Soluble Eugenol Formulation for Managing Ball Moss on Ornamental Trees

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses an important horticultural challenge: controlling Tillandsia recurvata (ball moss) in ornamental trees. The authors propose a soluble eugenol formulation (ESL) as a botanical alternative to conventional methods (notably baking soda), and present results from laboratory, field, and treatment trials. The work is well-motivated, novel in its formulation approach, and has practical implications for arboriculture, landscape management, and environmental sustainability. Overall, the study is suitable for publication pending revisions to strengthen clarity, rigor, and contextualization.

 

 

The manuscript demonstrates several notable strengths. It addresses a widespread horticultural and ecological problem with both applied and theoretical significance, while introducing a novel strategy through steviol glycoside–based solubilization of eugenol to enhance efficacy and bioavailability. The experimental design is comprehensive, moving systematically from laboratory screening and dose–response studies to outdoor seasonal trials and field applications on live oak trees, thereby providing a convincing progression from proof-of-concept to practical implementation. Seasonal testing further strengthens the work by revealing phenological sensitivity and treatment constraints. Importantly, the study situates its findings within an environmental and practical context, comparing eugenol with baking soda in terms of phytotoxicity, ecological impact, and application feasibility, while acknowledging the limitations of different delivery methods such as direct spraying versus broadcasting, which is highly relevant for real-world horticultural management.

     

• Replication and sample size details are limited in the manuscript. For instance, in the live oak field trials, only two trees were tested—one receiving direct hand-spraying and the other broadcast spraying (lines 217–233). Such a small sample size makes it difficult to generalize the conclusions across diverse environments and host trees. Additional biological replicates would strengthen the validity of the findings. Statistical methods are briefly mentioned, with ANOVA and t-tests applied (lines 200–202), but key statistical outputs such as degrees of freedom, F-values, or exact p-values are not consistently reported.
• Figures (e.g., Figure 2, lines 278–284) show treatment effects but lack detailed statistical annotation, error bars clarity, and proper legends. Control treatments also raise concerns: ethanol alone caused approximately 80% injury to ball moss (lines 251–252), making it challenging to disentangle solvent effects from the activity of eugenol and thymol. While the Discussion acknowledges this (lines 371–373), a deeper quantitative interpretation would clarify the extent of solvent interference.
• The claim that ESL is “comparable” to baking soda requires moderation. For example, in autumn field trials, ESL produced 98.8% injury—close to the 100% injury caused by baking soda (lines 313–315). However, in spring trials, ESL only achieved 53.7% injury (line 309), and in broadcasting trials on live oaks, ESL showed no significant effect (lines 334–336). Thus, while ESL demonstrates strong potential, it does not consistently match the efficacy of baking soda across all contexts. Furthermore, mechanistic explanations—such as nano-micelle penetration and “glassification” effects (lines 389–411)—are speculative.
• The study focuses on short-term injury outcomes with a two-week observation period (lines 191–199, 235–236), but does not evaluate long-term persistence of effects or potential ball moss re-establishment. Assessing regrowth over longer intervals would be critical for practical management recommendations. Host plant safety is mentioned, with observations of leaf curling in young oak leaves (lines 331–332) and phytotoxicity from baking soda in fall trials (lines 320–323). However, these assessments are qualitative. Quantitative measures of host plant injury, such as leaf necrosis percentages, growth impacts, or recovery dynamics, would greatly improve reliability.

Some revisions, and comments to be addressed

1. Provide detailed replication and statistical reporting (including degrees of freedom, F- or t-values, and exact p-values).

2. Reframe conclusions: present ESL as a promising and safer alternative, but not yet fully comparable to baking soda under all conditions.

3. Clarify mechanistic discussion as speculative hypotheses until further evidence is obtained.

4. Expand limitations to include long-term control efficacy, quantitative host phytotoxicity assays, and considerations of cost-effectiveness and scalability.

5. Improve clarity of figures/tables, streamline the Discussion to reduce redundancy, and enhance the reference list with recent work on essential oil formulations and bioherbicides.

6. Consider including a schematic figure summarizing application methods, outcomes, and limitations to aid practical understanding for readers and practitioners.

Author Response

Please see the attachment.

Comments 1: Provide detailed replication and statistical reporting (including degrees of freedom, F- or t-values, and exact p-values).

Response 1: We provided these details throughout the results section and the revisions are highlighted.   

Comments 2: Reframe conclusions: present ESL as a promising and safer alternative, but not yet fully comparable to baking soda under all conditions.

Response 2: This is a good comment. Thank you. We have reframed the conclusions.

Comments 3: Clarify mechanistic discussion as speculative hypotheses until further evidence is obtained.

Response 3: This is a good point. We added a sentence at the end of hypotheses to indicate this.

Comments 4: Expand limitations to include long-term control efficacy, quantitative host phytotoxicity assays, and considerations of cost-effectiveness and scalability.

Response 4: Thank you! These are great comments.

Comments 5: Improve clarity of figures/tables, streamline the Discussion to reduce redundancy, and enhance the reference list with recent work on essential oil formulations and bioherbicides.

Response 5: We revised based on these comments. We clarified the figures and tables, reduced some degree of redundancy, and enhanced the reference list. A more recent (2025) work on a water-soluble formulation of eugenol and on bioherbicides were included.    

Comments 6: Consider including a schematic figure summarizing application methods, outcomes, and limitations to aid practical understanding for readers and practitioners.

Response 6: We submitted a graphic abstract summarizing these points. We hope that provides a schematic tool for readers and practitioners to understand this study better. 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The main problem of manuscripts is that it is not clear whose effects were studied – whether thymol and eugenol as pure compounds, or thyme and clove essential oils containing these compounds, respectively. The methodology is written one way, in one Figure or Table, another way, and in other Figures or Tables – still another way. It is necessary to rewrite the entire manuscript so that the reader understands clearly all the experiments and results.

Some more specific notes:

1) Since the effect of eugenol was not the only one studied, it is necessary to change the title to something more representative of the content of the study.

2) Figure 1 and Figure 4 must be deleted; they are completely unnecessary.

3) Table 1: The information provided here is very confusing, because it is not clear what was used - whether with pure thymol and eugenol, or essential oils of thyme and clove.
4) Table 1: What do "ai Concentration in product" and "ai concentration in spray solution" mean? And whose concentrations are indicated everywhere - are they concentrations of thymol and eugenol, or concentrations of thyme essential oil and clove essential oil?

5) The methodology (lines 114-116) states that “... copper fungicide, a monocot herbicide, baking soda, oleander leaf or twig alcoholic extract, thymol (isolated from thyme essential oil), and eugenol (isolated from clove essential oil)” were used as candidate chemical agents. However, according to the results presented in Figure 2, Table 2, it is understandable that not thymol and eugenol, but thyme essential oil and clove essential oil, respectively, were used in experiments. Late, in Tables 3 and 4, the results with the effects of pure compounds (not essential oils) have already been presented... Complete confusion... What was used: pure compounds thymol and eugenol, or thyme and clove essential oils? This is a very big difference, because thymol and eugenol are pure compounds, and essential oils are mixtures of a lot of compounds.

6) Figure 2: here, in axis X, must be indicated concrete/exact concentrations of treatment, but not "Clove-0", "Clove-1", etc.

7) Figure 2: The title of the figure indicates that a t-test was used (it can be understood that each concentration was compared with soda). However, according to the letter marking above the columns, not t-test was used... It is necessary to mark the letters correctly, because now the letters do not correspond to what is declared in the title of the table.

Author Response

Please see the attachment.

Comments and Suggestions for Authors

The main problem of manuscripts is that it is not clear whose effects were studied – whether thymol and eugenol as pure compounds, or thyme and clove essential oils containing these compounds, respectively. The methodology is written one way, in one Figure or Table, another way, and in other Figures or Tables – still another way. It is necessary to rewrite the entire manuscript so that the reader understands clearly all the experiments and results.

Response: Thank you for pointing this out. We have cleared this confusion throughout the manuscript.

Some more specific notes:

1) Since the effect of eugenol was not the only one studied, it is necessary to change the title to something more representative of the content of the study.

Response: We considered your comment on the title. Even though eugenol was not the only one studied, it was the focus of the study. We would like to keep this focus. The screening was a part of the study, and the graphic abstract would show readers that other things have been examined.  We hope you agree with us.

2) Figure 1 and Figure 4 must be deleted; they are completely unnecessary.

Response: We agree with you about removing Figure 1. However, Figure 4 is important, especially for practitioners because it showed how different ways of application made a life-or-death difference. We hope you agree with us on keeping Figure 4 (now Figure 3 after removing Figure 1).     

3) Table 1: The information provided here is very confusing, because it is not clear what was used - whether with pure thymol and eugenol, or essential oils of thyme and clove.

Response: We apologize for this confusion and appreciate your catching these errors. We have cleared them.

 
4) Table 1: What do "ai Concentration in product" and "ai concentration in spray solution" mean? And whose concentrations are indicated everywhere - are they concentrations of thymol and eugenol, or concentrations of thyme essential oil and clove essential oil?

Response: This clearly caused confusion. We corrected errors and inconsistency in Table 1.  

5) The methodology (lines 114-116) states that “... copper fungicide, a monocot herbicide, baking soda, oleander leaf or twig alcoholic extract, thymol (isolated from thyme essential oil), and eugenol (isolated from clove essential oil)” were used as candidate chemical agents. However, according to the results presented in Figure 2, Table 2, it is understandable that not thymol and eugenol, but thyme essential oil and clove essential oil, respectively, were used in experiments. Late, in Tables 3 and 4, the results with the effects of pure compounds (not essential oils) have already been presented... Complete confusion... What was used: pure compounds thymol and eugenol, or thyme and clove essential oils? This is a very big difference, because thymol and eugenol are pure compounds, and essential oils are mixtures of a lot of compounds.

Response: We totally agree and apologize for the confusion it has caused. Only single compounds eugenol and thymol were used in the study. No essential oils of clove or thyme were used. We have corrected this and hopefully the confusion.  

6) Figure 2: here, in axis X, must be indicated concrete/exact concentrations of treatment, but not "Clove-0", "Clove-1", etc.

Response: We’ve changed this to Eugenol-0, Eugenol-3, … and described what it indicates.

7) Figure 2: The title of the figure indicates that a t-test was used (it can be understood that each concentration was compared with soda). However, according to the letter marking above the columns, not t-test was used... It is necessary to mark the letters correctly, because now the letters do not correspond to what is declared in the title of the table.

Response: Thank you for pointing this out. We have provided detailed statistical reporting and hopefully addressed your comment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Slade et al. have made substantial revisions based on my suggestions, and the paper is now worthy of publication. Congratulations on your hard work and commitment.

Author Response

Thank you so much for your expertise and we appreciate your constructive suggestions and comments. They certainly enhanced the quality of our paper and readability for practitioners. 

Reviewer 2 Report

Comments and Suggestions for Authors

1) Please correct numeration of Figures. At present manuscript version it is incorrect.

2) Lines 200-203: in the firs manuscript version you indicated that statistical analysis was performed by SAS (Cary, NC). In the last manuscript version you declare already that only Microsoft Excel (2025, Version 2507) was used for statistical analysis. Really your performed analysis of variance, Bonferroni corrected and t-test by Excel? Absolutely not. Therefore, please correctly (i.e. without lying) indicate the statistical program that was used to perform all statistical analysis of the data.

3) Table 1: What do "ai Concentration in product" and "ai concentration in spray solution" mean?

4) Figure 2: In the title of the figure you indicate that a t-test was used here. However, based on the letter designation above the bars, a t-test was not used. Therefore, please indicate the statistical analysis method that was actually used to perform the statistical analysis of the data presented in this graph.

Author Response

1) Please correct numeration of Figures. At present manuscript version it is incorrect.

 Response: Thank you for catching that! We’ve had it corrected. It was Figure 4 that should have been Figure 3.

2) Lines 200-203: in the firs manuscript version you indicated that statistical analysis was performed by SAS (Cary, NC). In the last manuscript version you declare already that only Microsoft Excel (2025, Version 2507) was used for statistical analysis. Really your performed analysis of variance, Bonferroni corrected and t-test by Excel? Absolutely not. Therefore, please correctly (i.e. without lying) indicate the statistical program that was used to perform all statistical analysis of the data.

Response: Thanks for noticing this and asking for this question. You are right; it was analyzed initially by SAS but we decided to do/re-do all the statistical analyses other than SAS. This is because of the required SAS annual license renewal, and that when installing on a new computer, IT geek must be involved as we experienced it. Using SAS can be a hassle. This is the first time we switched from SAS to Excel and we hope more people who read this paper will know Excel can do the stat analyzes just like SAS. To answer your question, YES, all of the analyses presented were performed by Excel, 100%! It not only can do one factor but also two-factors ANOVA, t-tests, and yes, the Bonferroni correction. If you would like to learn more about these analyses on Excel, the corresponding author would be happy to show you how with all respects. However, you would have to agree to re-track the pre-mature “Absolutely not” conclusion.

3) Table 1: What do "ai Concentration in product" and "ai concentration in spray solution" mean?

Response: we regret the confusion, We added the following in yellow highlight to clarify the meaning.  Table 1 provides treatment information including the concentration of active ingredients (ai) in a product or formulation if known, solvent used for preparing a spray solution, and the ai concentration in the spray solution.

4) Figure 2: In the title of the figure you indicate that a t-test was used here. However, based on the letter designation above the bars, a t-test was not used. Therefore, please indicate the statistical analysis method that was actually used to perform the statistical analysis of the data presented in this graph.

Response: We agree you are right. The t-tests were performed (again on Excel) but the letters were not corrected. We have corrected these and added the Bonferroni corrected alpha in the figure caption.  We thank you for catching this error!