Effect of the Inoculation Method on the Potential Plant Growth-Promoting Activity of a Microbial Synthetic Consortium
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsComments and suggestions for Authors:
In this manuscript, a microbial consortium obtained from the phyllosphere of Pistacia lentiscus was used to inoculate Lactuca sativa plants in different ways (leaf, root, or combined inoculation) under nutrient-limiting conditions in a hydroponic system. The effects of these treatments on lettuce growth were evaluated through analysis of plant biomass, structure, and leaf traits. Although the manuscript is interesting and well-written, some improvements are needed.
Lines 96-98: It would be helpful to explain in the Introduction why Pistacia lentiscus was chosen as the source of bacteria, beyond its location on the margins of Sardinia. Although this is discussed in the Discussion section (lines 380-381), brieflymentioning it in the Introduction would be beneficial.
Lines 127-137: For a better understanding of the methodology section, consider adding a description of purpose of why a particular medium was selected for the experiment.
Line 189: Why was a final concentration of 4x108 consortium cells/ml selected for plant inoculation? If this concentration is commonly used in such plant treatments, this should be mentioned it in the appropriate place in the manuscript.
Lines 418-421: Please add one or two additional examples that illustrate how the plant-plant and/or plant-environmental interactions contribute to shaping the plant-associated microbiome.
Author Response
Reviewer 1: In this manuscript, a microbial consortium obtained from the phyllosphere of Pistacia lentiscus was used to inoculate Lactuca sativa plants in different ways (leaf, root, or combined inoculation) under nutrient-limiting conditions in a hydroponic system. The effects of these treatments on lettuce growth were evaluated through analysis of plant biomass, structure, and leaf traits. Although the manuscript is interesting and well-written, some improvements are needed.
Dear Reviewer, we express our sincere gratitude for your insightful comments and constructive suggestions, which allowed us to significantly improve the quality of this manuscript.
Comment 1: Lines 96-98: It would be helpful to explain in the Introduction why Pistacia lentiscus was chosen as the source of bacteria, beyond its location on the margins of Sardinia. Although this is discussed in the Discussion section (lines 380-381), briefly mentioning it in the Introduction would be beneficial.
Authors: Following the Reviewer's recommendation, the introduction was enhanced to provide a more robust rationale for selecting P. lentiscus as a source of plant growth-promoting isolates. This involved incorporating citations to strengthen the justification for this choice and the statement from the discussion section, as suggested (lines 95-110).
Comment 2: Lines 127-137: For a better understanding of the methodology section, consider adding a description of purpose of why a particular medium was selected for the experiment.
Authors: As suggested by the Reviewer, we included a detailed description of the growth media used in this study in the manuscript (Lines 149-158).
Comment 3: Line 189: Why was a final concentration of 4x108 consortium cells/ml selected for plant inoculation? If this concentration is commonly used in such plant treatments, this should be mentioned it in the appropriate place in the manuscript.
Authors: A high-density stock culture of the synthetic bacterial community (SynCom) (4x108 cells ml-1) was prepared to achieve a final concentration of 2x106 cells ml-1in the hydroponic tank. This SynCom concentration was 10-fold higher than the total aerobic bacterial population in the control tank (2x105 cells ml-1). A diluted SynCom suspension (2x 106 cells ml-1) was used for foliar application to maintain consistency across treatments.
Following the Reviewer’s suggestions, we clarified this point in the revised manuscript and modified the text accordingly (lines 223-232).
Comment 4: Lines 418-421: Please add one or two additional examples that illustrate how the plant-plant and/or plant-environmental interactions contribute to shaping the plant-associated microbiome.
Authors: Following the Reviewer’s suggestion, a subparagraph with the requested examples was added (lines 458-465 in the revised manuscript).
Reviewer 2 Report
Comments and Suggestions for AuthorsThe article researched the effect of different inoculation method on plant growth by SynCom bacteria of 5 N-fixing bacteria. The result showed reference value for the application of microbial fertilizers on actual agriculture production. I have a few question before the decision.
1）The nitrogen form affects the colonization and function of phyllosphere and rhizosphere microorganisms，the nitrogen form of plant culture media should be shown.
2) line 181-195,did the final concentration of 4*108 cell per ml means each 5 strains concentration or the total SynCom bacteria? the dosage of microbial consortium should be consistent，please explain the why the different dosage used in the article, and what the final concentration of bacteria on RL treatment.
3) All the tables should be three-line table. And strongly recommend checking the result of statistical analyses.
Author Response
Reviewer 2. Comments and Suggestions for Authors:
Dear Reviewer, we express our sincere gratitude for your insightful comments and constructive suggestions, which allowed us to improve the quality of this manuscript significantly.
The article researched the effect of different inoculation method on plant growth by SynCom bacteria of 5 N-fixing bacteria. The result showed reference value for the application of microbial fertilizers on actual agriculture production. I have a few question before the decision.
Comment 1: 1）The nitrogen form affects the colonization and function of phyllosphere and rhizosphere microorganisms，the nitrogen form of plant culture media should be shown.
Authors: The nitrogen source (calcium and ammonium nitrate salts) was added to the text (line 198 in the revised manuscript) as suggested by the Reviewer.
Comment 2: 2) line 181-195, did the final concentration of 4*108 cell per ml means each 5 strains concentration or the total SynCom bacteria? the dosage of microbial consortium should be consistent,please explain the why the different dosage used in the article, and what the final concentration of bacteria on RL treatment.
Authors: The final concentration of 4x108 cells ml-1 is the concentration of the pooled strains (the total SynCom). The SynCom was diluted in the water tank (for root application, in both R and RL) and in the cell suspension for leaf application (L and RL) up to a final dosage of 2x106 cells ml-1. We better clarify this point in the text (lines 223-232 in the revised manuscript). Moreover, we corrected the typing error on the concentration of the cell suspension used for foliar application (line 232 in the revised manuscript).
Comment 3: 3) All the tables should be three-line table. And strongly recommend checking the result of statistical analyses.
Authors: In response to reviewer feedback, table lines were adjusted as suggested, and the statistical analysis described in section 2.8 was corrected to reflect the test employed accurately. Furthermore, the statistical analysis was re-evaluated, confirming the original results. To improve data visualization and interpretation, the following statement was added to Figure 3 and Table 2: “Distinct uppercase letters denote statistically significant variations (p < 0.05) among treatments for individual parameters, assessed independently for each parameter (n = 5)”. These revisions aim to address the reviewers' concerns and enhance the overall quality of the manuscript.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe paper named ” Effect of the Inoculation Method on the Potential Plant Growth-Promoting Activity of a Microbial Synthetic Consortium” try to enhance plant resilience and productivity in terms of bacterial consortium uses, without fertilizers.
Before publishing the paper, I ask the authors to explain some reasoning:
1. How do they decide the consortium type used?
2. Please explain the benefits for the environmental impact, in contrast with fertilizers use for plant growth.
3. How is this implementation possible in real life of plants? What is the impact of the consortium according with the soil biota?
Author Response
Reviewer 3.
Dear Reviewer, we express our sincere gratitude for your insightful comments and constructive suggestions, which allowed us to improve the quality of this manuscript significantly.
The paper named ” Effect of the Inoculation Method on the Potential Plant Growth-Promoting Activity of a Microbial Synthetic Consortium” try to enhance plant resilience and productivity in terms of bacterial consortium uses, without fertilizers.
Before publishing the paper, I ask the authors to explain some reasoning:
Comment 1: How do they decide the consortium type used?
Authors: As reported in lines 99-102 and 139-140 (original manuscript), we included in the SynCom all the N-fixing bacteria isolated from the different sites. To better clarify this point, we added the sentence “Recognizing the critical role of nitrogen in plant growth and development and the environmental effects associated with intensive use of synthetic N-fertilizers [36], the SynCom was constructed using free-living diazotrophs with varying salt tolerance and phosphate solubilizing capacities, which can represent a key natural source of N in natural and agricultural ecosystems [37].” (lines 111-115 in the revised manuscript). To answer the Reviewer’s request, we added extra information regarding sampling choice (lines 95-110), which can also probably clarify the request of Reviewer 3
Comment 2: Please explain the benefits of the environmental impact, in contrast with fertilizers used for plant growth.
Authors: We modified the sentence on line 41-45 on the original manuscript with the following: “Thus, the development of effective microbial biofertilizers, i.e., products containing beneficial microorganisms (mainly fungi and bacteria) that can be applied to the plant [4], represents a promising and sustainable approach for enhancing soil fertility and promoting plant growth, contributing to minimize excessive synthetic fertilizer application, thus mitigating greenhouse gas emissions, water pollution, and soil degradation [5]” (lines 40-45).
Comment 3: How is this implementation possible in real life of plants? What is the impact of the consortium on soil biota?
Authors: To address the Reviewer’s comment, we modified the original manuscript by adding the following sentence at the beginning of the discussion: “The implementation of PGPR in plant production systems can be achieved through various methods, including seed coating, root dipping, leaf spraying, and soil drenching. The choice of method depends on the crop species, the specific SynCom composition, and the environmental conditions [45]” (lines 378-381 in the revised manuscript). Moreover, we also added a sentence at the end of the discussion: “While this study focused on a hydroponic system to evaluate SynCom's efficacy under controlled conditions, it is important to investigate its performance in soil-based systems, considering its application in different plant systems. Further research is therefore needed to fully understand the long-term effects of the SynCom on plant health and productivity in soil-based cultivations and soil biota and the implications of inoculation on the overall sustainability of agricultural practices” (lines 527-532). Before commercialization, all PGPR must be tested in the field and on several plant species.
Round 2
Reviewer 2 Report
Comments and Suggestions for Authorsthe article have been carefully revised. I still have a question that I want to discuss with the authors. The final concentration of RL treatment is 4x108 cells ml-1 and 2x106 cells ml-1, which is higher than the R or L treatment. Suggest design different bacteria dosage for forward work, to identify whether the key actors is the dosages of bacteria or the inoculation ways。
Author Response
Comment 1.
Response to Comment 1: Dear Reviewer, As reported in the previous version, the foliar application was done with the diluted SynCom cell suspension to maintain consistency across treatments. We revised the text of paragraph 2.6 to better clarify the inoculation strategy we used (lines 216-226).
