Enhancing Tomato (Solanum lycopersicum L.) Resistance Against Bacterial Canker Disease (Clavibacter michiganensis ssp. michiganensis) via Seed Priming with β-Aminobutyric Acid (BABA)
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript explores the effect of BABA-seed priming on Clavibacter michiganensis ssp. michiganensis (Cmm), which causes bacterial cancer in tomato (Solanum lycopersicum L.), was investigated. Furthermore, it has been shown that the use of BABA as a spray or water-priming application gives better protection than the control. The qRT-PCR results showed that compared with the control group, the expression of NPR1 gene significantly increased after BABA-Priming treatment. BABA-seed priming has a beneficial effect on plant protection against Cmm invasion. However, the manuscript still has some issues that need further improvement.
1. Sections 2.1 and 2.2 contain some duplicated descriptions. Please integrate and remove the duplicated content.
2. The descriptions of L159-L166 and L168-L175 in Section 2.4 contain a significant amount of duplicate content. Please revise and remove the duplicate content.
3. It is recommended to unify the abbreviated names of each processing group in the chart to improve the readability of the article, such as using "NP+Cmm" to represent "Non Primed+Cmm" uniformly.
4. The caption in Figure 3.4 does not indicate "LSD: 0. xxx" in the legend. Please add it.
5. It is recommended to renumber all figures and tables in the manuscript according to journal requirements and standards.
Author Response
Responses to Reviewer 1
This manuscript explores the effect of BABA-seed priming on Clavibacter michiganensis ssp. michiganensis (Cmm), which causes bacterial cancer in tomato (Solanum lycopersicum L.), was investigated. Furthermore, it has been shown that the use of BABA as a spray or water-priming application gives better protection than the control. The qRT-PCR results showed that compared with the control group, the expression of NPR1 gene significantly increased after BABA-Priming treatment. BABA-seed priming has a beneficial effect on plant protection against Cmm invasion. However, the manuscript still has some issues that need further improvement.
We are grateful for the inputs and suggestions of the Reviewer 1. We carefully edited entire manuscript based on the inputs and suggestions of all three reviewers. Please find our responses to each query below.
- Sections 2.1 and 2.2 contain some duplicated descriptions. Please integrate and remove the duplicated content.
- Thank you for your valuable feedback. We have carefully reviewed Sections 2.1 and 2.2 and integrated the duplicated content to avoid redundancy. Now, the descriptions are consolidated appropriately under their respective headings to improve clarity and flow.
- The descriptions of L159-L166 and L168-L175 in Section 2.4 contain a significant amount of duplicate content. Please revise and remove the duplicate content.
- Thank you for your feedback. The duplicate content between lines L159-L166 and L168-L175 in Section 2.4 has been revised and the redundancy has been removed.
- It is recommended to unify the abbreviated names of each processing group in the chart to improve the readability of the article, such as using "NP+Cmm" to represent "Non Primed+Cmm" uniformly.
- Thank you for your suggestion. The abbreviated names of the treatment groups have been revised and standardized throughout the manuscript and figures to improve clarity and consistency (e.g., "Non Primed+Cmm" is now uniformly represented as "NP+Cmm").
- The caption in Figure 3.4 does not indicate "LSD: 0. xxx" in the legend. Please add it.
- Thank you for your comment. The LSD value (LSD: 0.001) has been added to the legend of Figure 3.4 to improve clarity, as recommended.
- 5. It is recommended to renumber all figures and tables in the manuscript according to journal requirements and standards.
- Thank you for the suggestions. All figure and table numbers have been revised and renumbered according to the journal’s formatting guidelines.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript by Nazlı Özkurt et al. explored effects of BABA inducing disease resistance in tomato seed against Clavibacter michiganensis. The results showed BABA served as a promising elicitor to induce tomato seed resistance against bacterial pathogen invasion. In my opinion, the manuscript can be accepted after minor revision. Please see my comments to improve the manuscript.
-Line 21, please revise 'water-priming' to 'water treatment'.
-Line 115, 1% is w/v?
-Line 117, how many seeds were in the same tube?
-Line 125, the provider should be introduced in a bracket.
-Line 126-127, how long were the seeds immersed in the BABA solution? What was the ratio between seeds and BABA solution?
-Line 133, what was the volume of the BABA solution sprayed on plant leaves? What was the exact amount of BABA substance received by each plant?
-Line 154, how to measure the lesion size with a ruler?
-The method section lacks replication information.
-Figure 3.1, the authors should provide the data analysis method in the caption. What is the meaning of the different letters labeled in the figure? What was the meaning of error bars?
-Figure 3.2, the lesion size should be revised to lesion area percentage. Also, What is the meaning of different letters labeled in the figure? What was the meaning of error bars? Similar issues also need to be revised in all figure and table captions.
-The authors need to measure the direct antimicrobial activity of BABA against cmm.
Author Response
Responses to Reviewer 2
The manuscript by Nazlı Özkurt et al. explored effects of BABA inducing disease resistance in tomato seed against Clavibacter michiganensis. The results showed BABA served as a promising elicitor to induce tomato seed resistance against bacterial pathogen invasion. In my opinion, the manuscript can be accepted after minor revision. Please see my comments to improve the manuscript.
We are grateful for the inputs and suggestions of the Reviewer 2. We carefully edited entire manuscript based on the inputs and suggestions of all three reviewers. Please find our responses to each query below.
-Line 21, please revise 'water-priming' to 'water treatment'.
- We are grateful for the suggestion. However, according to previous literature it is commonly called as “water priming” or “hydropriming” So, we did not change the naming. But, if it is still suggested by the reviewer(s) or editor, we will revise it as suggested.
- https://onlinelibrary.wiley.com/doi/full/10.1155/2015/859212
- https://journals.ekb.eg/article_64853.html
- https://doi.org/10.1002/jpln.200420425
-Line 115, 1% is w/v?
- The 1% sodium hypochlorite solution was prepared as a volume/volume (v/v) dilution, using 1 mL of commercial liquid bleach (NaClO) and 99 mL of distilled water, making a total of 100 mL.
-Line 117, how many seeds were in the same tube?
- Thank you for the input. In line 117, 35 seeds were placed in each tube.
-Line 125, the provider should be introduced in a bracket.
- Thank you for the input. The study used β-aminobutyric acid (BABA) (Sigma, USA) to prime seeds.
-Line 126-127, how long were the seeds immersed in the BABA solution? What was the ratio between seeds and BABA solution?
- Thank you for the query. The seeds were immersed in 12 mM BABA solution for 72 hours in 1.5 mL sterile Eppendorf tubes, with 35 seeds and 1.5 mL of solution per tube. Afterwards, the seedlings’ leaves were sprayed with 2 mM BABA solution.
Line 133, what was the volume of the BABA solution sprayed on plant leaves? What was the exact amount of BABA substance received by each plant?
- Thank you for the query. Each plant’s leaves were sprayed with 10 mL of 2 mM BABA solution, meaning each plant received 20 µmol of BABA.
-Line 154, how to measure the lesion size with a ruler?
- Thank you for the query. The plant stems were cut longitudinally, and the darkening of the vascular bundles is identified as the start and end points. The lesion length was measured in centimeters with a ruler between these two points.
-The method section lacks replication information.
- Each group consisted of 3 replicates, with 7 pots in each replicate, totaling 21 pots per group. We added relevant information to Statistical analyses section as “Each treatment group is replicated three times with seven pots (plants) per replication.”
-Figure 3.1, the authors should provide the data analysis method in the caption. What is the meaning of the different letters labeled in the figure? What was the meaning of error bars?
- We are thankful for the input. We have added meanings of the letters on figure and table captions as “Letters indicate significant differences between treatments at p < 0.05.”
-Figure 3.2, the lesion size should be revised to lesion area percentage. Also, What is the meaning of different letters labeled in the figure? What was the meaning of error bars? Similar issues also need to be revised in all figure and table captions.
- Thank you for the valuable input. We apologize for the letter labels. We added the meanings of letters as “Letters indicate significant differences between treatments at p < 0.05.” We have corrected all figures and tables for the same issue.
-The authors need to measure the direct antimicrobial activity of BABA against cmm.
- Thank you for the valuable input. According to Baysal et al. (2003), BABA does not have a direct anti microbial effect against Cmm. Since we already knew this from the literature and our aim was not the direct anti-microbial effect, we did not run the in vitro test. Our aim was to see if BABA induces systemic resistance. And if so, which defense related genes are involved. Please find Baysal et al. link below.
- Baysal, Ö.; Soylu, E.M.; Soylu, S. Induction of defence‐related enzymes and resistance by the plant activator acibenzolar‐S‐methyl in tomato seedlings against bacterial canker caused by Clavibacter michiganensis michiganensis. Plant Pathol. 2003, 52, 747-753.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript titled – Enhancing Tomato (Solanum lycopersicum L.) Resistance Against Bacterial Canker Disease (Clavibacter michiganensis ssp. michiganensis) via Seed Priming with β-Aminobutyric Acid (BABA) by Nazlı Özkurt et al., describes the protective effects of various seed priming and spray treatment combinations incorporating β-Aminobutyric Acid against tomato bacterial canker disease caused by Clavibacter michiganensis ssp. Michiganensis.
Overall, the manuscript is relevant and interesting in terms of research topic and content.
Comments and concerns:
Readability of the manuscript can be improved by addressing the English language usage and grammar issues.
“causes bacterial cancer in tomato” THE TERM CANCER (IN PLACE OF CANKER) IS USED THROUGHOUT THE MANUSCRIPT.
USE OF APPROPRIATE TECHNICAL / PLANT PATHOLOGY TERMS IS HIGHLY RECOMMENDED
Page 3 L 118 -119
Seeds germinated on moist filter paper in a petri dish for 7 days at room temperature (25-27 °C). They were then placed in plastic pots filled with a 2:1 peat/perlite combination.
WHICH TREATMENT GROUP DOES THESE SEEDS BELONG TO? NOT CLEAR.
Page 9
Table 3. 1. RESULTS LOOKS ODD.
WHY IS PLANT BIOMASS LOW IN TREATMENT RECEIVING BABA-Priming+BABA-SP+Cmm (IN SPITE OF LOW LESION SIZE AND LOW DISEASE SEVERITY)? NEEDS TO BE PROPERLY DISCUSSED.
Page 12 L 86 -87
Although these two earlier investigations were against fungus, we demonstrated in this study that the protective effect of BABA is not only against fungi but also against bacteria.
NOT TRUE, BABA PROTECTIVE EFFECT AGAINST BACTERIAL PATHOGEN HAVE BEEN REPORTED.
Page 14 L 172 -173
According to the findings, INA protects (???) Cmm by stimulating WRKY33b, whereas DPMP does not activate the WRKY33b gene.
Comments on the Quality of English LanguagePlease refer to comments and suggestions for authors
Author Response
Responses to Reviewer 3
The manuscript titled – Enhancing Tomato (Solanum lycopersicum L.) Resistance Against Bacterial Canker Disease (Clavibacter michiganensis ssp. michiganensis) via Seed Priming with β-Aminobutyric Acid (BABA) by Nazlı Özkurt et al., describes the protective effects of various seed priming and spray treatment combinations incorporating β-Aminobutyric Acid against tomato bacterial canker disease caused by Clavibacter michiganensis ssp. Michiganensis.
Overall, the manuscript is relevant and interesting in terms of research topic and content.
- We are grateful for the inputs and suggestions of the Reviewer 3. We carefully edited entire manuscript based on the inputs and suggestions of all three reviewers. Please find our responses to each query below.
Comments and concerns:
Readability of the manuscript can be improved by addressing the English language usage and grammar issues.
- We are thankful for the input. We carefully edited entire manuscript for the grammatical and language issues.
“causes bacterial cancer in tomato” THE TERM CANCER (IN PLACE OF CANKER) IS USED THROUGHOUT THE MANUSCRIPT.
- We apologize for the typo. The term has been corrected from "cancer" to "canker" throughout the manuscript.
USE OF APPROPRIATE TECHNICAL / PLANT PATHOLOGY TERMS IS HIGHLY RECOMMENDED
- We are thankful for the suggestion. We have carefully checked and edited entire manuscript for the terminology.
Seeds germinated on moist filter paper in a petri dish for 7 days at room temperature (25-27 °C). They were then placed in plastic pots filled with a 2:1 peat/perlite combination.
- The seeds in this sentence belong to the control group that did not undergo priming and were transferred to pots after germination.
WHICH TREATMENT GROUP DOES THESE SEEDS BELONG TO? NOT CLEAR.
- Our treatment groups include BABA seed priming, water seed priming, and a control group without priming; each group is further divided based on whether BABA spray was applied or not. Please refer to Table 2 for the detailed discriptions of the experiement.
Table 3. 1. RESULTS LOOKS ODD.
WHY IS PLANT BIOMASS LOW IN TREATMENT RECEIVING BABA-Priming+BABA-SP+Cmm (IN SPITE OF LOW LESION SIZE AND LOW DISEASE SEVERITY)? NEEDS TO BE PROPERLY DISCUSSED.
- The lower plant biomass observed in the BABA-Priming + BABA Spray + Cmm treatment, despite reduced lesion size and disease severity, may be due to the energy and resources allocated by the plant toward activating defense mechanisms. BABA is known to induce systemic resistance, which can lead to metabolic costs affecting growth. This trade-off between defense and growth likely explains the reduced biomass in this treatment group. Further discussion and analysis of this balance should be included to clarify these findings.
Although these two earlier investigations were against fungus, we demonstrated in this study that the protective effect of BABA is not only against fungi but also against bacteria.
NOT TRUE, BABA PROTECTIVE EFFECT AGAINST BACTERIAL PATHOGEN HAVE BEEN REPORTED.
- We apologize for the misleading information. We have corrected it in the manuscript. The protective effect of BABA has been previously reported not only against fungi but also against bacterial pathogens. In this study, we further confirm its efficacy against bacteria.
According to the findings, INA protects (???) Cmm by stimulating WRKY33b, whereas DPMP does not activate the WRKY33b gene.
- Thank you for the valuable comment. We aimed to compare our result with the Bektas 2021s results. We corrected the sentence as “Based on their results, INA enhances plant defense against Cmm by stimulating the WRKY33b gene, while DPMP does not activate WRKY33b gene expression”.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI am very satisfied with the modifications made by the author based on the review comments.
Reviewer 3 Report
Comments and Suggestions for AuthorsNA
