Bacterial Community Structure and Environmental Adaptation in the Endorhizosphere and Rhizosphere Soils of Aeluropus sinensis from Saline Lands Across Coastal and Inland Regions of China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments

Please proofread for minor errors such as tense and typos.

In the materials and methods, how were the samples transported to the lab, and what was the time lapse between collection and sample processing?

In the PCR protocol, did you include a no-template control to ensure that there was no contamination? If yes, then you must indicate this, especially given that you did not include an extraction blank in your DNA extraction.

The results section is crowded with a repetition of what was done in the methods. You do not have to repeat what was done. Once you have the subheading, go straight into the results. Secondly, all explanations of the results must be carried to the Discussion section. E.g., Lines 276-279, 290-292, etc. Check for all instances in the manuscript.

 

Specific comments

Line 20: affected...

Line 21: dominated

Line 22: correlated

Line 24: Italicize scientific name

Line 26-28: "A salt-tolerant strain, Arthrobacter bergerei (KF_254744.1), was isolated from a maize irrigation experiment." This sentence is random. It suddenly appears without any previous mention of any experiment.

Lines 43-46: Within this distinctive habitat, plant-associated rhizosphere bacterial communities perform essential functions, such as influencing plant growth and developmental processes, soil nutrient cycling, environmental adaptation, and restoring degraded ecosystems [5].

Line 72: italicise genus name

Lines 73-74: It can grow normally in soils with salt content as high as....

Line 77-79: Please provide some references for the "previous studies"

Lines 93-95 are a repetition of Lines 43-46.

Lines 193 and 199:  You talk of deer grass (Muhlenbergia rigens) and not A. sinensis, which is your study plant. Please clarify.

Comments on the Quality of English Language

The manuscript must be proofread to eliminate minor errors.

Author Response

Responses to Reviewer 1’s comments:

General comments

Please proofread for minor errors such as tense and typos.

Responses: We sincerely thank the reviewer for constructive criticisms and valuable suggestions. For improving the quality of the manuscript, we have made the following modifications to the manuscript:

 

1. In the materials and methods, how were the samples transported to the lab, and what was the time lapse between collection and sample processing?

Responses: Rhizosphere soil and plant tissue samples were collected in the field and transferred to the laboratory using liquid nitrogen tank. Sample submission for sequencing was performed on Sep, 2024. The interval between sample collection and sequencing was approximately one month, ensuring sufficient preservation of nucleic acid integrity under continuous cryogenic storage conditions. Please check in the revised manuscript:

Line 138-139

Rhizosphere soil and plant tissue samples were collected in the field and transferred to the laboratory using liquid nitrogen tank.

Line 171

Sample submission for sequencing was performed on Sep, 2024.

 

2. In the PCR protocol, did you include a no-template control to ensure that there was no contamination? If yes, then you must indicate this, especially given that you did not include an extraction blank in your DNA extraction.

Responses: In this study, a no-template control was included during the DNA extraction process to monitor potential contamination and ensure the reliability of downstream amplification results.

Please check in the revised manuscript:

Line 171-173

In this study, a no-template control was incorporated during the DNA extraction process to monitor for potential contamination and ensure the integrity and reliability of downstream amplification results.

3. The results section is crowded with a repetition of what was done in the methods. You do not have to repeat what was done. Once you have the subheading, go straight into the results. Secondly, all explanations of the results must be carried to the Discussion section. E.g., Lines 276-279, 290-292, etc. Check for all instances in the manuscript. Responses:  We gratefully acknowledge the reviewers' constructive suggestions regarding the results section. All indicated sentences have been either removed or relocated and integrated into the discussion section, thereby improving the clarity and overall readability of the manuscript.

Line (Old version) 276-279

These findings suggest that Mo_DY represents a typical saline habitat, Mo_HB is characterized by high salinity combined with elevated nutrient levels, No_DY and Mi_TJ represent low-salt but high-alkalinity habitats, and No_JS is a low-salt environment with relatively high nutrient availability.

Line 285-286

We investigated the bacterial community structure in rhizosphere soils across seven sampling sites of A. sinensis, revealing significant variations in α-diversity among samples (Table 2).

Line 290-292

These findings suggest that the bacterial community at the No_DY_Rh site had reduced evenness, potentially due to the dominance of specific taxa.

Line 298-301

Overall, varying soil environments exert significant influences on the diversity of endophytic and rhizospheric bacterial communities associated with the A. sinensis; however, the nature and extent of these influences differ between the two microbial habitats.

Line 302-304

We constructed PCoA plots for both rhizospheric and endophytic bacterial communities based on Bray-Curtis dissimilarity to assess their community structure variations.

Line 311-313

These findings suggest that rhizospheric bacterial communities are significantly influenced by salinity, with medium-salinity samples exhibiting greater similarity among themselves and distinctness from low-salinity counterparts.

Line 320-321

, suggesting a unique endophytic community structure shaped by local environmental conditions

Line 337

Following the analysis of species annotation results,

Line 341

Notable variations in phylum-level bacterial abundance were observed across samples with different soil salinity levels.

Line 350-352

The dominant bacterial phyla in the root endosphere were broadly similar to those found in the rhizosphere; however, significant differences were observed in terms of abundance and community composition.

Line 356-358

These findings suggest that Proteobacteria may play a crucial role in mediating the root's response to salt stress in A. sinensis. In contrast, Actinobacteria were more abundant in samples from low-salinity and no-salinity environments.

Line 360-361

indicating a potential ecological advantage of this phylum in the root endosphere under non-saline conditions

Line 363-367

We employed redundancy analysis (RDA) to explore the relationships between soil physicochemical properties and the composition of bacterial communities in the rhizosphere and endorhizosphere (Fig.3 E and F), with the aim of identifying the key environmental factors that most effectively explain variations in bacterial community structure.

Line 377-379

These findings suggest that rhizosphere bacterial communities in A. sinensis are predominantly driven by salinity-related environmental factors, whereas endophytic communities are more strongly influenced by soil nutrient availability.

Line 380-382

Linear regression analysis was performed to evaluate the distance-decay similarity patterns of bacterial communities in both the rhizosphere and intraradical compartments in response to environmental and soil physicochemical factors (Fig. 4).

Line 390-392

These findings suggest that rhizosphere bacteria are more responsive to variations in soil salinity and pH, whereas intraradical bacteria are more influenced by soil nutrient availability.

Line 440-442

The results indicated that Aes2-treated plants exhibited significantly better growth, including a higher number of leaves and reduced leaf yellowing and wilting, compared to the salt-stressed control group (CK-Salt).

 

4. Specific comments

Line 20: affected...

Line 21: dominated

Line 22: correlated

Line 24: Italicize scientific name

Line 26-28: "A salt-tolerant strain, Arthrobacter bergerei (KF_254744.1), was isolated from a maize irrigation experiment." This sentence is random. It suddenly appears without any previous mention of any experiment.

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. We have corrected the grammatical errors in the abstract. As:

Soil salinity significantly affected rhizosphere bacterial diversity, with moderate levels increasing richness. Proteobacteria dominated both root and rhizosphere microbiomes across habitats. The endorhizosphere community strongly correlated with soil nutrients such as available phosphorus (AP) and total nitrogen (TN). Co-occurrence analysis reveals that chemoheterotrophic microbes in the A. sinensis rhizosphere employ distinct adaptive strategies across gradients, and ammonia-oxidizing bacteria (AOB) may support nitrogen cycling in the Yellow River Delta saline-alkaline ecosystem.

5. Lines 43-46: Within this distinctive habitat, plant-associated rhizosphere bacterial communities perform essential functions, such as influencing plant growth and developmental processes, soil nutrient cycling, environmental adaptation, and restoring degraded ecosystems [5].

It has been modified as suggested. As:

Line 42-44

Within this distinctive habitat, plant-associated rhizosphere bacterial communities perform essential functions, such as influencing plant growth and developmental processes, soil nutrient cycling, environmental adaptation, and restoring degraded ecosystems [5]

 

6. Line 72: italicise genus name

7. Lines 73-74: It can grow normally in soils with salt content as high as....

It has been modified as suggested. As:

Line 70-72

Aeluropus sinensis (Debeaux) Tzvelev, a perennial herb endemic to China and belonging to the genus Aeluropus within the Poaceae family, is widely distributed in saline-alkali soils along northern coastal and inland regions [4]. It can grow normally in soils with salt content as high as 1% [16].

 

8. Line 77-79: Please provide some references for the "previous studies"

It has been modified as suggested. As:

Line 76-78

Previous studies have demonstrated that under mild salt stress conditions, A. sinensis effectively reduces rhizosphere soil pH while increasing key nutrient contents such as total nitrogen and available phosphorus [4-6].

 

9. Lines 93-95 are a repetition of Lines 43-46.

According to the suggestion, we deleted this sentence.

 

10. Lines 193 and 199:  You talk of deer grass (Muhlenbergia rigens) and not A. sinensis, which is your study plant. Please clarify.

We are very grateful to the reviewers for pointing out this error, and we have made the correction. "Deer grass" has been changed to A. sinensis.

Reviewer 2 Report

Comments and Suggestions for Authors

Please refer to the attached document

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Please refer to the attached document

Author Response

Responses to Reviewer 2’s comments:

1. Language, grammar, typographic and formatting errors may be eliminated with a careful reading of the manuscript by a native English speaker. Some of the points in this regard include (for example only):

Responses:

We sincerely thank the reviewer for constructive criticisms and valuable suggestions. For improving the quality of the manuscript, we have made the following modifications to the manuscript:

 

a. Line 24: Italicize “A. sinensis” to make the pattern uniform.

It has been modified as suggested. As:

Line 23-35

Co-occurrence analysis reveals that chemoheterotrophic microbes in the A. sinensis rhizosphere employ distinct adaptive strategies across gradients

 

b. Line 48: Give spaces before and after “[6]”

It has been modified as suggested. As:

Line 45-48

A comprehensive understanding of how halophytes facilitate their own survival and promote ecosystem development through the regulation of soil microbial composition is critical for elucidating the underlying mechanisms of plant salt tolerance and for formu-lating scientifically robust and practically effective ecological conservation strategies [6].

 

c. I would prefer writing “salt-tolerance” to “salt tolerance”

We have replaced “salt tolerance” with “salt-tolerance” in the manuscript. As:

Line 13-16

This study examines how rhizosphere and endophytic microbes associated with Aeluropus sinensis—a salt-excreting halophyte—contribute to its salt-tolerance across saline-alkali environments.

 

d. Line 63: Replace “Patel et al.” with “Patel and Archana”. Similarly, in Line 580, prefer “Aslam and Ali” to “Aslam et al.”

It has been modified as suggested. As:

Line 62-64

Patel and Archana [15] isolated numerous endorhizosphere bacterial strains with plant growth-promoting potential from Poaceae species, including maize, rice, sorghum (Sorghum bicolor), and wheat.

Line 548-550

Aslam and Ali [55] isolated multiple Arthrobacter strains from the rhizosphere of Suaeda glauca and demonstrated that inoculation significantly enhanced plant growth under high-salt conditions, as indicated by increased chlorophyll content, greater aboveground and root biomass, and improved water use efficiency.

 

e. Line 74-75: Replace “asexual reproduction” with “vegetative propagation”

It has been modified as suggested. As:

Line 73-75

This species exhibits strong vegetative propagation capacity and can stabilize sandy soils through dense stolons and well-developed root systems, contributing significantly to the ecological restoration of degraded coastal and salt lake margin ecosystems [17].

 

f. Line 139: What is meant by “maizeer points”?

This is a mistake in writing. Sampling points were established at the four corners and at the central intersection of the diagonals within each sample plot.

Line 135-136

Within each 4 m² plot (2m × 2m), sampling points were established at the four corners and at the central intersection of the diagonals within each sample plot.

g. Line 225: Prefer “was” to “were” as soil was suspended, not grams.

It has been modified as suggested. As:

Line 225-227

Rhizosphere soil was suspended in 50 mL of sterilized high-salt LB liquid medium and incubated on a shaker at a constant temperature of 30°C and 150-250 rpm for 72 hours.

 

h. Line 238-239: Replace “electrophoretic ally” with “electrophoretically”.

It has been modified as suggested. As:

Line 239-240

Following amplification, the PCR products were electrophoretically analyzed on a 1% agarose gel to confirm successful amplification.

 

2. Line 74: Consider the statement, “This species…ecosystems”. The species (A. sinensis) claimed by authors with attributes contained in the statement is entirely different from the one (A. littoralis) that has been referred to in the Reference No 17.

We apologize for the error in the literature citation. The reference has been corrected to an article focusing on Aeluropus sinensis, a species discussed in the context of coastal grassland ecosystems.

         Yang, C.; Zhu, F.; Guo, K.; Feng, X.; Liu, X.; Bezemer, T. M. Spatial patterning and species coexistence: A case study using concentric circular vegetation patches in saline land. Science of The Total Environment 2024, 951, 175483 , doi: 10.1016/j.scitotenv.2024.175483.

 

3. Line 77-79: Consider the statement, “Previous studies…phosphorus.”: The statement needs some references-at least more than one-to support the observation.

It has been modified as suggested. As:

Line 76-78

Previous studies have demonstrated that under mild salt stress conditions, A. sinensis effectively reduces rhizosphere soil pH while increasing key nutrient contents such as total nitrogen and available phosphorus [4-6].

 

4. Table 1: The “Altitude” in the last column needs to be specified. Is it altitude from MSL?

Responses: Yes. We have stated this point in the document.

Line 132

Altitude refers to the height difference from mean sea level (MSL).

 

5. Was moisture level of the soil kept into account before taking rhizosphere soil samples? If yes, please mention.

Responses: This study assessed soil moisture content by comparing the dry and fresh weights of soil samples. The variation in moisture content across different sampling points was less than 10% of the mean value, indicating consistent water content distribution. Furthermore, microbial diversity indices showed no significant correlation with soil moisture content.

 

6. The standard protocol followed for extraction and sequencing of soil total DNA may be referred to by suitable reference(s).

Responses: We gratefully acknowledge the suggestion. A more appropriate reference has been substituted to better illustrate the DNA extraction methodology. As:

Hong, B.Y.; Driscoll, M.; Gratalo, D.; Jarvie, T.; Weinstock, G.M. Improved DNA Extraction and Amplification Strategy for 16S rRNA Gene Amplicon-Based Microbiome Studies. Int. J. Mol. Sci. 2024, 25, 2966. https://doi.org/10.3390/ijms25052966.

 

7.  Line 193, 199: Is word “deer grass" used for Aeluropus sinensis? If yes, this common name could be used in the Introduction section followed by botanical name in parenthesis when first cited.

Responses: We are very grateful to the reviewers for pointing out this error, and we have made the correction. "Deer grass" has been changed to A. sinensis.

 

8. Line 225, 427: The composition of high-salt LB liquid medium and LB medium may be given or referred to with suitable reference(s) or brand/Manufacturer.

Responses: We gratefully acknowledge the suggestion. A more appropriate reference has been substituted to better illustrate the high-salt LB liquid medium and LB medium.

Zhou, L.; Liu, Y.H.; Niu, H.J.; Sun, H.Y.; He, Y.X.; Zhang, X.X.; Zhang, Z.Y.; Huang, Y.L. Isolation and identification of salt-tolerant growth-promoting bacteria capable of remediating secondary salinized soils of greenhouses. Microbiology China 2024, 51, 3454-3467, doi: 10.13344/j.microbiol.china.231022.

 

9. Section 2.7: Please specify the “salt stress” in pot experiments under controlled conditions.

Responses: Thank you for the reviewers' suggestions. We have supplemented the detailed description of the plant salt stress treatment methods. As：

Line 253-255:

In the control experiment, Hogland nutrient solution was applied to each pot; in the salt stress treatment, a modified Hogland nutrient solution supplemented with 200 mM NaCl was used to impose salinity stress.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is improved