Draft genome sequences of four Rhizobium spp. isolates, including one potential new species, from tropical legume plants in Costa Rica

  1. Donifer Campos-Parra
  2. Andres Blanco-Picado
  3. Jimena Herrera-Quesada
  4. Bradd Mendoza-Guido
  5. Keilor Rojas-Jimenez

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Abstract

We present the draft genomes of four strains of Rhizobium isolated from root nodules of Cojoba arborea , Lonchocarpus felipei , Mimosa pigra and Calliandra haematocephala in Costa Rica. Through comparative genomics, including the estimation of the average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH) and phylogenomic analysis, we determined that Rhizobium sp . LEGMi-135b represents a potentially novel species near Rhizobium hainanense (ANI: 94%; dDDH: 53.2%). The strain LEGMi-12c was associated with R. hainanense (ANI: 98%; dDDH: 87%), strain LEGMi-166a to Rhizobium altiplani (ANI: 100%; dDDH: 99.3%) and strain LEGMi-198b was related to Rhizobium cerradonense (ANI: 98%; dDDH: 81.4%). Using functional annotation tools, we also determined in silico the presence of genes related to nodulation and nitrogen fixation, such as fix , nif , nod , nol and ntr , in each strain. With this work, we provide valuable genomic resources of the genus Rhizobium that will be useful for future studies on rhizobial taxonomy, symbiosis and ecology in tropical ecosystems.

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  1. Version published to 10.1099/acmi.0.001012.v3 on Access Microbiology
  2. Access Microbiology

    Thank you for submitting your revised manuscript to Access Microbiology introdcuing the proposed amendments. I am pleased to inform you that your manuscript is now accepted for publication. Congratulations!

  3. Version published to 10.1099/acmi.0.001012.v2 on Access Microbiology
  4. Access Microbiology

    Thank you for submitting your manuscript to Access Microbiology. Your study provides valuable genomic data on Rhizobium isolates from tropical legumes and supports the designation of a novel species. The reviewers found the work scientifically sound and of potential interest to the journal’s readership. However, several minor but important clarifications and editorial improvements are required before the manuscript can be accepted. Please revise the manuscript carefully, addressing the following comments point by point. • Clarify reference selection: Expand the description in Methods (L. 94–97 and Fig. 1) to specify how reference genomes were chosen from GenBank, emphasizing inclusion of type strains where available. • Indicate type strains: In Figure 1, mark all type strain genomes with a superscript “T” for clarity. • Core gene information: State the number of single-copy core genes used in the phylogenomic analysis, the alignment length, and include bootstrap support percentages for key nodes. • While ANI and dDDH provide robust species-level resolution, the reviewers recommend adding a comparative 16S rRNA analysis to support your taxonomic assignment. • Add a new row in Table 1 titled “Closest 16S rRNA match – accession” and indicate the top BLAST hit and accession number. • Explicitly state the accepted species thresholds (≥95–96% ANI and ≥70% dDDH). Confirm that comparisons included all available type strains in GenBank. • Clearly affirm that LEGMi-20j and LEGMi-135b represent the same novel species based on the full genomic evidence. • Consider supplementing your taxonomic conclusion by running GTDB-Tk to confirm the closest classified relative. • The manuscript currently infers nitrogen-fixation and nodulation potential solely from genomic data. • Clarify that these functional annotations are based on in silico predictions, and that no phenotypic (e.g., nodulation or acetylene reduction) assays were performed. • Revise the Discussion to moderate claims about functionality and note that validation of gene activity remains to be performed in future work. • If possible, briefly comment on whether nod gene profiles correlate with host plant taxonomy to address potential host-specificity. • Include completeness and contamination metrics for each genome (e.g., from CheckM or BUSCO) in Table 1. • Add genome accession numbers for all assemblies in the Data Availability section. • Specify: o The library preparation kit (vendor and catalog details). o The version of Canu used for PacBio assembly. o Detailed parameters for the hybrid assembly in SPAdes, including how Illumina and PacBio reads were integrated. • In Table 1, correct inconsistencies: o LEGMi-135b N50 value “571,44” appears to be a typo—verify and correct. o LEGMi-166a and LEGMi-20j genome lengths are identical; confirm values from QUAST. o Use consistent decimal separators (periods, not commas). o Group functional genes into three categories: nodulation, nitrogen fixation, and regulatory genes for clarity. • In Table 1 and throughout, italicize all genus/species names and gene names consistently. • Strengthen the Introduction by explicitly stating how characterization of Rhizobium strains from tropical tree hosts advances current knowledge. Clarify the ecological or taxonomic gap this study addresses relative to R. hainanense and R. altiplani. • Add a statement confirming that biological sample collection was performed under appropriate local or institutional permits, and include any biosafety compliance statement for environmental isolates. • Perform a thorough grammatical and stylistic review to ensure clarity and consistency in terminology and formatting throughout the manuscript. Please ensure uniform editorial style and correct the following: • Cycloheximide (not “Cycloheximid”). • DNeasy PowerSoil Kit (Qiagen) (not “DNEasy”). • Canu (default parameters; genome size: 7 Mb) – remove stray “t”. • eggNOG-mapper v2.1.6 and RAST tools (not “Rast softwares”). • Ensure genus/species names (e.g., Rhizobium hainanense, R. altiplani) are italicized throughout. • Provide consistent formatting of all references (journal abbreviations, years, punctuation). • Ensure all references follow the journal’s citation format and include full bibliographic information. o Reference 5 (“Li H. 2025. lh3/seqtk. C.”) appears incomplete—please provide a full citation or remove if software-only. o Reference 11 (“Mendoza B. 2024. GenFlow...”) lacks a DOI or public access; provide a preprint link or include methodological details in the Supplementary Information. o Ensure consistent journal abbreviation style (e.g., iMeta, Genome Res.).

  5. Access Microbiology

    Comments to Author

    This manuscript reports the genomes of five distinct Rhizobium strains, with genomic metrics and phylogenetic analyses supporting the designation of a novel Rhizobium species. I provide a few comments for the authors' consideration. L 94 -97 and Fig 1. "We manually selected reference genomes from GenBank, prioritizing type strains and ensuring inclusion of the closest available Rhizobium species genome (identified via literature, TYGS results, and when available, reference genomes). In some cases, we found only non-reference genomes available." I recommend specifying in the phylogeny which genomes correspond to type strains, typically indicated by a superscript T. L. 37 - 39 and table 1. "The NCBI accession numbers of the 16S rRNA gene sequences of the isolates are: PP439182 (LEGMi-12c), PP439189 (LEGMi-20j), PP439210 (LEGMi-135b), PP439229 (LEGMi-166a) and PP439245 (LEGMi-198b)". The authors have made the 16S gene sequences of the isolates available. While dDDH and ANI provide a robust taxonomic assignment, comparative 16S analysis could further support the results, particularly for this genus, which lacks genome sequences for some type strains. I recommend adding a row to Table 1 for "Closest taxonomic assignment - accession (16S rRNA)," similar to dDDH and ANI.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  6. Access Microbiology

    Comments to Author

    1. Italicize all genus/species names throughout, including in Table 1. 2. Italicize all gene names, consistent across strains. 3. Cycloheximide, not cycloheximide. 4. Use DNeasy PowerSoil Kit (Qiagen), not "DNEasy". 5. Canu (default parameters; genome size: 7 Mb), remove stray "t" 6. eggNOG-mapper v2.1.6 and RAST software/tools" (not "Rast softwares"). 7. The genome features table is informative. -571,44 in N50 for LEGMi-135b seems like a formatting typo. If it is correct why N50 of LEGMi-135b is very short. -GC content uses mixed decimal separators (comma vs. period). -LEGMi-166a shows exactly the same genome length as LEGMi-20j (6,776,822 bp), likely a copy error, please confirm from QUAST and correct. 8. Functional genes list in Table 1 are long. please consider grouping them into nodulation, nitrogen fixation, and regulatory genes for readability. 9. Some reference formatting is inconsistent. 10. The "Acknowledges" section heading should be "Acknowledgements." 11. The researcher should firmly confirm that LEGMi-20j and LEGMi-135b are the same novel species.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  7. Access Microbiology

    Comments to Author

    This manuscript presents draft genome sequences of five Rhizobium spp. isolates from tropical legume trees in Costa Rica, including a putative novel species. The work generates valuable genomic resources and applies standard comparative‐genomic techniques (ANI, dDDH, phylogenomics, functional annotation).However, a minor recommendation, clarification needed in strengthening the manuscript to meet required standards. In the Introduction, more clearly articulate how characterization of tropical‐tree symbionts advances taxonomy, symbiovar delineation, or ecological understanding beyond existing work on R. hainanense and R. altiplani.What gap in Rhizobium taxonomy or ecology does this study uniquely fill? The manuscript uses ANI 

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  8. Access Microbiology

    Comments to Author

    Manuscript need attention to the following points. Please include the genome accession numbers in the Data Availability section. Please include the completeness and contamination metrics for each genome in Table 1. Please describe the library preparation process or specify the library preparation kit used in this study, including vendor details. Please provide the version number of the Canu software used for the PacBio assembly. The author should provide detailed information on how the hybrid assembly was performed using SPAdes, including the specific parameters used to integrate Illumina and PacBio reads, along with a clear explanation of the approach. I recommend running the GTDB-Tk tool to determine the closest relative or to achieve species-level identification of the genome assembly.

    Please rate the manuscript for methodological rigour

    Good

    Please rate the quality of the presentation and structure of the manuscript

    Satisfactory

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  9. Access Microbiology

    Comments to Author

    The ms by Fernández-Vargas et al describes the genomic sequences of 4 new Bradyrhizobium strains from the tropical forest in Costa Rica, which are compared to other Bradyrhizobium species suggesting they probably represent new species

    Please rate the manuscript for methodological rigour

    Very good

    Please rate the quality of the presentation and structure of the manuscript

    Very good

    To what extent are the conclusions supported by the data?

    Strongly support

    Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

    No

    Is there a potential financial or other conflict of interest between yourself and the author(s)?

    No

    If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

    Yes

  10. Version published to 10.1099/acmi.0.001012.v1 on Access Microbiology