Changes in Acetylene Reduction Activities and nifH Genes Associated with Field-Grown Sweet Potatoes with Different Nursery Farmers and Cultivars

Round 1

Reviewer 1 Report

This manuscript has a few major flaws from which I found that it is not recommended to be considered for publication. 

First, the results do not come with appropriate test statistics. To say 'significantly different', the authors should perform statistical significance tests such as t-test, ANOVA with post hoc analysis, or any others corresponding to experimental design. Perform statistical analysis and report the statistics with the results in separate tables or annotate the stats in figures with asterisks. Add 'Statistical analysis' sub-section in the Materials and Methods section and describe how you perform the analyses.

Second, one of the main factor in variations of the examined parameters is the geological locations of the experiment -- different farms -- but any information about these study sites cannot be found in the manuscript. Although the authors set the identical experimental plot, other environmental factors or edaphic factors can hugely affect the responses of the plants even in the same cultivar. In fact, the authors found deviations by location. 

Third, since the surface sterilization was not performed on the plant tissues, I do not agree with using 'endophytes' in the text. It seems reasonable that those nifH containing bacteria isolated from the plants in the experiment are known endophytes in previous studies from others but this could potentially mislead the results. I would rather not use the term endophytes, but use diazotrophic growth promoting bacteria instead.

Other minor concerns are included in the attached file. 

Comments for author File: Comments.pdf

Author Response

Rev.1

First, the results do not come with appropriate test statistics. To say 'significantly different', the authors should perform statistical significance tests such as t-test, ANOVA with post hoc analysis, or any others corresponding to experimental design. Perform statistical analysis and report the statistics with the results in separate tables or annotate the stats in figures with asterisks. Add 'Statistical analysis' sub-section in the Materials and Methods section and describe how you perform the analyses.

Response: A sub-section “Statistical analysis” was added in Materials and methods, and the results were incorporated in the revised MS.

Second, one of the main factor in variations of the examined parameters is the geological locations of the experiment -- different farms -- but any information about these study sites cannot be found in the manuscript. Although the authors set the identical experimental plot, other environmental factors or edaphic factors can hugely affect the responses of the plants even in the same cultivar. In fact, the authors found deviations by location. 

Response: We prepared an additional table for presenting some information on the sites. We agree with the reviewer’s opinion, and the results should be evaluated considering the large deviation.

Third, since the surface sterilization was not performed on the plant tissues, I do not agree with using 'endophytes' in the text. It seems reasonable that those nifH containing bacteria isolated from the plants in the experiment are known endophytes in previous studies from others but this could potentially mislead the results. I would rather not use the term endophytes, but use diazotrophic growth promoting bacteria instead.

Response: We revised MS according to the reviewer’s suggestion. We carefully distinguished between “plant-associated microbes” in this study and “endophyte” reported in the previous study.

L33

After reading the manuscript to the end, I realized that the introduction needs to be revised since the experiment isolated both endophytic and epiphytic bacteria from non-surface sterilized tissues.

Instead, you might want to mention just diazotrophic growth promoting bacteria that contain endo- and epi-phytic microorganisms both together in the meaning.

Response: We revised MS according to the reviewer’s suggestion.

L67

Any information about these locations or farms? There found variations in the measured variables from the same cultivar grown in different nursery farms. I believe these variations were probably from different characteristics of the soils, climate, and other abiotic or biotic factors in each region. Providing information about these study sites will definitely help guide readers.

Response: We prepared an additional table for presenting some information on the sites.

L102

From the ARA assay. Correct?

Response: The samples were taken after the ARA assay. We revised MS.

L110

No statistical analysis was performed on the data, so conclusions cannot be made with this report.

Response: A sub-section “Statistical analysis” was added in Materials and methods.

L124

The error bars

Response: We revised MS according to the reviewer’s suggestion.

L241

To support this claim, provide climate data in Sep and Oct of the study sites.

Response: We prepared the supplementary table for this information.

L247

This is the reason why you cannot use the term, endophytes, in the Introduction section.

Response: We revised MS according to the reviewer’s suggestion. Most referred papers dealt with endophytes.

L253

It is difficult to understand this sentence. Rephrase it.

Response: We revised MS according to the reviewer’s suggestion.

Reviewer 2 Report

The writing style and the nature of arguments discussed in this manuscript are satisfactory. However only with additional analyses and experiments together with modification(s)/ corrections, the manuscript can be made acceptable for the scientific community.

Comments for author File: Comments.pdf

Author Response

Rev.2

The authors themselves admit that there have been many examples of isolation of the diazotrophic endophytes in sweet potato. However they consider this work to be interesting as few reports are available in the literature dealing with their ecological nature through the cultivation of the plant in fields such as changes in their communities and nitrogen-fixing activities, and their differences among nursery farms and cultivars. I personally find this current work quite interesting. However the goal of this research and its contribution in a broader perspective in the community ecosystem is not clearly described. 

Response: We revised Introduction according to the reviewer’s suggestion.

One difficulty in the point of understanding of this work is the term endophyte.  Endophytes live inside the plant tissue without causing any disease symptoms. However the study done here deals with the community externally attached to plant tissue surface together with the endophyte community. I don’t understand how the nitrogen fixation from the surface attached bacterial community could contribute fixed nitrogen to promote sweet potato tuber growth. Better clarify these points and use the term “sweet potato-associated community”. 

Response: We considered the role of the sweet potato-associated microbial community as a whole, but we carefully distinguished between “plant-associated microbes” and “endophyte” in the revised MS.

Moreover communities of the other compartments like bulk soil (soil without any plants) were not analyzed at all from experimental field. This is important to understand the shift of community. Moreover plating dilution(s) of the bulk soil from the experimental field could give an idea of the richness of the community already existing in the soil of the experimental field. 

Response: We understand that analysis of microbial communities in soil and rhizosphere is important and interesting. However, it is expected that the microbial communities would be rather diverse and not evenly distributed in soil, and that some specific microbial communities would be enriched in rhizosphere, but environmental factors including soil and plant conditions would affect on the communities, which makes experimental conditions difficult for this type of research. We would like to consider it as a future study.

The PCR approach to identify the plant associated community is not at all a very robust method to cover the total community. As already mentioned by the authors PolR/PolF have only 25% nifH sequence coverage from community. Therefore as already suggested by the authors in discussion, a clone library approach would give the best answer to cover the sweet potato associated community in the experimental field. However from the available nifH sequences of the sweet potato community a phylogenetic tree could be constructed for a meaningful interpretation of the strains. Moreover a parallel approach to PCR amplify 16S rDNA of the associated community by universal degenerate primers (using a modified method to isolate microbial DNA from plant tissue) and phylogenetic tree could identify novel species (if any) which could be important for the association with the sweet potato tubers in the experimental field.

Response: We agree with the reviewer that meta-genomic analysis of nif and 16S rRNA genes has great advantage for the analysis of microbial community. Even though there were some limitations in our method, we think that our results would provide useful information on characteristics of microbial community associated with sweet potato. According to the reviewer’s suggestion, the phylogenetic tree of nifH was presented in the revised MS, however, analysis of 16S rDNA could not be applicable at present.

The authors added chemical fertilizer during planting in the experimental field with combined nitrogen (N), (line 72). Will this not inhibit the richness of diazotrophs?

Response: Effects of the fertilizer on diazotrophs have not been examined. Because of poor growth in the previous year, we added smaller amounts of the nitrogen-containing fertilizer than usual to minimize the unexpected effects on diazotrophs.

In Figure 3, “Nursery” must be replaced by June

Response: We revised Fig.3.

The conclusion “nitrogen fixation could enhance the growth” (Line 235) might not be the only reason of the enhancement of tuber growth. Other plant growth promoting microbes in the community associated with tubers can be responsible for the increase of sweet potato tuber weight.

Response: We revised MS according to the reviewer’s suggestion.

A gap is present (Line 235).

Response: We could not find the gap.

Reviewer 3 Report

This manuscript describes a study seeking to find the contribution of fixed nitrogen from the microbes to the growth of field-grown sweet potatoes. In this work, the authors analyzed plant weights and nitrogen contents of three cultivars (P, Q, B) in four locations (Sa, Sh, Mi, Ka) and found increase in fresh weight in Sa-B and Mi-B near harvesting period and higher nitrogen content in Sa-Q. Moreover, the authors executed the acetylene reduction and 15N dilution assay as proxies for biological nitrogen fixation from different parts of sweet potatoes and observed ethylene production and decrease in 15N content from tubers of the plants from September albeit high variation from sample to sample. Lastly, the authors identified nifH fragments from tubers to show the presence of endophytic diazotrophic bacteria in the tubers.

While the study sought to prove a direct connection between plant growth promotion and nitrogen fixation by microbes, it is hard to draw a conclusion based on the study. The authors should address appropriately the following points to be suitable for publication.

Majors:

-       The nitrogen contents (Fig. 2) does not correlate with plant growth (Fig. 1). Sa-B and Mi-B showed increase in plant weight but these two showed no increase in their nitrogen contents from the aboveground parts. Sa-Q showed higher nitrogen content compared to others at September, but the authors fail to show the growth improvement at October over September due to the technical difficulty. The reviewer would advise that the authors could include the nitrogen contents in belowground parts (i.e., roots and tubers) to find out the correlation between growth and nitrogen contents.

-       There is no statistical analysis especially for ARA or 15N assay exhibiting high variation, which makes it difficult for an accurate assessment of nitrogenase activity and the delivery of fixed nitrogen to sweet potatoes. 

-       To relate ethylene production to nitrogenase activity, the authors should perform the control experiments under the same conditions without the acetylene injections to show ethylene was not originated from plant parts. Generally, plants produce ethylene as a phytohormone to control growth and senescence especially under stress, which can complicate the quantification of ethylene reduced by microbes. 

Minors:

-       The reviewer would advise to provide correlation coefficients for Fig. 7, and the significance of any such correlations when mentioned (L170-175)

-       Approaches for dissecting nifH variants from PCR amplified fragments are not clear. Do the authors use llumina-sequencing (or other types of next-generation sequencing) to assess nifH variants? As the reviewer’s understanding based on the manuscript, the amplified nifH fragments with different sizes were gel-purified and sent for Sanger-sequencing, which cannot discriminate individual nifH variants. Even though the nifH fragments are the same size, their sequences can vary at the single nucleotide level. The authors should be careful when showing relative ratios of the microbes carrying the nif cluster based on this analysis. Presenting detailed methods in a Methods section would be helpful to follow the analysis.

-       Detecting the nifH fragments from DNA isolated from plant samples can be a first step to identify nitrogen fixing bacteria but cannot be a proof of their ability to fix nitrogen. The reviewer would suggest the authors to isolate some of bacteria (at least most abundant ones) to test their ability to fix nitrogen at least in free living conditions using ARA.

-       L34 needs references.

Author Response

Rev.3

The nitrogen contents (Fig. 2) does not correlate with plant growth (Fig. 1). Sa-B and Mi-B showed increase in plant weight but these two showed no increase in their nitrogen contents from the aboveground parts. Sa-Q showed higher nitrogen content compared to others at September, but the authors fail to show the growth improvement at October over September due to the technical difficulty. The reviewer would advise that the authors could include the nitrogen contents in belowground parts (i.e., roots and tubers) to find out the correlation between growth and nitrogen contents.

Response: We think that the nitrogen contents (%) seem not to contribute to the total weight. Because the nitrogen contents in the belowground parts are generally much lower than the aboveground parts, we did not measure them in this study.

There is no statistical analysis especially for ARA or 15N assay exhibiting high variation, which makes it difficult for an accurate assessment of nitrogenase activity and the delivery of fixed nitrogen to sweet potatoes. 

Response: A sub-section “Statistical analysis” was added in Materials and methods, and the results were incorporated in MS.

To relate ethylene production to nitrogenase activity, the authors should perform the control experiments under the same conditions without the acetylene injections to show ethylene was not originated from plant parts. Generally, plants produce ethylene as a phytohormone to control growth and senescence especially under stress, which can complicate the quantification of ethylene reduced by microbes. 

Response: We confirmed no detectable ethylene production without acetylene under the same conditions. This was incorporated in the revised MS.

The reviewer would advise to provide correlation coefficients for Fig. 7, and the significance of any such correlations when mentioned (L170-175)

Response: A sub-section “Statistical analysis” was added in Materials and methods, and the results were incorporated in the revised MS.

Approaches for dissecting nifH variants from PCR amplified fragments are not clear. Do the authors use llumina-sequencing (or other types of next-generation sequencing) to assess nifH variants? As the reviewer’s understanding based on the manuscript, the amplified nifH fragments with different sizes were gel-purified and sent for Sanger-sequencing, which cannot discriminate individual nifH variants. Even though the nifH fragments are the same size, their sequences can vary at the single nucleotide level. The authors should be careful when showing relative ratios of the microbes carrying the nif cluster based on this analysis. Presenting detailed methods in a Methods section would be helpful to follow the analysis.

Response: As the reviewer mentioned, the amplified nifH fragments with the same size, containing various fragments, were applied to the sequencing, Even in the procedures, most results showed the high similarity with the known sequences in the database, suggesting that the gene fragments consisted of dominant one (Table 1). As we analyzed 20 sweet potato samples for each farmer-cultivar combination, or 30 samples for each sampling time, relative ratios of the microbes (nifH gene) were presented as percentage among the samples.

Detecting the nifH fragments from DNA isolated from plant samples can be a first step to identify nitrogen fixing bacteria but cannot be a proof of their ability to fix nitrogen. The reviewer would suggest the authors to isolate some of bacteria (at least most abundant ones) to test their ability to fix nitrogen at least in free living conditions using ARA.

Response: We agree with the reviewer’s comment. We tried to isolate them, but unfortunately, we could not yet.

L34 needs references.

Response: We revised MS according to the reviewer’s suggestion. The presence of endophytic diazotrophs described in the following paragraph also supports this statement.

Round 2

Reviewer 1 Report

Many points made by the reviewers have been well addressed in the revision. The current form of the manuscript is overall satisfactory, so I would recommend the work for publication. 

However, there is room for improvement to confirm the conclusions drawn in the manuscript. For example, since the correlation between N-fixation (ARA) and FW increase was such low and insignificant, how would the authors improve their experiment to increase statistical power? Would other beneficial effects like phytohormone production rather than N-fixation had contributed to the FW increase? If so, how would the authors separate the N-fixation effects on growth promotion from other beneficial effects? This remains to the authors for future studies. 

Author Response

Rev.1

However, there is room for improvement to confirm the conclusions drawn in the manuscript. For example, since the correlation between N-fixation (ARA) and FW increase was such low and insignificant, how would the authors improve their experiment to increase statistical power? Would other beneficial effects like phytohormone production rather than N-fixation had contributed to the FW increase? If so, how would the authors separate the N-fixation effects on growth promotion from other beneficial effects? This remains to the authors for future studies.

Response: We agree with the reviewer’s comments. We revised MS considering the reviewer’s suggestion (L266). Actually, we think that evaluation of the each contribution of the N-fixation and other beneficial effects is challenging. Inoculation experiments would be one of the ways, but it would be difficult to eliminate the effects of the other microbes.

Reviewer 2 Report

The manuscript in its present form is quite improved.

I still have two concerns:

Line 20: There should be a tendency of negative correlation with ARA values in September and N15 in September: please rectify this

Fig S1: Mention Bootstrap values on the nifH phylogenetic tree nodes, at least those above 50.

Author Response

Rev.2

Line 20: There should be a tendency of negative correlation with ARA values in September and N15 in September: please rectify this

Response: We revised MS according to the reviewer’s suggestion.

Fig S1: Mention Bootstrap values on the nifH phylogenetic tree nodes, at least those above 50.

Response: We revised MS according to the reviewer’s suggestion.

Reviewer 3 Report

The authors have addressed my major concerns and have improved the manuscript. However, as the authors agreed in their response (“We think that the nitrogen contents (%) seem not to contribute to the total weight”), the authors should not conclude that “L257; nitrogen fixation could be one of the reasons for enhancing the growth”. Plant growth promotion was not related to nitrogen contents (Fig. 2). The authors need more data to make a conclusion regarding the contribution of nitrogen fixing bacteria on the plant growth, and the current study does not demonstrate the delivery of biologically fixed nitrogen to the plant by nitrogen fixing endophytes.

Author Response

Rev.3

The authors have addressed my major concerns and have improved the manuscript. However, as the authors agreed in their response (“We think that the nitrogen contents (%) seem not to contribute to the total weight”), the authors should not conclude that “L257; nitrogen fixation could be one of the reasons for enhancing the growth”. Plant growth promotion was not related to nitrogen contents (Fig. 2). The authors need more data to make a conclusion regarding the contribution of nitrogen fixing bacteria on the plant growth, and the current study does not demonstrate the delivery of biologically fixed nitrogen to the plant by nitrogen fixing endophytes.

Response: We are sorry for confusing the reviewer. In our previous response, we wanted to comment that the nitrogen contents (%) seem not to relate to the total weight in the process of the nitrogen fixation. We suppose that the nitrogen contents (%) would temporarily increase by the nitrogen fixation, but they would decrease along with the plant growth thereafter. We agree with the reviewer’s comment that the contribution to the plant growth was not certain due to the large deviation (Fig. 7), but we think that results of ARA and N15 content suggested incorporation of atmospheric dinitrogen into the plants.