Biogenic Zinc Oxide Nanoparticles Protect Tomato Plants Against Pseudomonas syringae pv. tomato
, Anna Scian, Daniele Del Buono, Michela Paglialunga, Ciro Tolisano, Dario Priolo, Chiaraluce Moretti * and Roberto BuonaurioRound 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe peer-reviewed article “Biogenic zinc oxide nanoparticles protect tomato plants against Pseudomonas syringae pv. tomato” is devoted to zinc oxide nanoparticles to protect tomato against the bacterial speck pathogen, caused by Pseudomonas syringae pv. tomato cv. Rio Grande. The authors confirmed previous studies on the protective effect of ZnO against Pseudomonas syringae pv. tomato (Elsharkawy, M., Derbalah, A., Hamza, A., & El-Shaer, A. (2020). Zinc oxide nanostructures as a control strategy of bacterial speck of tomato caused by Pseudomonas syringae in Egypt. Environmental Science and Pollution Research, 27, 19049-19057.). Researchers assess the level of gene expression by qPCR revealed the involvement of the systemic-acquired resistance (SAR) pathway such as pathogenesis-related protein 1b1 (PR1B1) and β-1,3-glucanase 192 (GluB) genes. Additionally, physiological and biochemical assessment of the treated tomato plants was carried out for some indicators, such as the content of hydrogen peroxide, malondialdehyde and antioxidants. The research paper is interesting and relevant, since it has both fundamental and applied significance. The manuscript is well written and illustrated. The results are reliable and confirmed by correct statistical processing of data. The manuscript has few minor comments and recommendations such as:
- I recommend supplementing the introduction and reference sections with several modern studies that are directly related to the effect of zinc-oxide particles against tomato pathogens/ such as
Fraga, F. S., Silva, A. C. A., Dantas, N. O., Tebaldi, N. D., & Luz, J. M. Q. (2021). Doped zinc-oxide nanocrystals for the control of tomato bacterial spot and Xanthomonas gardneri in seeds. Tropical Plant Pathology, 46, 406-413.
Khan, R. A. A., Tang, Y., Naz, I., Alam, S. S., Wang, W., Ahmad, M., ... & Li, Y. (2021). Management of Ralstonia solanacearum in tomato using ZnO nanoparticles synthesized through Matricaria chamomilla. Plant Disease, 105(10), 3224-3230.
- L 148. Inoculation → It is not clear how many plants were in each replication? (n=?).
- The conclusion section is too redundant. Authors are required to provide specific fundamental or practical recommendations based on their findings and those available in the scientific literature.
Author Response
I recommend supplementing the introduction and reference sections with several modern studies that are directly related to the effect of zinc-oxide particles against tomato pathogens/ such as Fraga et al., 2021 and Khan et al., 2021.
Our reply: References have been added in Introduction section. Reference number: 42 and 43.
L 148. Inoculation → It is not clear how many plants were in each replication? (n=?).
Our reply: Eight plants were used for each experiment, as stated in the Materials and Methods section.
The conclusion section is too redundant. Authors are required to provide specific fundamental or practical recommendations based on their findings and those available in the scientific literature.
Our reply: We agree with the Reviewer and Conclusion has been modified accordingly.
Reviewer 2 Report
Comments and Suggestions for AuthorsThis study aims to introduce function and application of biogenic zinc oxide nanoparticles for resisting Pseudomonas syringae in tomato. Approximately 100 ppm zinc oxide nanoparticles could reduce symptom severity and bacterial growth, and not affect the flavonoids and anthocyanins, indicating a valuable, safe, and environment friendly tool for tomato planting. The main comments,
- Would give any description about the purpose of DUCKWEED in the method?
- Provide the rationale for selecting what concentration for ASM, please.
- The Introduction should briefly describe the source (e.g., plant species, database accession numbers) and known biological functions (e.g., pathogenesis-related proteins, ROS-scavenging enzymes) of the resistance-related genes studied.
- Expand the Introduction to explain the mechanistic relevance of the measured physiological indicators (e.g., antioxidant enzymes, phenylpropanoid metabolites) to disease resistance processes.
- Formatting revisions
Please check the sentences and grammars in full text.
Line 148: Correct "treatment Inoculation" to "treatment inoculation" (lowercase).
Line 202: Define "TCA" at first mention as "tricarboxylic acid (TCA) cycle."
Line 352: Use subscript formatting for "H2O2."
Comments on the Quality of English LanguageNeed check the sentences and grammars in full text.
Author Response
Would give any description about the purpose of DUCKWEED in the method?
Our reply: Details have been added in the Materials and Methods section.
Provide the rationale for selecting what concentration for ASM, please.
Our reply: Details have been added in the Materials and Methods section.
The Introduction should briefly describe the source (e.g., plant species, database accession numbers) and known biological functions (e.g., pathogenesis-related proteins, ROS-scavenging enzymes) of the resistance-related genes studied.
Our reply: The tomato plants used in the experiments belong to the Rio Grande cultivar, as reported in the study. As for the resistance genes analyzed in the Supplementary Information, the sequences of the primers used and the corresponding bibliographic references have been provided.
Expand the Introduction to explain the mechanistic relevance of the measured physiological indicators (e.g., antioxidant enzymes, phenylpropanoid metabolites) to disease resistance processes.
Our reply: Details have been added in the Introduction section.
Formatting revisions
Our reply: Done.
Please check the sentences and grammars in full text.
Our reply: As Reviewer suggested the manuscript has been checked for English.
Line 148: Correct "treatment Inoculation" to "treatment inoculation" (lowercase).
Our reply: We did.
Line 202: Define "TCA" at first mention as "tricarboxylic acid (TCA) cycle."
Our reply: We did.
Line 352: Use subscript formatting for "H2O2."
Our reply: We did.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe article entitled as “Biogenic zinc oxide nanoparticles protect tomato plants against Pseudomonas syringae pv. tomato" discusses the importance of control of bacterial plant diseases. In this study authors have investigated the efficacy of biogenic zinc oxide nanoparticles (ZnO-NPs) in protecting tomato plants against Pseudomonas syringae pv. tomato (Pst), the causal agent of bacterial speck disease.
According to this study ZnO-NPs exhibited significant in vitro antibacterial activity (EC95 = 17.0±1.1 ppm) against the pathogen. Furthermore, when applied to the foliage of tomato plants at 100 ppm before or following Pst inoculation, they induced significant reductions in symptom severity and bacterial growth in planta, which were comparable to those shown by plants treated with acibenzolar-S-methyl, a plant defence inducer.
Other results such as gene expression assessed by qPCR revealed the involvement of the systemic-acquired resistance (SAR) pathway in tomato plants treated with ZnO-NPs before the inoculation, suggesting that the protection could be due to a priming effect.
Finally, infected plants showed oxidative stress with higher Hâ‚‚Oâ‚‚ and malondialdehyde (MDA) contents. ZnO-NPs reverted this effect, containing the content of the above molecules, and stimulated the production of metabolites involved in dealing with oxidative perturbations (carotenoids and phenols) while unaffected flavonoids and anthocyanins.
Overall, I found the paper very well written, clear and informative. I could follow the threat of thought and discovery easily and with interest throughout the paper and also enjoyed the well written discussion of the findings.
Minor points
Introduction
Line 34-35 “this crop is a natural source of A, C and K vitamins,”
Please write the actual words of A, C and K vitamins rather than just abbreviations
- Results and Discussion
Figure 3. Please enhance the size of letters on graphs in this figure.
Figure 4. Please make the writings on X-Axis and Y-Axis into Bold and also enhance the size of the alphabets on the graphs.
Figure 5. Please follow Figure 4 instructions.
Line 352 “a significant increase in H2O2 levels”
Please make 2 smaller in H2O2
Conclusions
Conclusion is too long, please make it shorter and just describe the mane findings of your study in conclusive remarks.
Line 403-406 “This is the first study that investigated the potential of biogenic ZnO-NPs in controlling Pst from the perspective of assessing their direct toxicity against the pathogen and their role as plant protectors.”
Please remove the above lines.
Line 407-409 “Furthermore, the biogenic material applied to plants via foliar spray at 100 ppm significantly reduced the severity of tomato bacterial speck symptoms, when the treatment was applied preventively (2 days before inoculation) or after the inoculation (5 days later).”
Please make it shorter and just mention the general results.
Overall, the conclusion is not written well. Please make it brief and concise.
Author Response
Line 34-35 “this crop is a natural source of A, C and K vitamins,” Please write the actual words of A, C and K vitamins rather than just abbreviations
Our reply: The actual words of A, C and K vitamins have been added in the Introduction section.
Figure 3. Please enhance the size of letters on graphs in this figure.
Our reply: The figure has been modified as suggested by the Reviewer
Figure 4. Please make the writings on X-Axis and Y-Axis into Bold and also enhance the size of the alphabets on the graphs.
Our reply: The figure has been modified as suggested by the Reviewer
Figure 5. Please follow Figure 4 instructions.
Our reply: The figure has been modified as suggested by the Reviewer
Line 352 “a significant increase in H2O2 levels”. Please make 2 smaller in H2O2
Our reply: The subscripts have been modified.
Conclusion is too long, please make it shorter and just describe the mane findings of your study in conclusive remarks.
Our reply: We agree with the Reviewer and Conclusion has been modified accordingly.
Line 403-406 “This is the first study that investigated the potential of biogenic ZnO-NPs in controlling Pst from the perspective of assessing their direct toxicity against the pathogen and their role as plant protectors.” Please remove the above lines.
Our reply: We agree with the Reviewer and we have removed this sentence.
Line 407-409 “Furthermore, the biogenic material applied to plants via foliar spray at 100 ppm significantly reduced the severity of tomato bacterial speck symptoms, when the treatment was applied preventively (2 days before inoculation) or after the inoculation (5 days later).”
Please make it shorter and just mention the general results.
Overall, the conclusion is not written well. Please make it brief and concise.
Our reply: We agree with the Reviewer and Conclusion has been modified accordingly.
Reviewer 4 Report
Comments and Suggestions for AuthorsA key concern with the manuscript is its limited general novelty, especially in the context of the broader research landscape. The use of nanoparticles, including zinc oxide nanoparticles (ZnO-NPs), for agricultural applications, particularly in disease control, is now an extensively studied area. Numerous studies have already explored the antimicrobial properties of nanoparticles, including their effects on various plant pathogens, as well as their role in enhancing plant immunity. As such, the application of pre-synthesized ZnO-NPs to manage bacterial speck disease in tomatoes does not present a significant breakthrough in the field. While the topic is certainly of interest, the research does not introduce any novel concepts or innovative approaches. The synthesis and characterization of ZnO-NPs used in this study were already published in a previous paper, and the current manuscript primarily re-applies these materials to a different host-pathogen system. Given that the effectiveness of nanoparticles in plant disease control has been widely demonstrated in existing literature, the manuscript lacks the necessary level of novelty to make a meaningful contribution to advancing the field. In order to offer a truly novel contribution, the manuscript would need to explore original aspects such as new nanoparticle formulations, modifications to enhance their efficacy, or a deeper investigation into novel mechanisms of action. Thus, the manuscript’s lack of novelty, combined with the growing body of research on nanoparticles in agriculture, means that it does not offer the level of scientific advancement expected for publication in a peer-reviewed journal. More innovative approaches or fresh perspectives are needed to elevate the research and make it stand out in this highly studied field.
Comments on the Quality of English LanguageThe English could be improved to more clearly express the research.
Author Response
A key concern with the manuscript is its limited general novelty, especially in the context of the broader research landscape. The use of nanoparticles, including zinc oxide nanoparticles (ZnO-NPs), for agricultural applications, particularly in disease control, is now an extensively studied area. Numerous studies have already explored the antimicrobial properties of nanoparticles, including their effects on various plant pathogens, as well as their role in enhancing plant immunity. As such, the application of pre-synthesized ZnO-NPs to manage bacterial speck disease in tomatoes does not present a significant breakthrough in the field. While the topic is certainly of interest, the research does not introduce any novel concepts or innovative approaches. The synthesis and characterization of ZnO-NPs used in this study were already published in a previous paper, and the current manuscript primarily re-applies these materials to a different host-pathogen system. Given that the effectiveness of nanoparticles in plant disease control has been widely demonstrated in existing literature, the manuscript lacks the necessary level of novelty to make a meaningful contribution to advancing the field. In order to offer a truly novel contribution, the manuscript would need to explore original aspects such as new nanoparticle formulations, modifications to enhance their efficacy, or a deeper investigation into novel mechanisms of action. Thus, the manuscript’s lack of novelty, combined with the growing body of research on nanoparticles in agriculture, means that it does not offer the level of scientific advancement expected for publication in a peer-reviewed journal. More innovative approaches or fresh perspectives are needed to elevate the research and make it stand out in this highly studied field.
Our replay: We do not agree with the Reviewer as the innovative aspect of this is work is to verify the effect of the biogenic zinc nanoparticles against Pseudomonas syringae pv. tomato (Pst). In this work we do not present the synthesis and characterization ZnO-NPs, which was already published by Del Buono et al., 2021 (see reference N. 52). Here, for the first time, we demonstrated, with a trans-disciplinary approach, that ZnO-NPs:
- exhibited significant in vitro antibacterial activity;
- reduce symptom severity and bacterial growth in planta, comparable to those shown by plants treated with the plant defence inducer acibenzolar-S-methyl;
- protect the plant through a priming effect;
- stimulate the production of metabolites involved in dealing with oxidative perturbations (carotenoids and phenols).
