Nitrogen Fertilizer Driven Bacterial Community Structure in a Semi-Arid Region of Northeast China
Round 1
Reviewer 1 Report
In this study, authors investigated the effects of the different levels of nitrogen fertilizer on the bacterial community structure. It was suggested that that the optimal N application rate in this study may be approximately 150 kg ha−1 on the basis of the variations of soil properties and bacterial community structure in semi-arid areas. The results will be beneficial for the improvement of nitrogen use efficiency.
Author Response
Thank you for your comment.
Reviewer 2 Report
The article is well prepared and I consider as a very good presentation of the results rendered.
Author Response
Thank you for your comment.
Reviewer 3 Report
The manuscript "Nitrogen Fertilizer Driven Bacterial Community Structure 2 in a Semi-arid Region of Northeast China" studied on the bacterial abundance, community composition, and diversity under five different nitrogen application rates. The authors identified the main factors influencing soil bacterial community variation and the mechanisms of bacterial diversity loss, and the optimal N fertilizer application rate of maize in water-limited areas. The study may be of an interest topic and fit the scope of Sustainability, however, I have following major concerns on the manuscript. First, the manuscript is not well written and needs carefully proofread. I suggest authors seeking the help of native English speaker to check the written. Second, the Illumine MiSeq sequences of the three replicates were pooled together, so there were no replicates for statistical analyses, for example in Figure 2. Third, some of the figures, e. g. Figure 6, were very hard to be read. Also, the table titles and figure captions/legends are not well explaining the presented results. Finally, nitrogen fertilizer effects on bacterial community are not novel studies, but on N functional bacteria may be a novel study. However, the reliability of using short Miseq sequencing to predict N functional bacteria needs further perfection. A lot of N-functional genes (such as AOA, AOB, nrxA, narG, nirK, nirS, nosZ …) are well studied and applied to monitor the N-cycling processes. It will be better to study these functional genes through quantitative-PCR (Q-PCR) to compare with the Illunina Miseq sequencing results. However, this part is missing in the manuscript.
Other comments:
Line 4, who is followed by “and”?
Line 24, what is AN which should be spelled out during the first time occurring in the manuscript.
Line 254-256, is there a statistical analysis (for example ANOSIM) for distinct bacterial community structure between low and high N application rate? Figure 5b on Weighted-Unifrac dissimilarity only tell the difference between N0 and N application (line 263).
Line 266-269, again, is there a statistical analysis to prove the enrichment of different genera under five N fertilizer levels?
Author Response
Comment 1: First, the manuscript is not well written and needs carefully proofread. I suggest authors seeking the help of native English speaker to check the written.
Response: Thank you for your comment. To improve the presentation and clarity of sentences, we have revised carefully the manuscript thoroughly. Furthermore, this manuscript has been additional edited by the Dr. with excellent English. We hope that our response is acceptable. We would also appreciate any further suggestions from the reviewer.
Comment 2: Second, the Illumine MiSeq sequences of the three replicates were pooled together, so there were no replicates for statistical analyses, for example in Figure 2.
Response: Thank you for your comment. In the revised manuscript, the Figure 2 and Figure 3 had been changed to three replicates.
Comment 3: Third, some of the figures, e. g. Figure 6, were very hard to be read. Also, the table titles and figure captions/legends are not well explaining the presented results.
Response: Thank you for your comment. In the revised manuscript, the Figure 6 had been changed to Linear discriminant analysis Effect Size (LEfSe) for different N gradients at different classification levels (a). The cladogram consists of phylum, class, order, family and genus levels in order from inside to outside. Red, green, blue, purple and cyan circles indicate taxa enriched in N0, N1, N2, N3, and N4, respectively, while the yellow circles represent the taxa without significant differences among the different N gradients. Histogram of the linear discriminant analysis (LDA) scores for differentially abundant genera (b). N0, N1, N2, N3, and N4 represent N fertilizer was used at a level of 0, 90, 150, 210, and 270 kg ha−1, respectively.
Comment 4: Finally, nitrogen fertilizer effects on bacterial community are not novel studies, but on N functional bacteria may be a novel study. However, the reliability of using short Miseq sequencing to predict N functional bacteria needs further perfection. A lot of N-functional genes (such as AOA, AOB, nrxA, narG, nirK, nirS, nosZ …) are well studied and applied to monitor the N-cycling processes. It will be better to study these functional genes through quantitative-PCR (Q-PCR) to compare with the Illunina Miseq sequencing results. However, this part is missing in the manuscript.
Response: Thank you for your comment. We understand that N-functional genes must be better to reveal the N-cycling processes. However, in the present study, we mainly focused on bacterial community structure. AOA, AOB, NOB, nifH, nirS genes is well studied in the paper that associate with nitrogen cycle, for future works, a lot of N-functional genes may be implemented to monitor the N-cycling processes.
Comment 5:Line 4, who is followed by “and”?
Response: Thank you for your comment. In the revised manuscript, we had delete and.
Comment 6:Line 24, what is AN which should be spelled out during the first time occurring in the manuscript.
Response: Thank you for your comment. In the revised manuscript, AN has been replaced by available nitrogen (AN).
Comment 7: Line 254-256, is there a statistical analysis (for example ANOSIM) for distinct bacterial community structure between low and high N application rate? Figure 5b on Weighted-Unifrac dissimilarity only tell the difference between N0 and N application (line 263).
Response: Thank you for your comment. In the revised manuscript, we have added the statistical analysis base on Weighted-Unifrac for distance of bacterial community beta diversity among different N gradients in the Table S3, and Weighted-Unifrac dissimilarity among different N gradients (N0, N1, N2, N3, and N4) in the Figure 5b.
Comment 8: Line 266-269, again, is there a statistical analysis to prove the enrichment of different genera under five N fertilizer levels?
Response: Thank you for your comment.We have added the statistical analysis and LDA score of different genera under five N fertilizer levels in the Table S4 .
Round 2
Reviewer 3 Report
The authorship format (Line 4) needs to be checked still. Who is after and?
