Gluconacetobacter diazotrophicus Inoculation of Two Lettuce Cultivars Affects Leaf and Root Growth under Hydroponic Conditions

Round 1

Reviewer 1 Report

It is an interesting manuscript that studies Gluconacetobacter diazotrophicus inoculation of two lettuce cultivars so as to observe the growth of leaves and roots in hydroponic conditions. It is a manuscript that has been worked on neatly, with many details.

However, I think that in the introduction it is necessary to add more information about the way G. diaz. acts on growth of plants.

The quality of the tables is visually low, I think the format of the tables needs to be revised.

Author Response

Comments and Suggestions for Authors

It is an interesting manuscript that studies Gluconacetobacter diazotrophicus inoculation of two lettuce cultivars so as to observe the growth of leaves and roots in hydroponic conditions. It is a manuscript that has been worked on neatly, with many details.

Thank you for recognizing the effort that we put into preparing this manuscript.

However, I think that in the introduction it is necessary to add more information about the way G. diaz. acts on growth of plants.

We appreciate this suggestion as many readers may not be familiar with G. diaz. We cited 23 new references in this revised version to provide a brief, but thorough background on G. diaz. as a growth promoting symbiotic organism. Published information specifically on the action of G. diaz in lettuce is lacking. That is one reason that our work is original, especially for a crop as important as lettuce. As a result of this revision, the manuscript is greatly improved. See revisions in the Introduction and References highlighted in red.

The quality of the tables is visually low, I think the format of the tables needs to be revised.

Yes, we have provided original tables in Word format to improve the quality.

Author Response File: Author Response.docx

Reviewer 2 Report

The subject of this study is well introduced and the manuscript is interesting. However, the data provided are insufficient to support the conclution. For instance, the authors mentioned that Black Seeded Simpson demonstrated a shifting of production towards aerial tissues, with significantly greater shoot production and reduced root production. However, the authors performed only two replicate experiments and, more seriously, the results of these two experiments were inconsistent. For BSS1, the SDM was elevated in group INOC compared to group NOC, while in SBB2, half of the results in group INOC were decreased. The results of SDM were also inconsistent. And I think there should be at least three replicates for each treatment experiment to be statistically significant and to really make conclusions. Thank you!

Author Response

Comments and Suggestions for Authors

The subject of this study is well introduced and the manuscript is interesting.

We are glad that you enjoyed the manuscript, and we hope other readers of Applied Sciences will also find the subject interesting.

However, the data provided are insufficient to support the conclution. For instance, the authors mentioned that Black Seeded Simpson demonstrated a shifting of production towards aerial tissues, with significantly greater shoot production and reduced root production. However, the authors performed only two replicate experiments and, more seriously, the results of these two experiments were inconsistent. For BSS1, the SDM was elevated in group INOC compared to group NOC, while in SBB2, half of the results in group INOC were decreased. The results of SDM were also inconsistent. And I think there should be at least three replicates for each treatment experiment to be statistically significant and to really make conclusions. Thank you!

We appreciate the reviewer’s attention to detail in regard to the data as presented in tables and figures. As described in the manuscript, this study was conducted in a growth chamber that accommodated 44 jars, which allowed 3 or 4 replications of each treatment in each of the four experiments (BSS 1, 2 and BIB 1, 2). The data were statistically analyzed within each experiment, and we report significant differences in 3 of the 4 experiments. We did not conduct statistics across experiments, and it would not be valid to do so. A third experiment would not allow for any different statistical design than was conducted for each individual experiment. The purpose for repeating the experiment for both cultivars was similar to that required for publishing results of field studies. A minimum of two years is required in order to verify that observed results in one year are not merely an aberration due to climate or other external factors. The reviewer is correct that we did not obtain significant differences in the BSS2 experiment. However, we did observe significant increases in shoot dry matter (SDM) in three of the four experiments. As we describe in the manuscript, “Two cultivars of lettuce were chosen for the study based on similar days to maturity, compact stature, and disease resistance.” Although the literature suggests that response to inoculation is not necessarily consistent even across cultivars of the same plant, based on significant results in one BSS experiment, significant results in both BIB trials, and the similarity of these cultivars, we think there is strong evidence that BSS does respond to inoculation with greater SDM production. In fairness, we present an honest description of these mixed results for BSS and allow the reader to judge whether the response is real. The conclusions that we arrived at for BIB are well supported. The conclusions for BSS are less well supported, but we feel that this should not prevent these data and results from being reported. We think the readers of this journal will appreciate the new information. We appreciate the reviewer’s high standards for publication, and we hope this clarification of the statistical design used in this study will be convincing.

Author Response File: Author Response.docx

Reviewer 3 Report

1. Table 1 is not good and must be re-constructed
2. The statics of Table 1 should be revised and clarified
3. In table 1: **: significant at alpha, Where **?
4. All the comments on Table 1 should be taken in consideration for Tables 2, 3, and 4
5. Discussion should be revised and improved
6. In Introduction: Lettuce in the 1st line should be re-written as complete latin name, this is the 1st writing of it.

Author Response

Comments and Suggestions for Authors

We appreciate the Reviewer’s constructive comments, and the resulting changes in the manuscript will greatly improve this publication.

1. Table 1 is not good and must be re-constructed

All Reviewers commented on Table quality, and revised tables in Word format are being submitted with the revised manuscript.

1. The statistics of Table 1 should be revised and clarified

What may not be clear to this Reviewer and other readers is that observations across all N levels for INOC were pooled and statistically compared to the pooled observations for NON allowing for 28 degrees of freedom. We modified the statistics section of the Methods to explain how the data were analyzed. This change is shown in blue type.

1. In table 1: **: significant at alpha, Where **?

In the statistical section of the Methods, we state “All significance tests were performed at alpha < 0.05 using PROC MIXED and PROC GLM for multi-factor ANOVA with type 3 tests of fixed effects and Tukey-Kramer HSD for least squares means differences.” All tables contain a footnote: ** = significant at alpha <0.01, * = <0.05. n/s = not significant. In Table 1, root dry matter is significant at **

1. All the comments on Table 1 should be taken in consideration for Tables 2, 3, and 4

Edits in the statistical section of the Methods pertain to all tables, and similar edits to improve quality were made to all tables.

1. Discussion should be revised and improved

Thank you for the suggestion. We added a section to the discussion: “Cross experiment observations and implications.” This section considers combined results across all 4 experiments and draws implications regarding possible mechanisms of plant response to inoculation. These changes are shown in blue in the revised manuscript.

1. In Introduction: Lettuce in the 1st line should be re-written as complete latin name, this is the 1st writing of it.

Thank you for catching that mistake. Latin name was added.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

I appreciate the authors for showing real data results, and present an honest description of these mixed results for BSS. Thereby, I think readers will be able to discern the accuracy of the results for themselves, and judge whether the response is real.

Author Response

I appreciate the authors for showing real data results, and present an honest description of these mixed results for BSS. Thereby, I think readers will be able to discern the accuracy of the results for themselves, and judge whether the response is real.

Again, thank you for your assessment of our data, its statistical analysis, and presentation of results. We value your opinion.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors should modify the discussion section with more clarify for the readers

Author Response

The authors should modify the discussion section with more clarify for the readers

We looked at the Discussion section more critically, and we realized that the writing style was overly concise, sometimes to the point of possibly confusing the reader. Although we did not change the meaning of any of the points that were made, we made edits through this section to relax the writing style and make it easier for the reader to understand our meaning. As a result, the manuscript is much improved. Thanks for the guidance.

Author Response File: Author Response.docx