Round 1

Reviewer 1 Report

I have reviewed the article and here are my comments/suggestion for the authors.

Comments for authors

The title of this manuscript not suited according to study results, it should be changed

Abstract

Introduction of subject experiment not given and explained. Author should explain briefly why this study required to conduct? Results presentations not in scientific terms, it should need to revise profoundly. Future directions are also not presented well overloaded text should need to removed.

Keywords. This sections also should need to revisit according scientific requirement

1.      Introduction/Background

 

2.       The overall introduction/background section is very confusing and it needs major revision as the authors have added a lot of irrelevant information. This section should be precise and to the point. Most of the literature is much old irrelevant to the experiment which needs to be addressed.

1.      There is several overlapping information in this section, authors are advised to remove such information.

2.      Remove all outdated references and keep the latest.

3.      Overall need more explanation and background in this section

4.      A lot of grammar errors in this section, should need to revise again

5.      References used in the introduction section are too old

Material and Methods

Experimental data and results should be from recent years, it should be repeated in recent year

Results

1.      There is no significance shown in all tables 2, data was not interpret properly 

2.      Results are described without heading, make sub-headings and describe information under those sub-headings.

3.      Result presentation is very rough, should need to rewrite

Discussion

1.      The discussion need to revised and need to make it more focused based on results.

2.      Remove the overlapping information/statements.

3.      Interpretation was not justifiable

4.      Irrelevent and extra data need to removed

References

1.      Update the reference section with latest findings keep only those old reference which are utmost important.

 

 

 

 

 

 

 

 

 

Author Response

Introduction was reduced and better organized,  but some of the old references were retained because the field started long ago and these are still valid. Results were organized in subsections and this was a very useful suggestion. Thanks.

Reviewer 2 Report

The paper shows a study of rhizobial diversity nodulating Inga vera and Lysiloma spp. The isolates were characterized with molecular techniques, such as rep-PCR and genome sequencing, and their relationships were evaluated using ANI and phylogenomics. Next, the authors focus on their analyses and discussion of a few genes, such as nodulation and uptake hydrogenase genes. Finally, the description of a new Bradyrhizobium species is proposed. 

 

The results are sound and worth publishing in "Diversity." Nevertheless, two points can be improved before publication:

 

(1) The introduction is confusing as it unites different subjects. It talks about the importance of legume seeds as a protein source, other legume species, such as species from Southern Europe, mentions nodulation genes, and legume inoculants in Brazil, among other subjects. In the end, the introduction is made up of fragmented ideas. Sometimes this also happens in the Results and Discussion section, such as in line 331 (see the comment below). Thus, the introduction must be improved and focus on the main subject of the paper. Likewise, the Results and Discussion section should be revised to remove fragmented ideas and make it more fluid;

 

(2) The main subject of the paper is the diversity of rhizobia associated with two legume species native to Mexico, but the authors also describe a new Bradyrhizobium species, which they call Bradyrhizobium lysilomae. Although the description of a new species can be related to rhizobial diversity, it seems to be disconnected from the paper's primary goal. Besides, the text focuses on the molecular characterization of the strains but does not say anything about the polyphasic characterization needed for describing a new species. For example, the morphological and physiological analyses used for species descriptions are not even described in the Material and Methods section. Furthermore, the proposal of a new species should follow the standards used by the international community, so additional analyses are needed. See Urquiaga et al. (2019) as a recent example https://www.microbiologyresearch.org/content/journal/ijsem/10.1099/ijsem.0.003697 Finally, usually, species descriptions are done in more specialized journals, so why is the particular reason for doing it in Diversity?

 

Additional comments:

 

The statement in lines 59-61 is not valid. There are other highly specific tree symbioses, such as the one between Sesbania virgata and Azorhizobium doebereinerae. S. virgata has an intimate relationship with his symbiont, is nodulated efficiently by A. doebereinerae only, and A. caulinodans, a related rhizobial species, cannot induce but the formation of pseudo-nodules on this legume. Likewise, A. doebereinerae induces efficient nodulation in S. vergata only. Please, see Moreira et al. (2006) S. Appl. Microbiol. 29: 197-206, Florentino et al. (2009). Sci. Agric., 66(5): 667-676, and Florentino & Moreira (2009) Arvore 33(2):215-226. As another example, Mimosa species are exclusively nodulated by Paraburkholderia or Rhizobium depending on their region of origin, thus showing some level of specificity, and this interaction can be affected by soil conditions (de Faria et al. 2020). Finally, the citation used to support the statements in lines 59-61 is from 1992, and rhizobial taxonomy has changed since then. Sequencing was not easy to do at that time, and taxonomy was based on other characters. 

 

Please, state the objectives and hypotheses of the study. The last sentence mentions what is shown in the paper without mentioning its goals. This seems to reflect the lack of focus on the introduction.

 

Lines 120-121:

Are not the samples from Mexico? Why were the seeds used to capture rhizobia from Edinburgh? This also makes the paper a little confusing since the justification focuses on studying the rhizobial diversity in Mexican soils and legumes.

 

Lines 229-230:

ARA is not a good measure to evaluate efficacy. Therefore, the method is considered as not suitable for quantitative analysis. In this case, plant biomass and its accumulated N are adequate variables.

See Unkovich et al. (2008) "Measuring plant-associated nitrogen fixation in agricultural systems."

 

Line 255:

The strain within the elkanii superclade is the B51278, not the B51279, as said here.

 

Fig. 5 is unreadable.

 

Line 255:

It is strain B51278, not B51279.

 

Lines 256-262:

Not necessary. This is Material and Methods.

 

Line 265:

"Some strains…"

Please, rewrite. I get what it means, but the part "not with other species" doesn't sound good.

 

Line 267:

"Species."

 

Line 270, Figure 3:

If you have genomic data available, why do an analysis of genes glnII, rpoB, recA, and gyrB? Isn't that redundant with the phylogenomic approach? It makes sense to compare it with the 16S rRNA gene since it is a standard gene used for bacterial taxonomy.

 

Line 293:

"… A novel species"

 

Lines 321-327:

Fragment of isolated ideas.

 

Line 331:

A fragment of an idea. The idea fits here, but it is important to link it better to your results and make a smooth transition. For example, you can interpret your results and say that they add evidence for horizontal transfer events in Bradyrhizobia, such as in Bradyrhizobium japonicum and elkanii in Brazil.

 

Line 333:

"…but it is found…"

 

Lines 333-344:

The authors explore the different nodulation genes to propose the types of nod factors. It would be helpful to present a discussion addressing the consequences of this. For example, what does it mean to have sulphation of a nod factor? How does having a nodX gene in your strain mean an oriental origin of this gene?

 

Lines 386-388:

The sentence doesn't make sense.

 

Lines 427-429:

And then? What are the consequences? Can they nodulate the other species? Yes, but not efficiently. Why? What does your data suggest?

 

 

Citation:

de Faria, S.M., de Carvalho Balieiro, F., Paula, R.R., Santos, F.M., Zilli, J.E. (2020). Biological Nitrogen Fixation (BNF) in Mixed-Forest Plantations. In: Bran Nogueira Cardoso, E., Gonçalves, J., Balieiro, F., Franco, A. (eds) Mixed Plantations of Eucalyptus and Leguminous Trees. Springer, Cham. https://doi.org/10.1007/978-3-030-32365-3_6

 

 

 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

In this work, Hernández-Oaxaca and collaborators analyse the diversity of rhizobia nodulating two tropical tree legumes, providing the genomic sequence of different isolates and describing a new Bradyrhizobium species.

Overall, the manuscript is clearly written, and the research has been properly conducted. Moreover, the results and conclusions obtained are appropriate for a journal such as Diversity, fully fitting its scope. There are, however, some issues that, in my opinion, should be addressed before this work would be accepted for publication in Diversity.

• Lines 318-344: The analysis of the set of Nod factors produced by each strain might be interesting and would contribute to an improvement of the work.
• Lines 377-381: the story about S. fredii nolBTUV genes is rather more complicated than the situation currently described in the manuscript.

- There are many more genes involved in the S. fredii T3SS: you can check it in Perez-Montaño et al. 2016. Sci. Rep. 6:31592. doi: 10.1038/srep31592, Supplementary Data 4: In S. fredii HH103 (and the situation is similar in USDA257) there are 35 genes (coding either for T3SS components or for effector proteins) distributed in 11 transcriptional units. The authors should check in their isolates the presence of other T3SS related genes apart from nolBTUV. In fact, nolBTUV are the first four genes of a large operon (comprising 14 genes in HH103)

- The authors claim that nolBTUV expression is driven by a nod box, and this is not true. All the transcriptional units related to T3SS in S. fredii are preceded by a tts box and their expression is activated by the transcriptional activator TtsI (whose expression is controlled by a nod box and activated by NodD and appropriate flavonoids). Please, correct this mistake in the text.

- The authors claim that the absence of nolB allows the nodulation of modern soybean cultivars. And this statement is true for USDA257 but not for S. fredii HH103. The latter strain naturally nodulates American (modern commercial varieties) soybean, and, on the contrary to USDA257, inactivation of HH103 rhcJ (=nolT) produces a partial impairment for nodulating soybean (de Lyra et al. 2006. Int Microbiol 9: 125-133). Interestingly, inactivation of nolT produces an improvement (as in USDA257) of the nodulation ability with Erytrhina. A relatively recent review adequately covering the role of T3SS in S. fredii strains symbioses with soybean can be found at Lopez-Baena et al. 2016. Int. J Mol. Sci. 17: 755 (please, check Table 2).

So, the effect of the T3SS on symbiosis depends on the specific interacting couple. Because of this, presence of the nolBTUV genes is some of the isolates studied in this work is a good indicative of control of symbiotic specificity but how it affects symbiosis with each host plant is not predictable at this very moment.

• Other interesting genes that should be discussed are the global regulators nolR and syrM, since, to the best of my knowledge, are unusual in Bradyrhizobium. These genes control the expression of hundreds of genes and are also involved in symbiosis specificity (you can check, for example:
• Acosta-Jurado et al. 2019. Env. Microbiol. 21: 1718-1739. Doi: 10.1111/1462-2920.14584
• Acosta-Jurado et al. 2020. Env. Microbiol. 22: 1718-1739. Doi: 10.1111/1462-2920.14897
• Fuentes-Romero et al. 2022. Microorganisms 10:139. doi: 10.3390/microorganisms10010139
•  
• Minor points:
  • Line 14: please, could you clarify whether Inga vera is common or scientific name? Most times it is written without normal characters, but occasionally (as in line 23) it is written in italics characters.
  • Line 15: Which host these isolates have been isolated from? Please, clarify.
  • Line 19: In my opinion, this sentence is ambiguous. Looking into the text I think that the message is: “none of the isolates harboured symbiotic plasmids, although two Inga isolates presented non-symbiotic plasmids”.
  • Line 70: You can find another good example of this in Yang et al. 2001. J Biotechnol 91: 243-255.
  • Lines 220-221: what was the criterium for selecting the third strain for whole-genome sequencing?
  • Line 235: who are “they”? Please, clarify.
  • Lines 338-344: please, provide a functional description of NodX.
  • Table 2: please, use italics for Bradyrhizobium, nod, nol, noe.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I am satisfied with the revision made by the authors