Functional Diversity of the Soil Culturable Microbial Community in Eucalyptus Plantations of Different Ages in Guangxi, South China

Round 1

Reviewer 1 Report

The manuscript is interesting and give some useful information about the effects of Eucalyptus plantations of different ages on the soil (culturable) microflora. But there are some important weak points that have to be corrected.

The title did not explain clearly that the studied soil microbial community was the only culturable fraction; thus I suggest this more clear title: Functional Diversity of the Soil Culturable Microbial Community in Eucalyptus plantations of Different Ages in Guangxi, South China; The 31 different carbon sources are not mentioned in the material and method section, and this makes more difficult to follow the description of the results (see the comments in the attached pdf). So, the table 2 must be put in the material and methods section; Figure 3 is incomplete: Where did the over-mature forest sample end up? Figure 6 legend is incomplete: what about the meaning of "TK"? Discussion 4.3. PCA and CCA of soil microbial utilization: The sentence “total nutrients to the community variation was greater than that of available nutrients” is internally contradictory: total nutrients have more weight on the microbial variation than the availability of the nutrients themselves. But, if the nutrients are not available for microorganisms, how can the community survive and vary? This point must be clarified, written in another way…; End of Discussion: about the sentence: “…if more carbohydrates are secreted by the plants into the soil, the capacity of the soil microbes to utilize the carbon substrates will become strong” - As this is a study on the only culturable fraction of soil microflora, the conditional is required. So, the sentence should change in: the capacity of the soil microbes to utilize the carbon substrates should become strong at least for the culturable fraction of the microflora.

Other suggestions are noted in the attached pdf.

The manuscript needs major revisions to be suitable for publication.

Comments for author File: Comments.pdf

Author Response

    Thank you very much for your careful comments on our manuscript, “Functional Diversity of the Soil  Culturable Microbial Community in Eucalyptus plantations of Different Ages in Guangxi, South China ”. We are very grateful for the constructive comments and suggested amendments. We have carefully studied the reviewers’ comments and have revised the manuscript accordingly. Because some charts cannot be shown here, please check the attachment, thank you.

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

In this work, authors selected five eucalyptus plantation sites in Guangxi, differing in age, from young to over-mature forest. The main objective of the project was to determine and compare the functional diversity of each soil, including Biolog EcoPlate method and physicochemical properties.  

Minor concerns:

Ln 52: authors mentioned that among the popular methods of measuring the microbial functional diversity, the Biolog EcoPlate assay is currently the most popular. I cannot agree with that, and more important there is no evidence / reference proving that statement is correct. This method is indeed very popular, but nowadays the high-throughput sequencing, especially shotgun sequencing of whole genomes, or rna-seq of whole transcriptomes is a leading method. Hover functional analysis such as Biolog, serve as a supplementary method. ln 60: author cited Grządziel and Gałązka (2017), [ref. 15], in the context of measuring microbial diversity in different soil types (NGS sequencing). I would recommend to include also another work of these authors, which include also Biolog EcoPlate analysis. Here is DOI of the abovementioned article: https://doi.org/10.3390/su11010056.

Major concerns:

Unfortunately, there is a huge methodological mistake, briefly:

Biolog EcoPlate assay was wrongly prepared: authors inoculated 93 wells containing appropriate substrate with diluted soil suspension (which is correct), but the negative controls were inoculated by 150 ul of sterile water (which is wrong). The negative controls in Biolog EcoPlate are designed to evaluate if the soil suspension does not contain any additional substrate which could be the carbon source for the microbial communities in wells. In other worlds, the same soil suspension should be placed in each of 96 wells (including negative A01, A05, and A09 wells).

 

There are situations were soil suspension is still rich in substrates (although it was diluted) and the color development in Biolog wells is a result of metabolizing the well’s substrate plus substrates from soil suspension. In that case there is no “one-substrate-metabolism” and even in “negative well”, where the manufacturer did not place any substrate (only redox dye) there could be color development.

 

Because the authors placed only sterile water in negative wells, there is no actual negative control of the experiment, but only negative control of the plate itself (for example if redox dye is not degrading, etc.).

 

This undermines the entire study and eliminates the possibility of publication in its present state. The only solution is to repeat the experiment in the right way.

 

Moreover, the whole 2.3.2 paragraph contains only one reference, while each procedure should be referenced. I have no grounds to decide whether the methodology was invented by the authors or repeated by other researchers.

 

Ln 125: what does it mean that suspension was diluted 1000-fold by 10-fold dilution ? It does not making any sense. Do you mean that suspension was diluted 1000-fold by the series of 10-fold dilutions ? Ln 128: This is unusual to incubate Biolog EcoPlate for 240 hours. The most of the researchers incubate plates for up to 120 h. After that time, the water from the wells evaporate and the results becoming disrupted. Ln 150-152: This sentence has no sense. It seems that the obtained results could be overinterpreted. First of all, the functional diversity (and the species composition) of soil microbiota depends on many different factors. The most differentiating factors are: pH, water, temperature, soil structure (porosity etc.), plants, environment etc. In this work, there is no soil characteristics, while in methods section authors mentioned about pH , TC, TN etc. measurements, there is no any table, graph or at least supplementary material presenting these values. There is no strong evidence that differences in functional diversity were caused by different plantation sites age. CCA representation is not enough to prove the correlations and the influence of each factor. For example if the pH of soils differs significantly, the vast differences in microbial activity would be observed and other factors (such as age) may have less influence.

 

Figure 4 has no legend (what the scale presents?), and no indication what hour of experiment was chosen for the data presentation.

 

Author Response

        Thank you very much for your careful comments on our manuscript, “Functional Diversity of the Soil  Culturable Microbial Community in Eucalyptus plantations of Different Ages in Guangxi, South China ”. We are very grateful for the constructive comments and suggested amendments. We have carefully studied the reviewers’ comments and have revised the manuscript accordingly. Because some charts cannot be shown here, please check the attachment, thank you.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors showed to understand my suggestions and they corrected the manuscript. Now the manuscript can be accepted for publication.

Author Response

Dear Professor:

Thank you very much for sending us the reviews and the editor's assessment of our manuscript “Functional Diversity of the Soil  Culturable Microbial Community in Eucalyptus plantations of Different Ages in Guangxi, South China” (forests-612699). We are very grateful to you of your constructive comments and suggested amendments. Your inputs have helped to improve the paper tremendously. We accepted your suggestions and we have  checked the full paper.

We hope that you will find our revisions satisfactory for publication in forests.

Hope all is well and have a nice day.

 

Yours sincerely,

Xiu Lan, on behalf of all co-authors

 

Xiu Lan

Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China.

Tel: +15577187164

Email: [email protected]

 

 

 

Reviewer 2 Report

The authors have responded to all my remarks. The work now includes the necessary corrections, including a more accurate and realistic description of the experience, proper signatures under the figures, and clarification of some sentences. I have no other objections.  

Author Response

Dear Professor:

Thank you very much for sending us the reviews and the editor's assessment of our manuscript “Functional Diversity of the Soil  Culturable Microbial Community in Eucalyptus plantations of Different Ages in Guangxi, South China” (forests-612699). We are very grateful to you of your constructive comments and suggested amendments. Your inputs have helped to improve the paper tremendously. We accepted your suggestions and we have carefully revised the full paper.

We hope that you will find our revisions satisfactory for publication in forests.

Hope all is well and have a nice day.

 

Yours sincerely,

Xiu Lan, on behalf of all co-authors

 

Xiu Lan

Key Laboratory of Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China.

Tel: +15577187164

Email: [email protected]