Effects of Nitrogen Application Rate on Rhizosphere Microbial Diversity in Oilseed Rape (Brassica napus L.)

Round 1

Reviewer 1 Report

A work of great interest to the reader, combining agriculture and microbiology of a novel nature. The manuscript must be completed with missing information, neatly prepared and concisely written. 

Abstract:

Lines 12-14: incorrect recording of units. Specify at what nitrogen dose (exact dose) lower and higher values of bacteria and fungi were found.  

Introduction

Line 30-31: Specify the area presently of Brassica napus L., countries in which this crop is cultivated and the yields achieved. 

Line 38-41: sentence too long, try to shorten; 

Line 45-46: "In plants, nitrogen is mainly in the new parts or 
the active parts of life, such as shoot tip and root tip". On the base of your results or it is citation? If citation, please add the reference. 

In the introduction, the authors repeat several times the effect of nitrogen on plant growth and development. Please avoid this. 

Materials and Methods

Line 117-118: Please explain what does it mean : The experimental 
soil was field soil (on the basis of which the soil nutrient content was determined? Please, describe high, low content of nutrients especially N). where soil analyses were carried out ?

pot experience? What conditions did you have? If outside, what weather conditions (temperature, precipitation). The authors did not indicate that the pots were standing in a greenhouse. Specify what the experience was. The authors mainly described the test factors: nitrogen fertilization, diamater of bowl or pot? At what stage was the oilseed rape transplanted? Was there a control object?

More information about nitrogen fertilization is needed (name of fertilizer, what is the base of doses of N divided in the experiment?, way of fertilization, when?

Methodology requires careful description. 

Results

Chao and Shannon index should be described in Methods section. how it is calculated ?

Figures are not clear. 

Reference

Extensive collection of publications cited in the manuscript. However, it requires improved citation according to journal requirements. 

Large disparities between chapters. 

Author Response

Dear Reviewer,

Thank you for your valuable advice, we have modified the article in the suggestions you provided.

First, the use amount of applied nitrogen fertilizer had been added to the part of Abstract.

Second, we have removed some repeated and unnecessary description on the effects of nitrogen on plant growth and development in introduction as suggested. We will conscientiously revise our expression and achieve publication standards.

Third, for your questions on line 117-118, we make the following explanation. The soil we used for the experiment is the tillage layer soil, which was collected by multi-point sampling method of the measurement of soil nutrition, from the 0-20 cm topsoil of farming field. The measurement of soil nutrition accounts for the background value of soil, which also reflects the real situation of the field soil. The results we have demonstrated that the content of nitrogen in soil is relatively high. In our experiment, the tested pots are placed outdoor, in which the climate of the growth period ofoilseed could account consistent as in field, the only variable is the transparent shading plate that used for waterproof. The oilseeds were sowed on October 11^th, and one robust seedling was transplanted in each pot on November 8^th. The average dosage of nitrogen fertilizer are170kgN/hm²foroilseed planting, which account as control group to carry out the research. As for the information on the fertilizer, we have updated the information as suggested. And for the flaw in Methods part, we have also modified it with more details included.

As for the problems with figures and reference, we will change and provide pictures and citations according to the standard in the reply. These changes had been made and uploaded to the system. Please let us know if there are any other problems.

Sincerely,

Reviewer 2 Report

The authors have explored the impact of nitrogen application rate on the rhizosphere microbial diversity in oilseed. The experiment consisted of two parts: a field study with different nitrogen treatments; a molecular biological part which consisted of large scale sequencing and evaluation by bioinformatics. A large amount of data is presented - which is not so easy to interpret but nevertheless some interesting conclusions can be made, however it is obvious that some new questions have been identified that will need to be followed up in future studies. This is a very straightforward, mainstream study with many details around sequence data. It becomes obvious that sequence data alone are not sufficient for deeper interpretations. I would welcome a bit more implementations of the pitfalls of interpretations of microbial diversity based on only gene sequencing. There is for one the issue around DNA and its lack of correlation with live versus dead bacteria, the fact that large scale gene sequencing may actually miss certain microbial taxa, the bias induced by just using one kind of DNA extraction protocol, the obmission of other microbial groups (Archaea, other Eukaryotes, viruses) that could influence the dynamics of the two target groups in this study (Bacteria ad Fungi). I would also appreciate to see some further comments on the soil properties - soil chemistry and how that may influence your studies etc. I do not suggest that you have to perform additional analyses - but I would recommend you to include some of the pitfalls in your straightforward elaborations of how relevant your findings of the microbial diversity are. Some minor issues to amend: Minor language errors ( e.g. line 75, plays should be play; line 123 - is it correct with use?; line 132, is sow correct? etc.) 2.3 Sampling: What is the volume of a POT? How many samples were processed in total (different experiments, plus replicates)? Line 141: did you use aseptic scissors?; volume of sampled soil - how was it stored? 2.6 High throughput sequencing. Have you submitted your sequence data to a public database - and if so, what is their accession number? Line 413: Rephrase microorganism to microbiome or similar line 424: change Exmples to examples - similar errro in line 483 (We should be we) Line 460-461: not quite understandable, rephrase.

Author Response

Dear Reviewer,

Thank you for taking time reviewing this study, we would like to make some explanations for the questions on your feedback.

First, about the question that brought up on the DNA of soil sample alone for analyzing bacteria and fungi, which indeed might miss some molecular biological parts and bring up issues on distinguishing DNA and its correlation with live versus dead bacteria. However, this application, as current study on the analysis of community structure of soil microorganisms, bacteria and fungi mainly based on, can be considered as a scientific method, which states the real changes of bacteria and fungi in the soil.

Second, though the omission of other microbial groups could influence the dynamics of bacteria and fungi, the aim at this study only concerns the impact of nitrogen application rate on the rhizosphere microbial diversity in oilseed, instead of further analysis of the impact of other factors.

For now, we have not taken further study on the influence of the soil property, but only objectively explored the impact of nitrogen application rate on the rhizosphere microbial diversity in oilseed.

Thanks again for your kind advice, we will consider more comprehensively and rigorously, and carry out an in-depth study with the interrelationship between various factors.

The following bullet points are the modificationsfor other minor issues:

• 3Sampling:The volume of the container is about 6 liters, the diameter of the upper edge is 28 cm, the diameter of the lower bottom is 20 cm, and the height of it is 18 cm. (I demonstrated it in 2.2)
• There are 4 types of nitrogen fertilizer treatments, which are sampled in 3 stages of seedling, flowering and maturation. In order to ensure 3 replicas of each stage, there are 15 pots of each fertilizer, and thus a total of 60 pots of processed samples.
• Line 141: We did use aseptic scissors for the entire study. Since we have only taken the small amount of sampled soil from the part that are closely attached to the root of oilseed, we stored them in aseptic bag in the lab.
• 6 High throughput sequencing. The sequence data have not yet been submitted to a public database, we are working on itand will provide the accession number in the article once complete.
• Line 413, line 424 and line 483 had been modified as required.
• Line 460-461: There are four types of nitrogen fertilizer treatments that repeated three times on each stage of seedling, flowering and maturation. Except for the differences on the dosage of nitrogen fertilizer, the use amount of phosphate fertilizer and potassium fertilizer are constant --- 90kg P₂O₅/hm² of phosphate and 120kg P₂O₅/hm² of potassium that are evenly applied into the pot as the form of base manure. Only fertilizer of carbamide (N 46%), superphosphate (P₂O₅12%), and muriate (K₂O 60%) have been used within the study.

Last, we apologize for the grammar problems, we will check and modify the whole article thoroughly and make sure there are no more mistakes.

The manuscript has been modified and uploaded to the system, please feel free to contact us if you have any other questions.

Best regards