Insights into Genetic and Physiological Characteristics of Clover Rhizobia in Afghanistan Soils
, Michiko Yasuda 2,*, Shafiqullah Aryan 3, Tadashi Yokoyama 4, Kalimullah Saighani 5 and Naoko Ohkama-Ohtsu 2,4Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn my opinion, this is a well-planned study and has a series of experiments. Overall, I think this work is publishable with a few minor comments.
In the introduction section: The author should add more discussion about the importance and significance of this study.
Line no 171: Reason for choosing this particular clove root nodules?
Line no 243: If a colony's usual growth was impeded by 2-4% NaCl. How much less than 2% is thus acceptable?
Please mention in conclusion: how much temperature, pH, and NaCl percentage used for this study
In the conclusion section or end of the manuscript: it is suggested to add a summary of the research including significant findings and also some qualitative results that could enhance the readability of this research
Author Response
Thank you very much for your kind comments.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors studied the genetic and physiological characteristics of clover rhizobia. They isolated 57 rhizobia from six soil samples and found that different genotypes significantly increased plant biomass. These findings will provide extensive insights into the development of biofertilizers in the context of Afghanistan. There are still several sections need further discussion. The below are some specific comments.
Line 20. The potential of nitrogen fixation was investigated in this study. How about the potential of nitrogen fixation?
Line 26. PC8, PC9. PC is an abbreviation for ?
Line 29. 16Sr RNA? Please check.
Line 114. 3.3 should be 2.3. 3.4~3.8 also should be revised.
Line147-150. I think this part should be written after the bioinformatics analysis (Line 146) rather than in a single paragraph.
Line 167. “In this study, six…..rhizobia” delete. Repeate with material and methods.
Line 171 clove should be clover
Line 89-91. Why these six soil samples were selected? I think the details of sampling sites in Table 1 should be displayed in material and methods.
Table 2. The properties of isolates at different pH, temperature and NaCl concentrations. Why they were displayed in table2 according to soil samples？
Author Response
Thank you very much for your kind comments.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript represents the first attempt on genetic diversity of Afghanistan's clover nodulating rhizobial which have potential improvement for growth of local clover. Therefore, the results are of important signification for the feed sources and stock farming in Afghanistan.
Totally, this manuscript is organized well, however, there are several problems should be addressed before publication.
1. Firstly, the authors should clarify how to avoid the influence of seed endophytic rhizobia on the soil rhizobia in this study.
2. Because geographical situation has a significant impact on the distribution of microorganisms, the author should provide altitude information of sampling sites in Table 1.
3. Authors exhibited two surprising phylogenetic trees in Figs.1 and 2. Firstly, there are many reports and sequence data of leguminous root module bacteria, e.g., Rhizobium, and the insufficient number of sequences (only 15 and 11 known sequences in Figs.1 and 2, respectively) of known related species included in this manuscript may limit the reliability of the analysis. Secondly, the author used only one species as outgroup, and it is not enough to reflect the phylogenetic background of the examined clover isolates. Thirdly, the species belonging to the same genus of the outgroup are distributed on different clades, it may also indicate that there is an error in the current selection of outgroup.
4. What is the total volume of the reaction system in PCR amplification in Section 3.5?
5. There are many unclear description and need to revise, such as, lines 71-72, 75-77,82, 117, 130, 135-136, 256-257...
Author Response
Thank you very much for your kind comments.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsAuthors obtained 19 new Rhizobium isolates in Afghanistan soils, and test their ability to increase the host biomass. However, the isolates were not identified to species level, and the other comments were listed below.
Line 36: Trifolium repens and Trifolium pratense are species, not varieties. The second genus named should be changed to T.
L 114: How many isolates were tested by abiotic stress?
L 117: A full stop is needed after (2011).
L 140: amplification conditions are needed herein.
L 149: Please list a table to show the species name and corresponding accession nimbers.
L 155: 10^8?
L 174: The latin name of previous crops is needed in the table.
L 176: the results of Rhizobial Tolerance for Abiotic Stresses is not stated reasonably. The factor should not be locations where isolates obtained, but the isolates themselves. Authors need to show the isolates numbers that can survive in the abiotic stress.
L 191: the method to construct phylogram is not mentioned.
L 192: why only 19 isolates were included?
L 202: phylogram based on 16S obviously cannot distinguish different species of Rhizobium. Hence the groups divided by authors are meaningless.
L 228: Please redo the phylogeny analyse and identify the isolates to species. Change the Rhizobium sp. to species names.
Comments on the Quality of English Language
The language is ok, but need carefully check during the revision process.
Author Response
Thank you very much for your kind comments.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors1. Abstract: I think the the potential of nitrogen fixation was studied and should be added to the abstract.
2. Why soil samples were clected from six sites (Nangarhar......)? Please add to the material and methods.
Author Response
Thank you very much.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe author provided good explanations to issues I concerned. However, there are still two minor questions in the revised manuscript. 1. No altitude information of the sampling points was found in Table 1 yet, 2. The conclusion in Lines 351-353 is inconsistent with the data in Table 2.
Author Response
Thank you very much.
Author Response File: Author Response.pdf
Reviewer 4 Report
Comments and Suggestions for AuthorsAuthors have well revised the manuscript.
Author Response
Thank you very much.
