Effect of Plant Growth-Promoting Rhizobacteria Synthetic Consortium on Growth, Yield, and Metabolic Profile of Lettuce (Lactuca sativa L.) Grown Under Suboptimal Nutrient Regime
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsDear Editor-in-Chief,
Thank you for giving me the opportunity to review the manuscript entitled “Effect of a PGPR synthetic consortium on growth, yield, and metabolic profile of lettuce (Lactuca sativa L.) grown under sub-optimal nutrient regime “.
The manuscript explores the impact of a PGPR synthetic consortium on lettuce growth, yield, and metabolic reprogramming under low-nutrient conditions in an aeroponic system. The study integrates plant physiology, anatomy, and metabolomics, providing a comprehensive view of PGPR effects.
1. The experimental design, data collection, and statistical analysis are robust. However, minor issues need attention: Outlier management in the figures (e.g., Figures 2–5) could be better clarified, particularly if they represent biologically meaningful variation or technical error.
2. Introduction: Slightly long; paragraphs discussing global food security and hydroponics could be streamlined.
3. Materials and Methods: Missed measurements: Root exudates could have been analyzed to better understand plant-microbe interactions.
4. The results are well-organized, with clear tables and figures. However: Figures 2–8: Legends could be more detailed, especially explaining statistical notations. Table 2: Including units for all parameters (e.g., stomatal density) would improve clarity.
5. Discussion: The metabolic shifts (e.g., changes in amino acids and antioxidants) are discussed, but further exploration of their physiological implications would strengthen the argument.
6. Conclusion summarizes the findings well but lacks emphasis on limitations and future research directions. For instance: Discussing scalability of PGPR applications in field conditions. Suggesting further studies on other crop species or stress conditions.
7. References: Some key citations on aeroponic systems and PGPR metabolomics could be added for completeness.
8. Language and formatting: The manuscript uses clear scientific language, but minor grammatical errors and formatting inconsistencies exist: Lines 107-108: "surface sterilized by 10 minutes immersion" → revise for clarity ("surface-sterilized by immersion for 10 minutes"). Punctuation: Ensure consistent use of commas and parentheses throughout.
Author Response
Dear Reviewer 1,
Thank you very much for your thoughtful comments and suggestions regarding our manuscript.
We have carefully considered each of your comments and have revised the manuscript accordingly. Please find below a point-by-point response to your concerns, outlining the changes we have made in the text.
Sincerely,
The authors
Reviewer 1 - 1. Outlier management in the figures (e.g., Figures 2–5) could be better clarified, particularly if they represent biologically meaningful variation or technical error.
Authors: The plants were grown under controlled conditions in the same growth tent. Light, aeration and temperature were uniform. Both outliers in Figure 2a and b were reported under non-inoculated treatment and were related to positive outliers. Removing them would have enhanced the difference between control and inoculated plants. The parameters reported in Figure 3 (stomatal conductance, electron transport rate and quantum yield of photosystem II) were measured contemporarily by Li-600, the instrument we used. The data were checked during measurements and if values shown on the screen on Li-600 were too far from the average measured values or if the instrument took too long to reach stability, the measurement was repeated and the previous data discarded. Moreover, we checked if outliers in one parameter coincided with outliers in the other parameters (being measured contemporarily), but they did not, thus we decided to keep the outliers as part of the analysis. In Figure 4, NAR was estimated by using the formula reported in materials and methods. In Figure 5, each sample was analyzed in triplicate and the mean value was taken into consideration. Also in this case, removing the outliers, which were only observed in the non-inoculated treatment would only have strengthen the differences between non-inoculated and inoculated plants, therefore not changing the results that we observed.
Reviewer 1 - 2. Introduction: Slightly long; paragraphs discussing global food security and hydroponics could be streamlined.
Authors: In response to Reviewer 1's feedback, we have streamlined the manuscript by removing the sentences related to food security (lines 33-37 in the revised manuscript). To address Reviewer 2's insightful suggestion, we had to incorporate information regarding hydroponics (lines 65-69 in the revised manuscript).
Reviewer 1 - 3. Materials and Methods: Missed measurements: Root exudates could have been analyzed to better understand plant-microbe interactions.
Authors: We have analyzed rhizosphere metabolites. However, we decided to use the data in a second manuscript. For this reason, the results were not included in the current manuscript.
Reviewer 1 - 4. The results are well-organized, with clear tables and figures. However: Figures 2–8: Legends could be more detailed, especially explaining statistical notations. Table 2: Including units for all parameters (e.g., stomatal density) would improve clarity.
Authors: The captions of the figures and Table 2 were modified by adding information related to the statistical analysis and including the missing units. Please find the modifications in the revised manuscript.
Reviewer 1 - 5. Discussion: The metabolic shifts (e.g., changes in amino acids and antioxidants) are discussed, but further exploration of their physiological implications would strengthen the argument.
Authors: As requested by the Reviewer, possible explanations for the metabolic shift in amino acid (lines 636- 640 in the revised manuscript) and antioxidants (particularly ascorbic acid; lines 670-683 in the revised manuscript) were added in the discussion.
Reviewer 1 - 6. Conclusion summarizes the findings well but lacks emphasis on limitations and future research directions. For instance: Discussing scalability of PGPR applications in field conditions. Suggesting further studies on other crop species or stress conditions.
Authors: According to reviewer’s suggestion, the following sentence was added in the conclusion: “In the future, it would be beneficial to explore ways to expand the use of PGPR consortia in field settings to assess their efficacy and cost-effectiveness in real-world agricultural scenarios. Additionally, investigating the effects of PGPR consortia on other crop species under various stress conditions, such as drought or salinity, will broaden our understanding of their potential for sustainable agriculture.” (lines 712-717 in the revised manuscript).
Reviewer 1 - 7. References: Some key citations on aeroponic systems and PGPR metabolomics could be added for completeness.
Authors: The references on PGPR inoculation in aeroponic systems and metabolomics are scarce. Following the reviewer’s suggestion, we added two citations related to research conducted in hydroponics on lettuce (lines 535-539 and lines 624-627 in the revised manuscript).
Reviewer 1 - 8. Language and formatting: The manuscript uses clear scientific language, but minor grammatical errors and formatting inconsistencies exist: Lines 107-108: "surface sterilized by 10 minutes immersion" → revise for clarity ("surface-sterilized by immersion for 10 minutes"). Punctuation: Ensure consistent use of commas and parentheses throughout.
Authors: The text (lines 124-125 in the revised manuscript) was modified according to reviewer’s suggestion. We also tried to formatting punctuation and better the quality of the manuscript as highlighted by the high number of modifications we inserted in the revised manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript horticulturae-3370294, titled “Effect of a PGPR synthetic consortium on growth, yield, and metabolic profile of lettuce (Lactuca sativa L.) grown under sub-optimal nutrient regime”, addresses an important paper investigating the effects of a plant growth-promoting rhizobacteria (PGPR) consortium on lettuce (L. sativa L.) cultivated in an aeroponic system under suboptimal nutrient conditions. However, in my opinion this paper must be revised in a major manner for reasons of forms and content.
Abstract
Can the authors clarify the main novelty of their study in the abstract? What unique contribution does this research make to the field of PGPR and soilless cultivation systems?
Introduction
The introduction provides a strong rationale for using PGPR. Could the authors expand on the specific gaps in knowledge this study aims to address compared to prior work in aeroponic systems?
The introduction mentions multiple benefits of PGPR, but does not detail why a consortium was used instead of single strains. Could the authors provide more background or justification for this choice?
Materials and Methods
Can the authors provide more details about the selection criteria for the eight bacterial strains included in the PGPR consortium? How were these strains optimized for use in aeroponic systems?
Was there any preliminary testing done to determine the optimal nutrient levels for lettuce under suboptimal conditions? If so, could the authors include this information?
The methods mention leaf metabolomic profiling but do not explain how the metabolite data were validated or compared with known standards. Could the authors clarify this?
Can the authors specify why certain parameters (e.g., electron transport rate, stomatal conductance) were measured on specific days (DAT8 and DAT13)? Was there a specific rationale behind these time points?
Results
The results mention a significant increase in biomass and leaf area in PGPR-inoculated plants. Can the authors explore whether these changes were consistent across all replicates or influenced by variability in plant response?
Can the authors elaborate on the implications of the observed changes in leaf anatomy, such as reduced leaf thickness and increased palisade parenchyma ratio, for photosynthetic efficiency?
The results show significant changes in leaf metabolites. Can the authors discuss whether these metabolic changes correlate directly with observed growth enhancements?
Discussion
The discussion suggests that the PGPR consortium affects leaf and root architecture. Can the authors propose mechanisms by which these architectural changes translate into enhanced nutrient uptake or stress resilience?
Can the authors provide a more detailed comparison of their findings with similar studies on PGPR in other soilless cultivation systems?
The manuscript highlights the decrease in stomatal density but an increase in stomatal aperture. Could the authors discuss the potential trade-offs between water use efficiency and carbon assimilation in this context?
Author Response
Dear Reviewer 2,
We greatly appreciate you taking the time to review our work and provide such constructive feedback.
We have carefully considered your suggestions and have revised the manuscript accordingly.
Please find below a point-by-point response to your concerns, outlining the changes we have made in the text.
Sincerely,
The authors
Reviewer 2: Can the authors clarify the main novelty of their study in the abstract? What unique contribution does this research make to the field of PGPR and soilless cultivation systems?
Authors: According to the Reviewer suggestion, we added the following sentence in the abstract: “These findings provide valuable insights into the intricate metabolic dialog between plants and beneficial microbes and demonstrate that the integration of soilless culture with the analysis of ecophysiological, anatomical and metabolomic plant responses can be a powerful approach to accelerate the design of new PGPR consortia for use as microbial biostimulants” (lines 21-24 in the revised manuscript).
Reviewer 2: The introduction provides a strong rationale for using PGPR. Could the authors expand on the specific gaps in knowledge this study aims to address compared to prior work in aeroponic systems?
Authors: To better highlight the gaps in the knowledge this study aims to address, the introduction was modified as requested by the Reviewer (lines 65-69 in the revised manuscript).
Reviewer 2: The introduction mentions multiple benefits of PGPR, but does not detail why a consortium was used instead of single strains. Could the authors provide more background or justification for this choice?
Authors: Following the reviewer’s suggestion, we added a sentence in the introduction to better explain the importance of using consortia rather than single strains (lines 81-84 in the revised manuscript). Other references were already present in the discussion (lines 485-503 in the revised manuscript). We hope this can be sufficient (Reviewer 1 asked us to reduce the length of the introduction).
Reviewer 2: Can the authors provide more details about the selection criteria for the eight bacterial strains included in the PGPR consortium? How were these strains optimized for use in aeroponic systems?
Authors: In response to the reviewer’s feedback, we have added a sentence in the Materials and Methods section (lines 169-173 in the revised manuscript) to explicitly highlight the motivations for the selection of the strains used as inoculants.
Reviewer 2: Was there any preliminary testing done to determine the optimal nutrient levels for lettuce under suboptimal conditions? If so, could the authors include this information?
Authors: We took as reference the Hoagland solution and the results reported by Sapkota et al. 2019 who tested the effect of different nutrient concentrations on the biomass production of lettuces grown in a hydroponic system. We added the reference and an explanation in paragraph 2.1 (lines 149-153 in the revised manuscript).
Reviewer 2: The methods mention leaf metabolomic profiling but do not explain how the metabolite data were validated or compared with known standards. Could the authors clarify this?
Authors: We specified in the text of the manuscript that we used paracetamol as an internal standard (lines in the revised manuscript). Moreover, the caption of Figure 8 was modified to include information related to the statistical analysis.
Reviewer 2: Can the authors specify why certain parameters (e.g., electron transport rate, stomatal conductance) were measured on specific days (DAT8 and DAT13)? Was there a specific rationale behind these time points?
Authors: We measured the parameters the first day after the second inoculation and the day before the conclusion of the experiment. We insert this information in the text (lines 259-260 in the revised manuscript).
Reviewer 2: The results mention a significant increase in biomass and leaf area in PGPR-inoculated plants. Can the authors explore whether these changes were consistent across all replicates or influenced by variability in plant response?
Authors: The observed changes in biomass in inoculated plants were consistent across replicates. We added a sentence to highlight this result (lines 336-337).
Reviewer 2: Can the authors elaborate on the implications of the observed changes in leaf anatomy, such as reduced leaf thickness and increased palisade parenchyma ratio, for photosynthetic efficiency?
Authors: Following the reviewer’s suggestions, we added implications for photosynthetic efficiency as a result of the observed modifications in leaf anatomy. The text of the discussion was, therefore, modified (lines 590-599 in the revised manuscript).
Reviewer 2: The results show significant changes in leaf metabolites. Can the authors discuss whether these metabolic changes correlate directly with observed growth enhancements?
Authors: Following the reviewer’s request, we discussed the correlation between the metabolic changes and the observed growth enhancement. The details are reported in the discussion (lines 596-599 and 669-682 in the revised manuscript).
Reviewer 2: The discussion suggests that the PGPR consortium affects leaf and root architecture. Can the authors propose mechanisms by which these architectural changes translate into enhanced nutrient uptake or stress resilience?
Authors: According to the reviewer’s comment, we modified the text of the discussion and proposed mechanisms that can explain the enhanced nutrient uptake or stress resilience induced by modifications in root architecture (lines 517-520, lines 616-617 and lines 624-626 in the revised manuscript).
Reviewer 2: Can the authors provide a more detailed comparison of their findings with similar studies on PGPR in other soilless cultivation systems?
Authors: Following reviewer’s suggestion, we added several references related to similar studies on PGPR in soilless cultures (lines 517-525, lines 532-539 and lines 624-626 in the revised manuscript).
Reviewer 2: The manuscript highlights the decrease in stomatal density but an increase in stomatal aperture. Could the authors discuss the potential trade-offs between water use efficiency and carbon assimilation in this context?
Authors: We modified the text according to the reviewer’s suggestions, adding information related to the implications of the observed leaf anatomical and physiological changes on water use efficiency (lines 600-611 in the revised manuscript).
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsNo more comments
Reviewer 2 Report
Comments and Suggestions for AuthorsI'am following up on my review of this manuscript.
Authors have replied to all comments, which improved the quality of their manuscript. However, i believe that the paper is much stronger and ready for further consideration.
