Assessing the Effect of Physicochemical Properties of Saline and Sodic Soil on Soil Microbial Communities

Round 1

Reviewer 1 Report

In my opinion, the article by Gao et al. is quite well written and contributes to enriching knowledge regarding the differences in microbial communities between saline and sodic soils with different physicochemical properties.

Only the following observation seems to me of no negligible relevance for the acceptance of the publication of the article: you talk about the method for determining the CEC, but the CEC values of the soils do not appear in the text. I never see the CEC considered among the various physicochemical properties reported in the various graphs. Why this?

For the rest, below a few minor changes are required, necessary to improve the quality and the understanding of the manuscript, which seems to be unclear in some parts:

Abstract:

• Lines 16 and 17: “The results…properties” this sentence is not very clear and there is no description of the physicochemical properties analysed.
• Lines 23-25: the last sentence is not understood, it has to be rewritten. What are the "significant differences"? What does it mean that they were more vital?

Introduction:

• Lines 30-32: this sentence has to be divided in two parts and has to be rewritten, that it cannot be understood.
• Line 33: “soil types…meadows” these are not soil types, but the different land uses.
• Line 39 and line 43: here it would be better to wrap.
• Line 42 and 43: insert a space between “4” and “dS/m”... Check this throughout the text.
• Lines 48 and 49: join these two sentences without wrapping.
• Lines 55, 57 and 66: the year of the reference is missing. “Joseph Frazer et al.” this reference is wrong, please insert the correct one including the year of publication. The numbers [19], [20] and [23] should be inserted next to the reference to which they refer and not at the end of the sentence.
• Line 57: use the abbreviation for EC directly, since you abbreviated it earlier in the Introduction section itself.
• Line 68: why do you use "meanwhile" here? I don't think there is any need...it doesn't seem to connect the previous sentence.
• Lines 68 and 69: I don't understand why you first write “saline and sodic saline” and then “saline-sodic”. This part is confusing, please rewrite it explaining better what you mean.
• Line 70: what does "pH stress" mean here... Do you mean high pH? I think "stress" should be removed.
• Line 72: “their” is wrong. Maybe you meant to write “the”.
• Lines 78-90: this part needs to be modified... Tt is very complex. You have repeated the same sentence and the same concept 3 times. Try to organise better and make it clearer what the aims of the study were.
• Line 82: insert in which country it is located.

Materials and Methods:

• Line 96: here it would be better to wrap.
• Line 102: perhaps “From” is missing at the beginning of the sentence?
• Line 110: use only the abbreviation for EC, that has already been abbreviated.
• Line 113: add something about how the samples were mineralised? About the various parameters and standards used? They are missing.
• Line 114: add a reference for the ammonium acetate method that was followed? And consider the initial comment regarding the CEC.
• Line 127: in the title “High-throughput sequencing” should not be reported, because you talk about sequencing in the next paragraph.
• Line 139: insert a space between template and 10 ng.
• Line 140: “the” before “30 s “must be removed.
• Line 143: explain why all PCR products were detected? to control quality?
• Line 160: the paragraph is 2.6

Results:

• Lines 191 and 192: the reference to Figure 1a should also be included.
• Line 193: “solid” is a mistake.
• Line 195: the unit of measurement is missing for the reported values and "receptively" is a mistake.
• Line 199: are they mg/kg or g/kg? Figure 1a shows g/kg. Figure 1b lacks the unit of measurement for the content of various ions in saline and sodic soils. Moreover, so far, the letter next to the figure number has always been written in lower case... Check this and standardise throughout the text. Finally, the Ca content was also lower in saline soils, as for the other ions reported.
• Line 200: the reference to Figure 1c is missing.
• Line 201: remove the full stop from the title of the paragraphs. Check this in the text.
• Line 210: specify that the “diversity indexes” are of bacteria and fungi and not of archaea.
• Line 211: put “Observed” in lower case.
• Line 217: “Figure 1” not in italics and check this throughout the text. Moreover, in Figure 1 before “content” on y axis, specify that it is mineral element content.
• Line 219: in the Table 1, "classifications" write it on one line only; “Archaea, Bacteria, and Fungi” write them in bold as it is not easy to see them. Finally, insert a space between” 585” and “±”.
• Line 220: remove the dot between SD.
• Line 228: the reference to “Figure 2b” is missing.
• Line 231: the reference to “Figure 2a and b” is missing.
• Line 241: the reference to “Figure 3a, c and e” is missing.
• Line 242: insert a comma before “except”.
• Line 248: remove the second dot at the end of the caption in Figure 3.
• Line 253: seems to be a sentence repeated earlier in line 251 and says nothing more.
• Line 262: insert a space between “age” and 13.81%”.
• Line 279: here it would be better to wrap.
• Line 282: “Furthermore” here its use is not appropriate, because you are talking about bacteria first and now fungi.
• Lines 291 and 292: this sentence should be written more clearly. It seems to me that one genus correlated significantly in the saline soil and one in the sodic soil.
• Lines 298-300: in what kind of soil for these two genera? On the graph, you reported g_Fusariella.
• Line 307: “were” I think it is a mistake and it should be removed.
• Line 311: this last part needs to be better explained. In which soils? You mean both? Please specify. Also, this part of the fungi needs to be better commented and expanded. I see that there are other negative correlations, but also positive ones.
• Line 315: insert after communities “at phylum level” also in accordance with Figure 6. Also, check the caption in Figure 6, which must be written in accordance with Figure 5; for example, "(b)" must be inserted before and not at the end, and “tests” must be written in singular, as in Figure 5. In Figure 5, there is a mistake in the second box: it is “bacterial phylum”. In Figure 5, insert the indication of saline and sodic soils, as in Figure 6. In Figure 6, improve reading quality... Tt is almost impossible to read the genera names of microbes.

Discussion:

• Line 330: check the meaning of this sentence, it doesn't sound good.
• Line 339: “did not be” is wrong in English. Correct this.
• Line 342: put “mantel” in capitals.
• Line 358: check the meaning of this sentence, it doesn't sound good.
• Line 400 and 401: check the meaning of this sentence, it doesn't sound good.
• Line 409: find a synonym for “improves”, there is “improvement” nearly and thus it doesn't sound good.

Concusions:

• Line 428: after “shed” you must add “light on” for the right meaning of the sentence.

Supplementary Materials:

• In Figure S1: “PCoA” is written twice.
• “Table S1”: there is no reference to this table in the text. Insert it, please. It is only mentioned in the list of the Supplementary Materials (line 434 of the manuscript).

Author Response

May. 23th. 2022
Agriculture
Dear editor, thank you and the reviewers for the valuable comments about the manuscript entitled “Assessing the effect of physicochemical properties of saline and sodic soil on soil microbial communities” for publication in Agriculture as an article. The submission ID for this manuscript is agriculture-1677092.
We have carefully revised the manuscript according to the comments and suggestions of you and the reviewers. The amendments have been highlighted with yellow color in the revised manuscript. We feel that the manuscript has been greatly improved based on these changes. The point-by-point responses to the comments are detailed below.
Thank you for your consideration. We urgently hope that it can be published in this
journal.
We look forward to hearing from you soon.
Sincerely yours,
Zhisheng Yu, Ph.D
Professor of College of Resources and Environment, University of Chinese Academy
of Sciences, 19 A Yuquan Road, Beijing 100049, P. R. China
E-mail: [email protected]

Response to Reviewer 1Comments

Responses to comments are as follows.
We are most grateful for the professional advice. Your suggestions have been highly
valuable for the improvement of our article. We hope you are satisfied with our reply.

• Point 1: Only the following observation seems to me of no negligible relevance for the acceptance of the publication of the article: you talk about the method for determining the CEC, but the CEC values of the soils do not appear in the text. I never see the CEC considered among the various physicochemical properties reported in the various graphs. Why this?

Response 1: Thanks for the reviewer giving us this precious opportunity and greatly positive suggestions, which guided this manuscript's improvement and clarity. According to the Formula in reference 32, we described the calculation process of ESP with CEC and exchangeable sodium. To avoid the confusing, we added CEC data in the supplement.

Response 1 revision: CEC as analyzed using the ammonium acetate method. Exchangeable sodium percentage (ESP, %) was calculated by the date of exchangeable sodium and CEC (Table S1) [33].

“Table S1” was added in the text. (Line 113)

For the rest, below a few minor changes are required, necessary to improve the quality and the understanding of the manuscript, which seems to be unclear in some parts:

Abstract:

• Point 2: Lines 16 and 17: “The results…properties” this sentence is not very clear and there is no description of the physicochemical properties analysed.

Response 2: Thanks. Thanks for the reviewer giving us this precious opportunity and greatly positive suggestions. According to the results of ESP and EC in the soil samples, the samples were divided into two categories (saline soil and sodic soil). The sentence in the abstract was used to illustrate this result.

Response 2 revision: The results of ESP and EC in the soil samples indicated that the soil samples were categorized as saline soil and sodic soil. (Line 18-19)

• Point 3: Lines 23-25: the last sentence is not understood, it has to be rewritten. What are the "significant differences"? What does it mean that they were more vital?

 Response 3: Thanks. Thanks for your suggestions. This sentence aimed to explain the proportions in sodic soil more strongly affecting soil microbiota. while, as you mentioned, the confusing sentence had to be rewritten. 

Response 3 revision: These results indicated that the proportions in sodic soil were more strongly affecting soil microbiota. (Line 25-26)

Introduction:

• Point 4: Lines 30-32: this sentence has to be divided in two parts and has to be rewritten, that it cannot be understood.

Response 4: Thanks. Thanks for your suggestions. The long sentence was not easily understood. Two short sentences were better.

Response 4 revision: The saline-alkali situation has been perceived as one of the serious reasons for causing soil degradation throughout the world. Saline-alkali soil reduces soil microbial diversity, activity, and soil quality. (Line 31-33)

• Point 5: Line 33: “soil types…meadows” these are not soil types, but the different land uses.

Response 5: Thanks. Thanks for your suggestions. I check the definition of ecosystem carefully again. Many references classified soil samples into different ecosystems such as agriculture ecosystem, forest ecosystem, and grassland ecosystem. So, “soil types” was not accurate. I think it could be replaced with soil ecosystems.

Response 5 revision: Saline-alkali soil distributes in more than 100 countries and affects various soil ecosystems such as agriculture, grassland, and wet meadows. (Line 34)

Supplemental references in response 4:

1) Frąc, M.; Hannula S.E.; Bełka, M.; Jędryczka, M. Fungal biodiversity and their role in soil health. Front Microbiol. 2018, 9, 707. https://doi.org/10.3389/fmicb.2018.00707

2) Barberan, A.; Bates, S.T.; Casamayor, E.O.; Fierer, N. Using network analysis to explore co-occurrence patterns in soil microbal communities. ISME J. 2012, 6, 343-351. https://doi.org/10.1038/ismej.2011.119

• Point 6: Line 39 and line 43: here it would be better to wrap.

Response 6: Thanks. Thanks for your suggestions. These two sentences were introducing the definition of saline and sodic soil. The two sentences were revised and wrapped.

Response 6 revision: According to the soil classification, saline-alkali soil is the general term of salinization and alkalization soil (based on the physicochemical properties of soil such as electrical conductivity, EC, dS/m, and exchangeable sodium percentage, ESP, %, saline-alkali soil can be classified as saline soil, EC < 4 dS/m, ESP < 15%, and sodic soil, EC < 4 dS/m, ESP > 15%) [8,9]. (Line 40-44)

• Point 7: Line 42 and 43: insert a space between “4” and “dS/m”... Check this throughout the text.

Response 7: Thanks. Thanks for your suggestions. As you mentioned, the two spaces were forgotten. They have been added.

Response 7 revision: … sodium percentage, ESP, %, saline-alkali soil can be classified as saline soil, EC < 4 dS/m, ESP < 15%, and sodic soil, EC < 4 dS/m, ESP > 15%) [8,9]. (Line 43-44)

• Point 8: Lines 48 and 49: join these two sentences without wrapping.

Response 8: Thanks. Thanks for your suggestions. As you suggested, I also considered the order of these sentences. The two sentences should be moved to the last part of the first paragraph.

Response 8 revision: Microbial communities mainly consist of archaea, bacteria, and fungi [15]. (Line 49)

• Point 9: Lines 55, 57 and 66: the year of the reference is missing. “Joseph Frazer et al.” this reference is wrong, please insert the correct one including the year of publication. The numbers [19], [20] and [23] should be inserted next to the reference to which they refer and not at the end of the sentence.

Response 9: Thanks. Thanks for your suggestions. The year of the reference in the article “Zhang et al.” and “Joseph Frazer et al.” have been added. Joseph Frazer has been revised by the author’s first name Banda. The numbers [19], [20], and [23] have been moved to the referred authors before.

Response 9 revision:

A study by Zhang et al. (2019) [19] found that bacterial alpha diversity in desert soil strongly negatively correlated with saline gradients using EC as an indicator of soil salinity. Banda et al. (2021) [20] demonstrated that the relative abundance of Euryarchaeota (archaeal phylum) in the saline lakes increased with rising salinity. (Line 55-59)

Zhao et al.(2018) [23] demonstrated that pH is equally the determinant of bacterial communities in soil across a salt lake shoreline. (Line 66-68)

• Point 10: Line 57: use the abbreviation for EC directly, since you abbreviated it earlier in the Introduction section itself.

Response 10: Thanks. Thanks for your suggestions. The abbreviation of electrical conductivity has been added. The same problem in response 8 has been revised.

Response 10 revision: A study by Zhang et al. (2019) [19] found that bacterial alpha diversity in desert soil strongly negatively correlated with saline gradients using EC as an indicator of soil salinity. (Line 55-57)

• Point 11:Line 68: why do you use "meanwhile" here? I don't think there is any need...it doesn't seem to connect the previous sentence.

Response 11: Thanks. Thanks for your suggestions. The previous sentence and this sentence were used to introduce the following two sentences. As you suggested, “meanwhile” should be deleted.

Response 11 revision: It was demonstrated that pH in the soil could increase due to saline ions exchange. (Line 68)

• Point 12: Lines 68 and 69: I don't understand why you first write “saline and sodic saline” and then “saline-sodic”. This part is confusing, please rewrite it explaining better what you mean.

Response 12: Thanks. Thanks for your suggestions. “saline and sodic saline” and “saline-sodic” should be revised more clearly. The second-word saline in “saline and sodic saline” should be revised to another word “soil”. “saline-sodic” should be revised to “saline and sodic soil”. To describe the changing factor of saline soil and sodic soil, this sentence was revised.

Response 12 revision:

Therefore, saline and sodic soil are closely related to occur concurrently. (Line 69)

The change of saline and sodic soil was associated with high Na, pH, and ESP [25]. (Line 70)

• Point 13: Line 70: what does "pH stress" mean here... Do you mean high pH? I think "stress" should be removed.

Response 13: Thanks. Thanks for your suggestions. "stress" should be deleted. The same problem in response 11 has been revised.

Response 13 revision: The change of saline and sodic soil was associated with high Na, pH, and ESP [25]. (Line 70)

• Point 14: Line 72: “their” is wrong. Maybe you meant to write “the”.

Response 14: Thanks. Thanks for your suggestions. “However, the differences between saline and sodic soil and their relative importance of saline and sodic soil in shaping the microbial community of soil are still incomplete, …”. There was a writing mistake in this sentence.

Response 14 revision: However, the differences between saline and sodic soil and the relative importance of saline and sodic soil in shaping the microbial community of soil are still incomplete, …” (Line 72-73)

• Point 15: Lines 78-90: this part needs to be modified... Tt is very complex. You have repeated the same sentence and the same concept 3 times. Try to organise better and make it clearer what the aims of the study were.

Response 15: Thanks. Thanks for your suggestions. The aims and confusing sentences of this part have been modified.

Response 15 revision: The feces of livestock can also result in soil with different levels of salinization and alkalization, and shapes different sizes of spots on the grassland [27]. In this study, we analyzed the effect of physicochemical differences between saline and sodic soil on soil microbiota using high-throughput sequencing in Maodeng pasture in Xilingol, in Inner Mongolia, China. Here, we present the aims of this study: (1) the differences in physicochemical properties in saline and sodic soil (2) the effect of the physicochemical properties differences between saline and sodic soil on soil microbiota (3) revealing the potential function change of in saline and sodic soil. The purpose of this study is to understand the differences between salinization and alkalinization and provide a direction for improving soil quality.  (Line 78-87)

• Point 16: Line 82: insert in which country it is located.

Response 16: Thanks. Thanks for your suggestions. The location of country has been added.

Response 16 revision: In this study, we analyzed the effect of physicochemical differences between saline and sodic soil on soil microbiota using high-throughput sequencing in Maodeng pasture in Xilingol, in Inner Mongolia, China. (Line 79-82)

Materials and Methods:

• Point 17: Line 96: here it would be better to wrap.

Response 17: Thanks. Thanks for your suggestions. The sentence has been revised.

Response 17 revision: The experiment was carried out at the field station located in Maodeng pasture (Research Station of Animal Ecology, Institute of Zoology, CAS, 44˚11'N, 116˚27'E), located in the Xilingol League of Inner Mongolia. (Line 93-95)

• Point 18: Line 102: perhaps “From” is missing at the beginning of the sentence?

Response 18: Thanks. Thanks for your suggestions.

Response 18 revision: Stones and residue were removed from five subsamples before they were mixed as a site soil sample. (Line 101-102)

• Point 19: Line 110: use only the abbreviation for EC, that has already been abbreviated.

Response 19: Thanks. Thanks for your suggestions. the abbreviation for electrical conductivity has been revised.

Response 19 revision: The basic soil physicochemical properties (including pH, EC, C/N ratio, exchangeable sodium, and cation exchange capacity (CEC)) and soil ion (including K, Ca, Na, Mg, Fe, Al, Sr, Si) were measured and analyzed. (Line 108-110)

• Point 20: Line 113: add something about how the samples were mineralised? About the various parameters and standards used? They are missing.

Response 20: Thanks. Thanks for your suggestions. We tested metal elements with ICP-OES. The principle of ICP-OES is to measure the emitted light energy of each wavelength and to calculate the concentration of metal elements in the sample. Therefore, the samples were not mineralized. The various parameters and standards referenced the standard liquid of the ICP-OES during the process of experiments.

• Point 21: Line 114: add a reference for the ammonium acetate method that was followed? And consider the initial comment regarding the CEC.

Response 21: Thanks. Thanks for your suggestions. The reference for the ammonium acetate method has been added

Response 21 revision: CEC as analyzed using the ammonium acetate method [32]. (Line 111-112)

• Point 22: Line 127: in the title “High-throughput sequencing” should not be reported, because you talk about sequencing in the next paragraph.

Response 22: Thanks. Thanks for your suggestions. The title “High-throughput sequencing” should be deleted.

Response 22 revision: 2.4. PCR amplification (Line 125)

• Point 23: Line 139: insert a space between template and 10 ng.

Response 23: Thanks. Thanks for your suggestions. The space has been added.

Response 23 revision: “…and DNA template 10 ng…” (Line 137)

• Point 24: Line 140: “the” before “30 s “must be removed.

Response 24: Thanks. Thanks for your suggestions. The word “the” in sentence has been deleted.

Response 24 revision: “…35 cycles of 95 ℃ for 30 s …” (Line 138)

• Point 25: Line 143: explain why all PCR products were detected? to control quality?

Response 25: Thanks. Thanks for your suggestions. As you mentioned, this step was used to detect the size of the amplified produces. The size of the archaeal produces was 434bp. The size of bacterial produces was 468bp. The size of fungal produces was 300bp. We detected the size of all the produces using 1% (w/v) agarose gels to make sure of the validity of the amplified size.    

• Point 26: Line 160: the paragraph is 2.6

Response 26: Thanks. Thanks for your suggestions. The wrong title number has been revised.

Response 26 revision: “2.6. Statistical analyses” (Line 158)

Results:

• Point 27: Lines 191 and 192: the reference to Figure 1a should also be included.

Response 27: Thanks. Thanks for your suggestions. According to the value of ESP and EC in Figure 1a, the soil samples were divided into two categories. Figure 1b should be replaced with Figure 1a. The lower case “a”should be revised.

Response 27 revision: “… (Figure 1A) …”  (Line 189-190)

• Point 28: Line 193: “solid” is a mistake.

Response 28: Thanks. Thanks for your suggestions. The word was wrong. It should be another word sodic.

Response 28 revision: “The basic physicochemical properties of sodic soil samples, including ESP, EC…”  (Line 191)

• Point 29: Line 195: the unit of measurement is missing for the reported values and "receptively" is a mistake.

Response 29: Thanks. Thanks for your suggestions. The unit of measurement was added. The word receptively was deleted.

Response 29 revision: The average of ESP in saline and sodic soil were 3.61% and 23.03% (Figure 1A). （Line 193）

• Point 30: Line 199: are they mg/kg or g/kg? Figure 1a shows g/kg. Figure 1b lacks the unit of measurement for the content of various ions in saline and sodic soils. Moreover, so far, the letter next to the figure number has always been written in lower case... Check this and standardise throughout the text. Finally, the Ca content was also lower in saline soils, as for the other ions reported.

Response 30: Thanks. Thanks for your suggestions. The unit of metal elements was mg/kg. The unit of Figure 1b was added in ordinate legend. All the figures in the text were revised with capital letters. Ca was supplemented in the results of metal elements contents.

Response 30 revision:

All the figures in text were revised with capital letters including Figure 1 (Line 215), Figure 2 (Line 246), Figure 3 (Line 249 ), Figure 5 (Line 332), Figure 6 (Line 339), Figure 7 (Line 343).

Ca, Mg, Fe, Al, Sr, and Si contents in saline soil were lower than sodic soil samples. (Line 197-198)

• Point 31: Line 200: the reference to Figure 1c is missing.

Response 31: Thanks. Thanks for your suggestions. Figure 1C was added.

Response 31 revision: Ca, Mg, Fe, Al, Sr, and Si contents in saline soil were lower than sodic soil samples (Figure 1C). (Line 197-198)

• Point 32: Line 201: remove the full stop from the title of the paragraphs. Check this in the text.

Response 32: Thanks. Thanks for your suggestions. All the full stop of the titles in the text were deleted.

Response 32 revision:

3.2. Sequencing data and alpha diversity of saline and sodic soil (Line 199)   

3.3. Beta diversity of saline and sodic soil (Line 218)

• Point 33: Line 210: specify that the “diversity indexes” are of bacteria and fungi and not of archaea.

Response 33: Thanks. Thanks for your suggestions. The confusing sentence has been revised.

Response 33 revision: Alpha diversity of the bacterial community was higher than archaea and fungi, and all the bacterial diversity indexes in saline soil were higher than in sodic soil. (Line 207-208)

• Point 34: Line 211: put “Observed” in lower case.

Response 34: Thanks. Thanks for your suggestions. The word has been revised.

Response 34 revision: The observed OTUs of the archaeal community in sodic soil samples were significantly higher than those in saline soil samples (t-test, P < 0.05). (Line 209-210)

• Point 35: Line 217: “Figure 1” not in italics and check this throughout the text. Moreover, in Figure 1 before “content” on y axis, specify that it is mineral element content.

Response 35: Thanks. Thanks for your suggestions. All the numbers of titles have been revised. The ordinate legend has been revised.

Response 35 revision:

“ Figure 1. (A). Basic physicochemical properties of soil samples (t-test, P < 0.05); (B, C). Soil contents of metal elements and differences in saline and sodic soil (t-test, P < 0.05).”  (Line 216-217). 

The ordinate legend was rewritten as “Metal element contents in soil samples (mg/kg)”. (Figure 1B.)

• Point 36: Line 219: in the Table 1, "classifications" write it on one line only; “Archaea, Bacteria, and Fungi” write them in bold as it is not easy to see them. Finally, insert a space between” 585” and “±”.

Response 36: Thanks. Thanks for your suggestions. The word "classifications" in Table 1 has been adjusted. The words “Archaea, Bacteria, and Fungi” have been rewritten in bold. The space has been added between “585” and “±”. (Table 1)

• Point 37: Line 220: remove the dot between SD.

Response 37: Thanks. Thanks for your suggestions. the dot between SD has been removed.

Response 37 revision: Values in this table indicate the means ± SD of each index.  (Line 243)

• Point 38: Line 228: the reference to “Figure 2b” is missing.

Response 38: Thanks. Thanks for your suggestions. The reference figure number “Figure 2B” has been added.

Response 38 revision: Further analysis revealed that the unique OTUs in the archaeal communities mainly belonged to phyla Thermmoplasmatota and unclassified_archaea (Figure 2B). (Line 222-224)

• Point 39: Line 231: the reference to “Figure 2a and b” is missing.

Response 39: Thanks. Thanks for your suggestions. The reference figure number “Figure 2A and B” has been added.

Response 39 revision: Although the unique OTUs in bacterial communities of sodic soil was lower than in saline soil, they belonged to more phyla (Figure 2A and B). (Line 226-227)

• Point 40: Line 241: the reference to “Figure 3a, c and e” is missing.

Response 40: Thanks. Thanks for your suggestions. The reference figure number “Figure 3A, C and E” has been added and supplemented to the sentence.

Response 40 revision: While, basic physicochemical properties and metal elements of saline were not significant correlation with microbial communities (Figure 3A, C and E). (Line 240-241)

• Point 41: Line 242: insert a comma before “except”.

Response 41: Thanks. Thanks for your suggestions. The comma before “except” has been added.

Response 41 revision: “…metal elements of sodic soil positively correlated with microbial communities, except for K.” (Figure 3B, D and F). (Line 238-240)

• Point 42: Line 248: remove the second dot at the end of the caption in Figure 3.

Response 42: Thanks. Thanks for your suggestions. The second dot in Figure 3 has been deleted. The lower cases in Figure 3 have been revised.

Response 42 revision: Figure 3. The relationship between basic physicochemical properties and soil metal elements in saline and sodic soil samples and archaeal (A and B), bacterial (C and D), and fungal (E and F) communities. (Line 250-251)

• Point 43: Line 253: seems to be a sentence repeated earlier in line 251 and says nothing more.

Response 43: Thanks. Thanks for your suggestions. The sentence mainly introduced the three dominant archaeal phyla. The word “Among” connected the before and after sentences.

Response 43 revision: The main three archaeal phyla in soil samples (relative abundance > 1%) belonged to Crenarchaeota, unclassified archaea, and Thermoplasmatota (Figure 5A). Among, most of the archaeal sequences of soil samples were fallen into Crenarchaeota (average relative abundance, 94.89%). (Line 251-254)

• Point 44: Line 262: insert a space between “age” and 13.81%”.

Response 44: Thanks. Thanks for your suggestions. The space between “age” and 13.81%” has been added.

Response 44 revision: “…(average, 13.81%), followed by Glomeromycota…” (Line 265)

• Point 45：Line 279: here it would be better to wrap.

Response 45: Thanks. Thanks for your suggestions. The sentence in Line279 described the correlation between saline soil properties and bacterial compositions. We added the word “However” to describe the correlation between sodic soil properties and bacterial compositions.

Response 45 revision: However, Proteobacteria and Chloroflexi positively correlated with K, Fe, Al, and Si in sodic soil, especially Chloroflexi strongly correlated with Al and Si (P < 0.01). (Line 283-285)

• Point 46：Line 282: “Furthermore” here its use is not appropriate, because you are talking about bacteria first and now fungi.

Response 46: Thanks. Thanks for your suggestions. As you mentioned, this word was not suit.

Response 46 revision:  “For the fungal community, low abundant phyla of fungal phyla were only significantly related to Si in saline soil…” (Line 285-286)

• Point 47：Lines 291 and 292: this sentence should be written more clearly. It seems to me that one genus correlated significantly in the saline soil and one in the sodic soil.

Response 4:7: Thanks. Thanks for your suggestions. The confusing sentence has been rewritten.

Response 47 revision: In the archaeal genera, both of g_Candidatus_Nitrocosmicus in saline soil and g_Methanocorpusculum in sodic soil significantly correlated with C/N (Figure 6B). (Line 295-296)

• Point 48：Lines 298-300: in what kind of soil for these two genera? On the graph, you reported g_Fusariella.

Response 48: Thanks. Thanks for your suggestions. The confusing sentence has been revised. We added the “In the sodic soil” for clarifying the soil environment of these two genera.

Response 48 revision: In sodic soil, fungal genus g_unclassified_f_Nectriaceae significantly correlated to EC, ESP, and C/N. g_unclassified_f_Microascaceae significantly correlated to pH (P < 0.01), and g_Fusarium was significantly correlated to EC (P < 0.01). (Line 301-304)

• Point 49：Line 307: “were” I think it is a mistake and it should be removed.

Response 49: Thanks. Thanks for your suggestions. As you mentioned, the word should be deleted.

Response 49 revision: “Bacterial metabolic pathways strongly positively correlated with Fe and Si in sodic soil samples except for genetic information processing…” (Line 312-314)

• Point 50：Line 311: this last part needs to be better explained. In which soils? You mean both? Please specify. Also, this part of the fungi needs to be better commented and expanded. I see that there are other negative correlations, but also positive ones.

Response 50: Thanks. Thanks for your suggestions. This part analysis of the bacterial and fungi has been expanded.

Response 50 revision: There were no significant correlation between physicochemical properties of saline soil and archaeal and bacterial metabolic pathways (Figure 7). For the fungal functional metabolic pathways, “Fungal Parasite Plant Pathogen Saprotroph” negatively correlated with Mg in saline soil (Figure 7C). “Animal Pathogen Endophyte Plant Pathogen Wood Saprotroph” in saline soil negatively correlated with Mg, but negatively correlated with pH in sodic soil. Only “Animal Pathogen Endophyte Plant Pathogen Wood Saprotroph” was positively correlated with Al in saline soil. “Animal Pathogen Endophyte Lichen Parasite Plant Pathoge Soil Saprotroph-Wood Saprotroph” negatively correlated with Mg and Sr in sodic soil. “Animal Pathogen-Dung Saprotroph-Endophyte-Epiphyte-Plant Saprotroph-Wood Saprotroph” negatively correlated with ESP, Na, and pH in saline and sodic soil (Figure 7C). “Animal Pathogen-Dung Saprotroph-Endophyte-Epiphyte-Plant Saprotroph-Wood Saprotroph” negatively correlated with Al and Fe in saline soil, but positively correlated with K. In total, the differences of correlation with fungal functins and different soil properties shown that both saline and sodic soil affected fungal functiona. “Animal Pathogen-Dung Saprotroph-Endophyte-Epiphyte-Plant Saprotroph-Wood Saprotroph” in fungal function was more negatively correlated with soil physicochemical properties. (Line 315-330)

• Point 51：Line 315: insert after communities “at phylum level” also in accordance with Figure 6. Also, check the caption in Figure 6, which must be written in accordance with Figure 5; for example, "(b)" must be inserted before and not at the end, and “tests” must be written in singular, as in Figure 5. In Figure 5, there is a mistake in the second box: it is “bacterial phylum”. In Figure 5, insert the indication of saline and sodic soils, as in Figure 6. In Figure 6, improve reading quality... Tt is almost impossible to read the genera names of microbes.

Response 51: Thanks. Thanks for your suggestions. Figure 5A has inserted “at phylum level” after communities. We compared with and revised the Figure 5 and 6. We improved the sharpness of the Figures 6.

Response 51 revision: Figure 5. (A). The relative abundance of microbial communities at phylum level, including archaeal (orange box), bacterial (pink box), and fungal (green box) communities; (B) Relative abundance of microbial communities at phylum level is related to soil environmental factors (saline soil at left and sodic soil at right) by partial Mantel test (phyla of low abundance represent archaeal microbial communities with relative abundance less than 1%, the relative abundance of bacterial and fungal microbial communities less than 5%), edge width corresponds to the Mantel’s r statistic for the corresponding distance correlations, and the edge color denotes the statistical significance based on 9999 permutations. (Line 333-336)

Figure 6. (A). Dominant microbial at genera level in saline soil (blue frame) and sodic soil (red frame) based on LEfSe identifies (LDA scores greater than 2.0). (B). Relative abundance of the dominant genus related to soil environmental factor by partial Mantel test, edge width corresponds to the Mantel’s r statistic for the corre-sponding distance correlations, and the edge color denotes the statistical significance based on 9999 permutations. (Line 340-341)

Discussion:

• Point 52：Line 330: check the meaning of this sentence, it doesn't sound good.

Response 52: Thanks. Thanks for your suggestions. The confusing sentence has been revised.

Response 52 revision: However, less studies consider the affecting of differences of saline soil and sodic soil on soil microbiota, respectively. (Line 348-349)

• Point 53：Line 339: “did not be” is wrong in English. Correct this.

Response 53: Thanks. Thanks for your suggestions. The grammer mistake has been revised.

Response 53 revision:  “Although the roles of soil factors for alpha diversity were still not clearly explained…”  (Line 357)

• Point 54：Line 342: put “mantel” in capitals.

Response 54: Thanks. Thanks for your suggestions. The word “Mantel” has been revised.

Response 54 revision: “The Mantel test results showed…” (Line 360)

• Point 55：Line 358: check the meaning of this sentence, it doesn't sound good.

Response 55: Thanks. Thanks for your suggestions. The repetitive and confusing sentence has been deleted.

Response 55 revision: However, unique OTUs were affected by differences in saline and sodic soil, e.g., Thermmoplasmatota was higher abundance in sodic soil, Basidiomycota was higher in saline soil, and a higher abundance of Actinobacteria was observed in sodic soil (Figure 2B). (Line 374-377)

• Point 56：Line 400 and 401: check the meaning of this sentence, it doesn't sound good.

Response 56: Thanks. Thanks for your suggestions. We read the reference and rewritten the sentence.

Response 56 revision: K dominant involves in balancing the osmotic of microbial cytoplasm in saline soil [61]. (Line 417-418)

• Point 57：Line 409: find a synonym for “improves”, there is “improvement” nearly and thus it doesn't sound good.

 Response 57: Thanks. Thanks for your suggestions. We selected a synonym for  “improves”.

Response 57 revision: “…might innovate soil improvement strategies in the future.” (Line 426)

Concusions:

• Point 58：Line 428: after “shed” you must add “light on” for the right meaning of the sentence.

 Response 58: Thanks. Thanks for your greatly suggestions. The grammer mistake has been revised.

Response 58 revision: “Our results shed light on the main factors in saline and sodic soil affecting…” (Line 445)

Supplementary Materials:

• Point 59：In Figure S1: “PCoA” is written twice.

Response 59: Thanks. Thanks for your suggestions. The second has been deleted. The lower cases in Figure S1 have been revised.

Response 59 revision: Figure S1 PCoA analysis of archaeal (A), bacterial (B), and fungal (C) communities based on Bray-Curtis distance matrix. (Line 450-451)

• Point 60：“Table S1”: there is no reference to this table in the text. Insert it, please. It is only mentioned in the list of the Supplementary Materials (line 434 of the manuscript).

Response 60: Thanks. Thanks for your suggestions. The mistake has been revised.

Response 60 revision: “Table S1 The data of exchangeable sodium and cation exchange capacity; ” (Line 451-452)

Author Response File: Author Response.docx

Reviewer 2 Report

My comments are in the body of the manuscript in PDF.

Comments for author File: Comments.pdf

Author Response

May. 23th. 2022
Agriculture
Dear editor, thank you and the reviewers for the valuable comments about the manuscript entitled “Assessing the effect of physicochemical properties of saline and sodic soil on soil microbial communities” for publication in Agriculture as an article. The submission ID for this manuscript is agriculture-1677092.
We have carefully revised the manuscript according to the comments and suggestions of you and the reviewers. The amendments have been highlighted with yellow color in the revised manuscript. We feel that the manuscript has been greatly improved based on these changes. The point-by-point responses to the comments are detailed below.
Thank you for your consideration. We urgently hope that it can be published in this journal.
We look forward to hearing from you soon.
Sincerely yours,
Zhisheng Yu, Ph.D
Professor of College of Resources and Environment, University of Chinese Academy
of Sciences, 19 A Yuquan Road, Beijing 100049, P. R. China
E-mail: [email protected]

Response to Reviewer 2 Comments

Responses to comments are as follows.
We are most grateful for the professional advice. Your suggestions have been highly valuable for the improvement of our article. We hope you are satisfied with our reply.:

Abstract:

• Point 1：Please enter the research objectives

Response 1: Thanks for the reviewer giving us this precious opportunity and greatly positive suggestions, which guided this manuscript's improvement and clarity. The object of this study has been supplemented.

Response 1 revision: “The object of this study is demonstrating the difference of salinization and alkalization soil and driving factors to affecting microbiota. In this study…” (Line 14-15)

Introduction:

• Point 2：Please clarify the research objectives

Response 2: The aims and confusing sentences of this part have been modified.

Response 2 revision: The feces of livestock can also result in soil with different levels of salinization and alkalization, and shapes different sizes of spots on the grassland [27]. In this study, we analyzed the effect of physicochemical differences between saline and sodic soil on soil microbiota using high-throughput sequencing in Maodeng pasture in Xilingol, in Inner Mongolia, China. Here, we present the aims of this study: (1) the differences in physicochemical properties in saline and sodic soil (2) the effect of the physicochemical properties differences between saline and sodic soil on soil microbiota (3) revealing the potential function change of in saline and sodic soil. The purpose of this study is to understand the differences between salinization and alkalinization and provide a direction for improving soil quality.  (Line 78-87)

• Point 3：Please check this value. Is it 27.6℃

Response 3: Thanks. Thanks for your suggestions. The annual temperature referenced relevant references in the text [28-31]. The character climate of Xilingol is cold and windy. The glaciation can last for five months. The highest temperature is about 21℃ in July and the lowest is about -20℃ in January. The reference has more detailed information on the climate of Xilingol. (Line 96)

Results:

• Point 4：It is sodic soil?

Response 4: Thanks. Thanks for your suggestions. The spelling mistake has been revised.

Response 4 revision: “The basic physicochemical properties of sodic soil samples, including ESP, EC…” (Line 191)

• Point 5：It is appropriate to use the content of each element. These elements are micro and macronutrients for plants and microorganisms. It is not appropriate to use the terminology of soil ions.

Response 5: Thanks. Thanks for your suggestions. As you mentioned, “the content of each element” was more accurate.

Response 5 revision: The contents of each element in saline and sodic soil samples showed a wide change (Figure 1B). (Line 194-195)

• Point 6：The table must appear complete on a single page

Response 6: Thanks. Thanks for your suggestions. The table has been adjusted.

Response 6 revision: Table 1 (Line 242).

• Point 7：The relative abundance of bacteria has an error. Data are presented at the phylum level. However, a fraction of the bar is being presented for bacteria domain.

Response 7: Thanks. Thanks for your suggestions. The data of the domain has been verified. The phyla have been modified.

Response 7 revision: Figure 2 (Line 246)

• Point 8：Please improve figure resolution

Relative abundance should be displayed as a bar graph

Response 8: Thanks. Thanks for your suggestions. The resolution of figure 5 has been improved. Bar graphs can present microbial relative abundance more clearly. Considering the graph size and correspondence between graphs A and B, we presented the relative abundance using the lines.

Response 8 revision: Figure 5 (Line 332).

• Point 9：The testural part of the graphic is unreadable. Improvs figure resolution and standardizes the font to arial or times mew roman

Response 9: Thanks. Thanks for your suggestions. The resolution of figure 6 has been improved. The font of the genus name has been revised times new roman.

Response 9 revision: Figure 6 (Line 339).