Evaluation of Foliar Application of Salicylic Acid for Plasmodiophora brassicae Infection in Brassica napus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of the article «Impact of foliar applied salicylic acid on Plasmodiophora brassicae infection in Brassica napus» by Emilee Storfie, Sheau-Fang Wang, and Stephen Strelkov

 This article presents a comprehensive analysis of weekly salicylic acid (SA) treatments on the reduction of clubroot development in rutabaga, assessed by dormant pathogen spore load, physiological parameters (height, phytotoxicity), and the accumulation of SA-responsive gene transcripts. Negative results were obtained in most cases (no statistically significant effects). I appreciated the logical use of color schemes in the figures; the detailed and clearly described methodology; and the honest description of the results (moderate effect where present; no effect where the effects are insignificant).

There are a number of comments and recommendations regarding this study:

1) As the authors themselves note, a very high pathogen level (1×10⁷ spores/ml) was chosen for the experiments. This is a major methodological limitation. In this case, using a spore concentration gradient would have been more informative. Therefore, it is recommended to conduct a pilot experiment with different inoculum levels.

2) The article presents a suboptimal treatment regimen – weekly spraying with fixed SA concentrations without regard to plant or pathogen developmental stage. Several studies show that the timing of treatment is critical for resistance induction. Treatments should be linked to key stages of infection (e.g., before inoculation and during primary/secondary infection).

3) Late sampling for molecular analysis. Sampling one week after the last treatment likely missed the peak expression of genes associated with SA signaling, which are often transient. Therefore, I recommend taking dynamic samples for gene analysis (e.g., 24, 48, and 72 hours after treatment).

4) Lack of data on endogenous SA levels. It would be crucial to measure the increase in SA concentration in the roots after foliar application. Without this data, the assertion of a systemic response remains indirect. Therefore, it is necessary to measure SA levels in the roots and leaves after application.

5) Although a 5 mM SA concentration provided the greatest numerical reduction in disease, the difference compared to the control was not significant. Relying heavily on this finding is risky. It is more of a guide for future research than a reliable recommendation.

6) Issue of reproducibility of conditions. The difference between the greenhouse and growth chamber (where the effect was weaker) demonstrates how dependent the results are on environmental conditions (light, humidity, stress). This reduces the hope of a consistent effect in the field.

7) A significant portion of the Discussion is a literature review on the role of SA, which goes somewhat beyond the immediate results of the study. It should be revised.

8) You also note in the discussion that early in infection, the SA response is induced in both resistant and susceptible plants. Subsequently, SA signaling is maintained in resistant plants, while it is suppressed in susceptible plants. In susceptible Laurentian plants, treatment with 5 and 10 mM SA resulted in a significant decrease in BnPR1 expression compared to the control. It appears that exogenous SA did not enhance, but rather suppressed, the expression of the key gene at the time of sampling (6 weeks post-inoculation). This apparent contradiction should probably be discussed in more detail in the discussion.

9) In light of the above, the title of the article with negative results should be changed, for example to "Limited efficacy of foliar SA against pathotype 3A..." or "Evaluation of...".

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments and Suggestions for Authors

The manuscript entitled “Impact of foliar applied salicylic acid on Plasmodiophora brassicae infection in Brassica napus” presents a well-designed study addressing an important and timely topic in clubroot management. The experimental approach is sound, the methodology is generally clear, and the discussion is well aligned with the results and relevant literature. However, there are still a few minor issues that should be addressed before the manuscript can be considered for publication.

In particular, some sections would benefit from improved clarity and conciseness, especially where results are described in parallel for greenhouse and growth chamber trials, to avoid redundancy.

Minor grammatical and typographical errors are present throughout the text and should be corrected with careful proofreading.

In addition, the statistical reporting could be slightly improved by consistently indicating significance levels in the text when referring to figures and tables. Addressing these minor points will further enhance the readability and overall quality of the manuscript.

Comments

1. Lines 11–15. The abstract clearly summarizes the experimental design and main findings; however, consider explicitly stating the number of biological replicates and experimental environments (greenhouse and growth chamber) to strengthen transparency.
2. Lines 17–21. The phrase “mostly not statistically significant” could be rephrased more precisely (e.g., “did not reach statistical significance in most cases”) to maintain a formal scientific tone.
3. Lines 34–39. While the role of SA crosstalk with other phytohormones is well described, briefly indicating how this crosstalk may influence clubroot development specifically would help strengthen the conceptual linkage to the study objective.
4. Lines 69–76. The discussion of previous SA application methods is informative; however, a short justification explaining why foliar application was prioritized over soil or root-based methods.
5. Lines 95–100. Please specify whether resting spore viability was assessed before inoculation.
6. Lines 122–124. It would be helpful to clarify whether a surfactant was used during foliar SA application and, if not, to briefly justify this choice, as it may influence uptake efficiency.
7. Lines 132–136. Consider adding a brief justification for the chosen DSI scale and citing its relevance or validation for B. napus–P. brassicae interactions to reinforce methodological rigor.
8. Lines 142–143. The manuscript states that pooled root samples were stored at −20 °C before processing; however, the rationale for selecting this storage temperature is not explained. Please clarify why −20 °C was considered appropriate for preserving both DNA and RNA integrity in root tissues, and indicate the duration for which samples were stored at this temperature before extraction. Providing this information is important for assessing sample stability. In addition, the authors are encouraged to cite relevant methodological references demonstrating that short- to medium-term storage of plant tissues at −20 °C does not compromise downstream DNA/RNA quality, or to justify this choice in comparison with −80 °C storage commonly used for RNA preservation.
9. Lines 197–200. Please clarify whether the assumptions of ANOVA (normality and homogeneity of variance) were tested before analysis. If not, briefly stating this as a limitation would improve statistical transparency.
10. Lines 248–250. The text notes a significant difference only for DSI in one treatment. For clarity, please explicitly identify this treatment in the text to better guide the reader when interpreting Figure 3.
11. Lines 259-260. The use of a single significance threshold (p ≤ 0.05) indicated by asterisks may oversimplify the interpretation of statistically significant differences. Please consider reporting exact P-values where possible, or distinguishing different levels of significance using multiple symbols (e.g., p ≤ 0.05, p ≤ 0.01, p ≤ 0.001). This would provide a more precise and rigorous representation of the statistical strength of the observed treatment effects.
12. Lines 355–357. The explanation regarding sampling time potentially missing transient transcriptional responses is reasonable; however, briefly suggesting an alternative sampling strategy (e.g., earlier time points after SA application) would strengthen the forward-looking aspect of the discussion.
13. Lines 383–392. The Conclusions section effectively summarizes the main findings of the study; however, it could be further strengthened by slightly moderating the statements to reflect the largely non-significant treatment effects observed more clearly.
14. Lines 383–392. Please ensure that the conclusions remain fully aligned with the statistical outcomes and avoid any potential overinterpretation of the modest reductions in disease severity and resting spore levels. In addition, briefly restating the key limitations of the study (e.g., high inoculum pressure, timing of SA application, or foliar delivery method) within the Conclusions would improve balance and rigor, while reinforcing the need for the proposed future research directions.

Overall, addressing these minor points will further improve clarity, reproducibility, and the interpretative strength of the manuscript without requiring additional experiments.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Minor grammatical and typographical errors are present throughout the text and should be corrected with careful proofreading.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf