimental conditions. For clarity we simply refer to PcO6 being on the root in Fig 1 and Fig 2 captions:

"Water loss from plants continually watered (W) or exposed to drought (D). All plant roots were colonized with PcO6, fertilized with..."

5. The conclusions is poorly and need to rewrite without repeating the results and discussion but what are you concluded from your study

> Conclusions now have additional content to provide what we conclude from the research:

"The absence of any detrimental impact on wheat or its microbiome support utilization of Pluronic F68 as an agricultural adjuvant. In an adjuvant role F68 may improve solubilization and delivery of active ingredients, including improved root ingress as demonstrated using fluorescein-F68. Whether F68 would transport encapsulated cargo into an intact root is unknown; however, that root-applied F68 increased shoot mass suggests that F68 improved nutrient acquisition in both sufficiently watered and drought stressed wheat. It is not believed that the plant directly utilizes F68 as a nutrient; indeed, neither PcO6 nor JunSE1 are able to utilized F68 as a carbon source. Thus, the designation of F68 as an inert GRAS compound appears to extend to agricultural settings.

With regard to its known role in cell membrane repair and anticipated benefits to plants under drought stress, where membrane leakage may occur, it was not possible to identify any benefit of F68 under the experimental conditions. This may reflect a need to increase the drought stress and / or F68 dosing levels. This is supported by the observation that root-applied glycine betaine, a protective osmolyte, did not benefit either watered or drought-stressed wheat under these experimental conditions. Transport of F68 and GB from roots to the shoots was not assessed, and considering the practicality of field-scale applications, wheat response following foliar delivery should be investigated. In sum, the observations with both the plant and their bacterial colonists support consideration of Pluronic F68 as an adjuvant for agricultural formulations."

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks to the authors for answering questions and correcting the manuscript. In the future, I recommend that authors highlight their edits in the text.

I recommend the publication of this paper in present form.

Author Response

Thanks to the authors for answering questions and correcting the manuscript. In the future, I recommend that authors highlight their edits in the text.

> The authors thank the reviewer for their suggestions. A track-changes PDF of the manuscript is attached to allow for easier review of the changes from the original submission.

I recommend the publication of this paper in present form.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I accept the response of the authors to my comments; the changes improved the manuscript.

Author Response

I accept the response of the authors to my comments; the changes improved the manuscript.

> The authors thank this reviewer for their comments to help improve the manuscript.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

I have one comments, as follows:

1. Write the conclusions in one paragraph

Author Response

I have one comments, as follows:

1. Write the conclusions in one paragraph

We have now combined the conclusions into a single paragraph. Thanks.