Arbuscular Mycorrhizae Contribute to Growth, Nutrient Uptake, and Ornamental Characteristics of Statice (Limonium sinuatum [L.] Mill.) Subject to Appropriate Inoculum and Optimal Phosphorus

Round 1

Reviewer 1 Report

Overall, the presented article is quite interesting, well-written, well-presented and should be of interest to readers. It provides valuable insights into the benefits resulting from AMF colonization in Statice, in terms of growth, nutrient uptake and ornamental characteristics. However, a few points (listed below) must be addressed before the manuscript can be accepted for publication.

L63-L70 - It would be interesting to include an experimental design of the treatments, number of replicates, and number of plants used per replicate, as the paragraph referring to this information is a bit confusing. It is just a suggestion, but I believe that a diagram or image included as supplemental material, for example, would benefit the understanding of the readers in general and those who want to replicate or conduct a similar experiment.

L83 - “…and growth medium with 50 ± 10 active organs per gram.” are “organs” really the term you want to use? Perhaps “functional units” or “colony-forming units”? Something similar. “Organs” doesn't seem like a good choice.

L87 - Why did the non-inoculated pots receive 25g of double-autoclaved inoculum? Why not simply add 25g of soil since they are supposed to be non-inoculated? Have you performed any control on this autoclaved inoculum, such as plating, to ensure that nothing actually grows, and it is effectively inactivated? I would like a brief explanation for this choice.

L97 and 105 - Was the Evaluation of Vegetative and Ornamental Characteristics carried out for all plants in all replicates? This information should be clearly presented in the text, as below in point 2.4, only 2 plants were used for the Assessment of Root Mycorrhizal Colonization. Regarding this point 2.4, why were only 2 plants considered for this evaluation? How were these 2 plants chosen? Randomly? Also, these details should be included in the methodology section.

L112 - “Oven dried materials were grounded to…” The word "materials" should be replaced with another since it refers to the plants (or parts thereof) that will be analysed. As presented, it seems that they are referring to things other than the plants.

L146 – “Flowering delayed with M1, M2, and M3 inoculation by 12, 9, and 7 days…” the days 9 and 7 are switched. According to the table analysis the correct order is “Flowering delayed with M1, M2, and M3 inoculation by 12, 7, and 9 days…”.

L205 - “Principal component analysis was performed while taking growth, ornamental, and nutrient…” The word "characteristics" is missing after "ornamental," as it doesn't make much sense otherwise.

L207 – “According to the PCA, the top two components accounted for 75.09% of the variation…” What is meant by "the top two components"? The variance values associated with PCA1 and PCA2 represent the proportion of total data variation. Together, these two components can be used to visualize the underlying data structure and to identify patterns and relationships among different variables.

Overall, the entire section 3.4. Principal Component Analysis should be reviewed. It is necessary to first understand the results of the analysis before they can be properly discussed. The caption of figure 2 should also be revised to make clear the analysis presented on the right and left and completed with the missing terms. The sentence "Treatment positions along PCA 1 and 2 are represented by symbols." does not make much sense in the caption. With careful analysis, it is easy to understand which treatments are more related to each other, which factors are involved in this relationship, and which are not, etc.

I would also appreciate clarification on the location of the Pearson correlations results table, as mentioned in the methodology, or where the discussion of these correlation results can be found.

Italics are missing in “et al.” references throughout the text.

Author Response

Reviewer #1

Response to Reviewers Comments

Authors (A): The authors would like to appreciate the kind evaluation of the manuscript by the editor and the reviewers and giving very useful comments. Below is the list of actions and responses carried out based on the comments issued by the reviewer 1.

Reviewer (R) Overall, the presented article is quite interesting, well-written, well-presented and should be of interest to readers. It provides valuable insights into the benefits resulting from AMF colonization in Statice, in terms of growth, nutrient uptake and ornamental characteristics. However, a few points (listed below) must be addressed before the manuscript can be accepted for publication.

We thank the reviewer for the positive comments.

1. R) L63-L70 - It would be interesting to include an experimental design of the treatments, number of replicates, and number of plants used per replicate, as the paragraph referring to this information is a bit confusing. It is just a suggestion, but I believe that a diagram or image included as supplemental material, for example, would benefit the understanding of the readers in general and those who want to replicate or conduct a similar experiment.
2. A) We thank the reviewer for the comment. The requested items are in section 2.1, adding information on the number of plants used for each measurement.

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1. R) L83 - “…and growth medium with 50 ± 10 active organs per gram.” are “organs” really the term you want to use? Perhaps “functional units” or “colony-forming units”? Something similar. “Organs” doesn't seem like a good choice.
2. A) The term "organs" was replaced by the more correct term "fungal structures".

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1. R) L87 - Why did the non-inoculated pots receive 25g of double-autoclaved inoculum? Why not simply add 25g of soil since they are supposed to be non-inoculated? Have you performed any control on this autoclaved inoculum, such as plating, to ensure that nothing actually grows, and it is effectively inactivated? I would like a brief explanation for this choice.
2. A) In order to work accurately and to provide the same growing medium between inoculation and non-inoculation treatments, 25 g of autoclaved inoculum was added to control pots (no inoculation). When the inoculum is autoclaved, it becomes completely inactive in terms of fungal structure, as a result, no colonization takes place. Also, to ensure the inactivity of the autoclaved inoculum, root colonization was checked in several control plants (inoculated with autoclaved inoculum) and no colonization was observed.

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2. R) L97 and 105 - Was the Evaluation of Vegetative and Ornamental Characteristics carried out for all plants in all replicates? This information should be clearly presented in the text, as below in point 2.4, only 2 plants were used for the Assessment of Root Mycorrhizal Colonization. Regarding this point 2.4, why were only 2 plants considered for this evaluation? How were these 2 plants chosen? Randomly? Also, these details should be included in the methodology section.
3. A) Thanks for the noting; we included the number of plants used in 2.3. Since it is necessary to harvest the roots and destroy the plant to check the colonization, 5 experimental units were considered for each replication so that there were enough plants for the analyses. Out of 5 experimental units of each replication, two of them were destructively used to estimate colonization (6 plants per treatment), and three of them were used to evaluate vegetative and ornamental traits and measure elements (9 plants per treatment). Details about the number of plants used in the different measurements were included in the Materials and Methods section (end of the first paragraph of 2.1; beginning of the first paragraph of 2.3 and 2.4).

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1. R) L112 - “Oven dried materials were grounded to…” The word "materials" should be replaced with another since it refers to the plants (or parts thereof) that will be analysed. As presented, it seems that they are referring to things other than the plants.
2. A) The sentence was corrected (first line of 2.5).

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1. R) L146 – “Flowering delayed with M1, M2, and M3 inoculation by 12, 9, and 7 days…” the days 9 and 7 are switched. According to the table analysis the correct order is “Flowering delayed with M1, M2, and M3 inoculation by 12, 7, and 9 days…”.
2. A) Thank you for noting. The order has been corrected.

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1. R) L205 - “Principal component analysis was performed while taking growth, ornamental, and nutrient…” The word "characteristics" is missing after "ornamental," as it doesn't make much sense otherwise.
2. A) Thank you for noting. "characteristics" was added to correct the content of the sentence.

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75. R) L207 – “According to the PCA, the top two components accounted for 75.09% of the variation…” What is meant by "the top two components"? The variance values associated with PCA1 and PCA2 represent the proportion of total data variation. Together, these two components can be used to visualize the underlying data structure and to identify patterns and relationships among different variables.
76. A) The word "top" was replaced by "first".

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3. R) Overall, the entire section 3.4. Principal Component Analysis should be reviewed. It is necessary to first understand the results of the analysis before they can be properly discussed. The caption of figure 2 should also be revised to make clear the analysis presented on the right and left and completed with the missing terms. The sentence "Treatment positions along PCA 1 and 2 are represented by symbols." does not make much sense in the caption. With careful analysis, it is easy to understand which treatments are more related to each other, which factors are involved in this relationship, and which are not, etc.
4. A) The results related to Principal Component Analysis were thoroughly revised. As for the figure caption, according to another reviewer's comment, the left and right figures were merged, so the figure caption now looks appropriate. The unnecessary sentence mentioned was removed.

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1. R) I would also appreciate clarification on the location of the Pearson correlations results table, as mentioned in the methodology, or where the discussion of these correlation results can be found.
2. A) The figure related to the correlation analysis results was added to the results section (Figure 3) and point 3.4 title now includes “and correlation”.

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1. R) Italics are missing in “et al.” references throughout the text.
2. A) According to the format of the journal, "et al" is not written in italics in the text.

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We tried our best to improve the quality of the manuscript as the reviewers commented. The authors hope the revised version of the manuscript meets the editor and reviewer’s expectations.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors

The following modifications are required:

Abstract

ü  Before describing the goal, the authors must define the issue in a single line and explain why they chose this approach to study this research.

ü  This section should include information about the experimental design and the components of factors.

ü  The authors should provide some measured quantitative values for studied traits.

ü  In the final line of the abstract, the authors should present a decisive conclusion derived from the research and provide a single line of future prospects.

Keywords

ü  The keywords did not accurately reflect the manuscript's content, and the words that comprised the title should not be used as keywords. As a result, the keyword structure should be altered.

Introduction

ü  The different species of Limonium genus should be provided

ü  The authors should insert some lines about the biofertilizer types.

ü  Some sentences about the different species of Arbuscular mycorrhizal fungi should be inserted

ü  No information about the importance of P on the growth and ornamental characteristics

ü  Some information about the chemical and beneficial properties of Limonium sinuatum should be included

ü  The authors should give some lines about the knowledge gap which their research has covered along with the hypothesis statement

ü  The authors should also include a novelty statement. In this study, what new things have the authors done or correlated in comparison to previous ones?

ü  The goal is not to reflect the study's content. The aim should include the name or type of studied parameter, such as the type of response.

Materials and Methods

ü  Why did the authors use the randomized complete block design rather than the randomized complete design when conducting the experiment in the greenhouse and in the pot?

ü  The number of plants per treatment should be clearly stated.

ü  This section should specify the experimental design and the component of factors.

ü  In section 2.1, Why didn't the authors use the P level of zero?

ü  The level of M0 should be added to the section 2.1

ü  Section 2.2: this section should be clearly detailed. The authors should be firstly shown the preparation of mycorrhizal inoculum and then the treatment of plants by the mycorrhizal inoculum and P.

ü  Section 2.3: the units should contain the units and support by the references

ü  Section 2.5: The types of materials used for analyzing should be provided

ü  Section 2.6: The type of ANOVA (One- or Two-way ANOVA) should determine. Furthermore, the name of the software used for data analysis should be included.

Results

ü  The authors just should the effects of the interaction between the studied factors. The main effects of each factor on the measured traits are not available in this manuscript.

ü  Section 3.1: The authors should provide some measured quantitative values for studied traits.

ü  The ± value should be defined. Also, the *, **, and *** should be specified in Table 2

ü  The standard deviation or error should be inserted to the Table 3

ü  Section 3.2: The authors should provide some measured quantitative values for studied traits.

ü  The caption of Figure 1 should be placed under the figure. Also, the letter m should be capitalized on this figure

ü   Section 3.3: The authors should provide some measured quantitative values for studied traits.

ü  Figure 2 (PCA):  It is preferable to combine the figures 2A and B to demonstrate clearly the PCA plot.

ü  L121: The authors mentioned that they performed the Pearson correlations, but the results of correlation are not available in the manuscript

The quality of English language should be improved

Author Response

Reviewer #2

Response to Reviewers Comments

Authors (A): The authors would like to appreciate the kind evaluation of the manuscript by the editor and the reviewers and giving very useful comments. Below is the list of actions and responses carried out based on the comments issued by reviewer 2.

Abstract

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1. A) Information about the experimental design and the number of replications was added to the abstract. Now lines 27-31 show the experimental design and the components of factors.

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1. R) The authors should provide some measured quantitative values for studied traits.
2. A) Quantitative values were added in the abstract.

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1. R) In the final line of the abstract, the authors should present a decisive conclusion derived from the research and provide a single line of future prospects.
2. A) The last sentence was modified so that the results were stated decisively. Also, another line was added with a view to the future.

Keywords

1. R) The keywords did not accurately reflect the manuscript's content, and the words that comprised the title should not be used as keywords. As a result, the keyword structure should be altered.
2. A) Keywords changed. Some were deleted, others were edited or added, so as to cover the content of the article and not appear in the title.

Introduction

1. R) The different species of Limonium genus should be provided
2. A) Information about the Limonium genus and its species was added to the introduction (L55-58).

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1. R) The authors should insert some lines about the biofertilizer types.
2. A) Biofertilizers and their types were well described in two paragraphs (lines 89-97) in the article.

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1. R) Some sentences about the different species of Arbuscular mycorrhizal fungi should be inserted
2. A) Information about types of Arbuscular mycorrhiza was added in lines 106-115.

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1. R) No information about the importance of P on the growth and ornamental characteristics
2. A) The importance of phosphorus and its effect on some ornamental plants was added in a paragraph to the introduction (Lines 79-88).

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1. R) The authors should give some lines about the knowledge gap which their research has covered along with the hypothesis statement
2. R) The authors should also include a novelty statement. In this study, what new things have the authors done or correlated in comparison to previous ones?
3. A) The items requested in the last two comments were done, and now the last paragraph of the introduction (lines 129-142) cover knowledge gaps, work done in the present research, and novelty.

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1. R) The goal is not to reflect the study's content. The aim should include the name or type of studied parameter, such as the type of response.
2. A) It was changed and improved. See the last paragraph of the introduction.

Materials and Methods

1. R) Why did the authors use the randomized complete block design rather than the randomized complete design when conducting the experiment in the greenhouse and in the pot?
2. A) Randomized complete block design was chosen due to the uncertainty of the uniformity of our greenhouse environment for all treatments.

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1. R) The number of plants per treatment should be clearly stated.
2. A) Done. We clarified the plants per treatment in lines L151-156.

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1. R) This section should specify the experimental design and the component of factors.
2. A) The experimental design and factors is better described in lines 146 to 151.

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2. R) In section 2.1, Why didn't the authors use the P level of zero?
3. A) The initial amount of P in the soil used for this study was 10 mg/kg and this was considered as a control. And in the subsequent treatments, P was added to the desired soil.

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2. R) The level of M0 should be added to the section 2.1
3. A) It is available in line 148-149 (non-mycorrhizal inoculation).

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2. R) Section 2.2: this section should be clearly detailed. The authors should be firstly shown the preparation of mycorrhizal inoculum and then the treatment of plants by the mycorrhizal inoculum and P.
3. A) Thank you for your comments, we acted accordingly in section 2.2.

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2. R) Section 2.3: the units should contain the units and support by the references
3. A) It was done.

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2. R) Section 2.5: The types of materials used for analyzing should be provided
3. A) It was done.

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2. R) Section 2.6: The type of ANOVA (One- or Two-way ANOVA) should determine. Furthermore, the name of the software used for data analysis should be included.
3. A) It was done.

Results

1. R) The authors just should the effects of the interaction between the studied factors. The main effects of each factor on the measured traits are not available in this manuscript.
2. A) For the traits where the interaction between AMF and P was significant, we reported the interaction effect, while for the traits where the interaction was not significant, we reported the main effects.

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3. R) Section 3.1: The authors should provide some measured quantitative values for studied traits.
4. A) It was done. Several quantitative values were added in 3.1, but also in 3.2 and 3.3

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1. R) The ± value should be defined. Also, the *, **, and *** should be specified in Table 2
2. A) The ± value was defined for all tables and the *, **, and *** were specified in tables 2 and 4.

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1. R) The standard deviation or error should be inserted into Table 3
2. A) The standard errors were inserted into the data of Table 3.

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1. R) The caption of Figure 1 should be placed under the figure. Also, the letter m should be capitalized on this figure
2. A) Figure 1 was replaced by the new figure (capitalized M). Also, the caption of the Figure was transferred to the bottom of the figure.

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3. R) Section 3.3: The authors should provide some measured quantitative values for studied traits.
4. A) Quantitative values were presented in the whole text (abstract, results and discussion).

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1. R) Figure 2 (PCA):  It is preferable to combine the figures 2A and B to demonstrate clearly the PCA plot.
2. A) Thank you for the suggestion. Figures were combined.

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1. R) The authors mentioned that they performed the Pearson correlations, but the results of correlation are not available in the manuscript
2. A) Thanks for your comment. The Pearson correlation table was added to the results (Figure 3).

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We tried our best to improve the quality of the manuscript as the reviewers commented. The authors hope the revised version of the manuscript meets the editor and reviewer’s expectations.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have made the changes and corrections as proposed to them. Below, I offer a few minor suggestions for some parts that have been modified and could be further improved:

L42-44 – Replace the current sentence with... “Limonium is a genus in the family Plumbaginaceae that includes approximately 15 to 20 cultivated species, including L. sinuatum (statice), hybrids of L. bellidifolium and L. latifolium, as well as L. sinense and L. perezii [2].” Is more accurate and less confusing.

L46 – Replace “…are eaten as food,” with “used for culinary purposes” or similar.

L91 – Replace “fungus” with “fungi”. In this context, the plural form should be used.

L94-99 – According to the most recent rules, all scientific names should be in italics, including names of phyla, families, and so on.

L307 – “… did not have a share in the two components.” This portion of the sentence is unclear, and its meaning is not understandable. I suggest rephrasing this final bit.

L412 – Replace the current sentence with... “Enhanced phosphorus absorption due to mycorrhizal inoculation is a widely recognized phenomenon.”

Author Response

Reviewer #1

Response to Reviewers Comments

The authors have made the changes and corrections as proposed to them. Below, I offer a few minor suggestions for some parts that have been modified and could be further improved:

1. R) L42-44 –Replace the current sentence with... “Limoniumis a genus in the family Plumbaginaceae that includes approximately 15 to 20 cultivated species, including  sinuatum (statice), hybrids of L. bellidifolium and L. latifolium, as well as L. sinense and L. perezii [2].” Is more accurate and less confusing.
2. A) We changed the sentence as per your request, although it is using the same gerund twice (changed).
3. R) L46 –Replace “…are eaten as food,” with “used for culinary purposes” or similar.
4. A) Thank you for suggesting a better wording. Now L47.
5. R) L91 –Replace “fungus” with “fungi”. In this context, the plural form should be used.
6. A) We made the replacement and then changed the verb conjugation because the plural term. Now L98.
7. R) L94-99 –According to the most recent rules, all scientific names should be in italics, including names of phyla, families, and so on.
8. A) Thank you for pointing out our mistake. We corrected the scientific names that were not in italics. We also did the same in the literature list.
9. R) L307 –“… did not have a share in the two components.” This portion of the sentence is unclear, and its meaning is not understandable. I suggest rephrasing this final bit.
10. A) We modified the wording trying to make it clearer. Now L327.
11. R) L412 –Replace the current sentence with... “Enhanced phosphorus absorption due to mycorrhizal inoculation is a widely recognized phenomenon.”
12. A) We made the requested change. Now L434.

We are thankful to the reviewer for helping us to improve the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors

Detail should be included in section 3.4, "Principal component analysis and correlation."  By displaying the findings of the four quarters of the plot, the PCA plot should be displayed in an understandable way. The type, value, and P-value of the correlation should be added for each trait pair.

Extensive correction should be performed.

Author Response

Reviewer #2

Response to Reviewers' Comments

Comments and Suggestions for Authors

3. R) Detail should be included in section 3.4, "Principal component analysis and correlation."  By displaying the findings of the four quarters of the plot, the PCA plot should be displayed in an understandable way. The type, value, and P-value of the correlation should be added for each trait pair.
4. A) Thank you for the comments, we revised them accordingly.

Comments on the Quality of English Language

1. R) Extensive correction should be performed.
2. A) We extensively had the English language correction for the first revision. We had again a thorough check and found corrections and revisions to be made. Specifically:

- Abstract: three small corrections.

- Introduction: extensive revisions made.

- Materials and Methods: revisions made.

- Results: extensive revisions made.

- Discussion: extensive revisions made

- Conclusions: revised.

We are thankful to the reviewer for enhancing the quality of our manuscript.

Author Response File: Author Response.pdf