Responses of Nitrogen-Fixing Bacteria Communities to Elevation, Season, and Slope Aspect Variations in Subtropical Forests of Yunnan, China
Round 1
Reviewer 1 Report
Dear Authors,
Your contribution has a valuable scientific output.
Some transformation of the text, additional data, explanations and corrections should be useful. The details are presented herewith below.
Using a literature sources, you have shown in the Introduction section that nitrogen-fixing bacteria in diverse ecosystems are significantly affected by the season, elevation, and slope of terrain. Then you informed the readers that the objectives of your study were to determine how nitrogen-fixing bacteria change with the altitude and seasons. This is a repeat of the previous studies. However, the Section 2 . Materials and Methods and Section 3 Results contain much more interesting information and data. This should be reflected in the Introduction and in the Abstract and Conclusions as well. In the Section 4 Discussion there is too much information about results of other authors. This shadows your own findings.
Lines 266-268
Our results also suggest that plant coverage is significantly negatively correlated with bacterial abundance, especially herb coverage.
This is an important result. Please reflect it in the Materials and Methods and in the Results.
Lines 270-272
...correlation analysis of plants showed that nitrogen-fixing bacterial abundance was significantly positively correlated with soil NH4 + content.
Maybe it should be better to discuss this result concerning a fact that N content in the soil is determined by nitrogen-fixing bacteria?
Lines 319-322
Furthermore, the effect of changes in soil moisture, near-surface air temperature, soil temperature, species distribution, and vegetation composition on the slope aspect gradient were similar to those in longitude and altitude gradients on a large scale [59].
It should be much more productive to show your own data here.
Lines 337-339
These findings provide evidence that the variation in the abundance and diversity of soil nitrogen-fixing bacteria is affected by multiple factors in the subtropical forests of Yunnan, China.
The findings you presented are well-known. You should present more convexly your original data and new patterns you revealed.
Author Response
Your contribution has a valuable scientific output.
Some transformation of the text, additional data, explanations and corrections should be useful. The details are presented herewith below.
Using a literature sources, you have shown in the Introduction section that nitrogen-fixing bacteria in diverse ecosystems are significantly affected by the season, elevation, and slope of terrain. Then you informed the readers that the objectives of your study were to determine how nitrogen-fixing bacteria change with the altitude and seasons. This is a repeat of the previous studies. However, the Section 2 . Materials and Methods and Section 3 Results contain much more interesting information and data. This should be reflected in the Introduction and in the Abstract and Conclusions as well. In the Section 4 Discussion there is too much information about results of other authors. This shadows your own findings.
Thank you for your pertinent suggestions, which have enhanced our article a lot. We have revised the entire article based on your comments, including the abstract, preface, methodology, and discussion. Please see the article for the specific changes. Thanks again!
Lines 266-268
Our results also suggest that plant coverage is significantly negatively correlated with bacterial abundance, especially herb coverage.
This is an important result. Please reflect it in the Materials and Methods and in the Results.
We have added the data in table, and analysis in the Materials and Methods and in the Results. and it can be found in the text.
Lines 270-272
...correlation analysis of plants showed that nitrogen-fixing bacterial abundance was significantly positively correlated with soil NH4 + content.
Maybe it should be better to discuss this result concerning a fact that N content in the soil is determined by nitrogen-fixing bacteria?
We have added the sentence in discussion.
It was found that the levels of C and N influenced the activity and distribution of nitrogen fixation bacteria [26]. In our study, nitrogen-fixing bacterial abundance was significantly positively correlated with soil NH4+ content, which indicated that the NH4+ content might be the key factors influencing the nitrogen-fixing bacterial abundance.
Lines 319-322
Furthermore, the effect of changes in soil moisture, near-surface air temperature, soil temperature, species distribution, and vegetation composition on the slope aspect gradient were similar to those in longitude and altitude gradients on a large scale [59].
It should be much more productive to show your own data here.
Thank you for your suggestions. We have added two sentences based our results in the discussion.
In different seasons, the abundance of nitrogen-fixing bacteria on the eastern and western slopes was similar in the order of winter > autumn > spring = summer. Especially, the abundance of nitrogen-fixing bacteria in autumn on the western slope was significantly higher than that on the eastern slope (P < 0.01).
Furthermore, there was no difference in the total abundance of soil nitrogen-fixing bacteria on the eastern and western slopes, while the diversity indices of nifH bacteria on the eastern and western slopes with elevation variation were similar.
Lines 337-339
These findings provide evidence that the variation in the abundance and diversity of soil nitrogen-fixing bacteria is affected by multiple factors in the subtropical forests of Yunnan, China.
The findings you presented are well-known. You should present more convexly your original data and new patterns you revealed.
The conclusion has been rewritten as your suggestion, as follows:
Our results showed that the abundance and diversity of soil nitrogen-fixing bacterial communities in subtropical forests are affected by elevation, seasonal variation, and slope aspect. The abundance of nitrogen-fixing bacteria was highest in winter and increased gradually in October, but decreased in April and July as the elevation gradient increased. In different seasons, the abundance of nitrogen-fixing bacteria on the eastern and western slopes was similar in the order of winter > autumn > spring = summer. Moreover, there was no difference in the total number of soil nitrogen-fixing bacteria on the eastern and western slopes. The results also demonstrated that the diversity of soil nitrogen-fixing bacteria was higher at low (H1, H2) and medium altitudes (H3, H4) but lower at high medium altitudes (H5, H6). Furthermore, the diversity indices of nifH bacteria on the eastern and western slopes with elevation variation were similar. The abundance of nitrogen-fixing bacteria was significantly affected by herb coverage and NH4+-N and the most influential factors in the soil nifH community were NH4+-N, NO3--N, and K. In addition, permutational multivariate analysis demonstrated that he abundance of nitrogen-fixing was most affected by season, whereas the diversity of nitrogen-fixing bacteria was most affected by altitude.
Reviewer 2 Report
Author Response
Dear authors, In the manuscript “Responses of nitrogen-fixing bacteria communities to elevation, season, and slope aspect variations in subtropical forests of Yunnan, China” submitted to Forests the authors studied abundance and diversity of soil nitrogen-fixing bacterial communities in subtropical forests. Overall, the presented rationale and methodology are scientifically well established. However, I have some major concerns about the methods and results presented by the authors. Most importantly there is no positive or negative control included in any experiment presented in the manuscript. Adding a house-keeping gene or ideally genes (eg 16S rRNA) that can be further used for identification is strongly advised. Minor comments are listed below:
Thank you for your positive comments and suggestions on the article. I know that the DGGE technique is somewhat backward as a traditional technique, and we are referring to some of the methods in the literature, which do not have to require negative and positive controls.
The specific steps of our quantitative fluorescence analysis are as follows：
The plasmid concentration was determined using a spectrophotometer (AA-6300C, Japan), and the copy number of the nifH gene was directly calculated from the extracted plasmid DNA concentration. Plasmids with known plasmid gene copy numbers were consecutively diluted 10 times to obtain a standard curve of more than seven orders of magnitude (6.54 × 103 to 6.54 × 109 copies) each time. A high amplification efficiency of 101.1%-103.4%, R2=0.99, and slope from −3.2-−3.5 was obtained. The threshold cycle (Ct) values obtained for each sample were compared with the standard curve to determine the initial copy number of the target gene. Each experiment was repeated three times.
Figure 3: Gel picture is not described in the legend and bands are hardly visible
We have tried our best to adjust the clarity of the images and added the legend.
Figure 3: What are “natural colours”
It is a mistake; we have corrected it.
Figure 3: What makes the authors think of Azotobacter? It’s the first and only time this term is used
It is a mistake; we have corrected it.
Table 4: A similarity to an uncultured bacterium clone doesn’t say a lot. Is this a full-length hit or only partial? Why didn’t the authors extract the nucleotide sequence of several nifH genes from public databases and conduct a phylogenetic analysis?
Thank you for your suggestions.
We have added the full names of uncultured nitrogen-fixing bacteria and made an evolutionary tree analysis.
