The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer

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This study presents the whole-genome sequence of B. velezensis B26, a newly identified strain isolated from chicken carcass soil in Udupi, India. The bacterium showed strong activity against fungal pathogens and exhibited diverse enzymatic activities. The whole-genome sequencing was executed using Illumina technologies. Assembly revealed that strain B26 possesses a genome of 3,946,698-bp with a G+C content of 46.3%. Genome annotation identified 3776 protein-coding genes, 1 rRNA gene, 50 tRNA genes, 5 ncRNA genes, and 59 pseudogenes. Functional analysis of the B. velezensis B26 genome revealed 216 genes involved in carbohydrate metabolism, 3 genes in potassium metabolism, 148 genes linked for cofactors, vitamins, prosthetic groups and pigments, 10 genes involved in phosphorus metabolism, 24 genes associated with iron acquisition and metabolism, 20 genes for nitrogen metabolism, 6 genes involved in sulfur metabolism, 6 genes in secondary metabolism, 12 genes associated with metabolism of aromatic compounds, 43 genes involved in stress response and 36 genes associated with virulence, disease and defence. The raw sequence data generated in this work have been deposited in the NCBI database and the genome sequence is available under the accession number JAYKOV000000000. These genomic data provide insight into the biocontrol ability and plant-growth promoting capabilities of B. velezensis B26." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/14-170/v1", "name": "The complete genome sequences of Bacillus velezensis B26: a promising..." } } ] } Home Browse The complete genome sequences of Bacillus velezensis B26: a promising... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Kamath B V, Mallya S, Mallikarjuna SV and Kolathur KK. The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.12688/f1000research.160546.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Genome Note The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] Venkatesh Kamath B 1 , Sandeep Mallya 2 , Subrahmanyam Volety Mallikarjuna 1 , Kiran Kumar Kolathur https://orcid.org/0000-0003-4173-0977 1 Venkatesh Kamath B 1 , Sandeep Mallya 2 , Subrahmanyam Volety Mallikarjuna 1 , Kiran Kumar Kolathur https://orcid.org/0000-0003-4173-0977 1 PUBLISHED 06 Feb 2025 Author details Author details 1 Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India 2 Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India Venkatesh Kamath B Roles: Conceptualization, Data Curation, Formal Analysis, Writing – Original Draft Preparation, Writing – Review & Editing Sandeep Mallya Roles: Writing – Original Draft Preparation, Writing – Review & Editing Subrahmanyam Volety Mallikarjuna Roles: Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Kiran Kumar Kolathur Roles: Conceptualization, Investigation, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Genomics and Genetics gateway. This article is included in the Manipal Academy of Higher Education gateway. Abstract Bacillus velezensis is a bacterium widely recognized for its biocontrol properties and ability to promote plant growth. This study presents the whole-genome sequence of B. velezensis B26, a newly identified strain isolated from chicken carcass soil in Udupi, India. The bacterium showed strong activity against fungal pathogens and exhibited diverse enzymatic activities. The whole-genome sequencing was executed using Illumina technologies. Assembly revealed that strain B26 possesses a genome of 3,946,698-bp with a G+C content of 46.3%. Genome annotation identified 3776 protein-coding genes, 1 rRNA gene, 50 tRNA genes, 5 ncRNA genes, and 59 pseudogenes. Functional analysis of the B. velezensis B26 genome revealed 216 genes involved in carbohydrate metabolism, 3 genes in potassium metabolism, 148 genes linked for cofactors, vitamins, prosthetic groups and pigments, 10 genes involved in phosphorus metabolism, 24 genes associated with iron acquisition and metabolism, 20 genes for nitrogen metabolism, 6 genes involved in sulfur metabolism, 6 genes in secondary metabolism, 12 genes associated with metabolism of aromatic compounds, 43 genes involved in stress response and 36 genes associated with virulence, disease and defence. The raw sequence data generated in this work have been deposited in the NCBI database and the genome sequence is available under the accession number JAYKOV000000000. These genomic data provide insight into the biocontrol ability and plant-growth promoting capabilities of B. velezensis B26. READ ALL READ LESS Keywords Genome sequencing, Biocontrol agent, Plant growth promotion, Pangenome analysis Corresponding Author(s) Kiran Kumar Kolathur ( [email protected] ) Close Corresponding author: Kiran Kumar Kolathur Competing interests: No competing interests were disclosed. Grant information: The author(s) declared that no grants were involved in supporting this work. Copyright: © 2025 Kamath B V et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. How to cite: Kamath B V, Mallya S, Mallikarjuna SV and Kolathur KK. The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.12688/f1000research.160546.1 ) First published: 06 Feb 2025, 14 :170 ( https://doi.org/10.12688/f1000research.160546.1 ) Latest published: 08 Jul 2025, 14 :170 ( https://doi.org/10.12688/f1000research.160546.2 )  There is a newer version of this article available. Suppress this message for one day. Introduction Bacillus velezensis is a valuable bacterium that is extensively used for several biotechnological applications to promote plant growth. For instance, the B. velezensis LT1 strain displayed antifungal activity against the soil-borne fungal plant pathogen Sclerotium rolfsii LC1. B. velezensis LT1 secretes a diverse antimicrobial compound that inhibit the growth of S. rolfsii LC1 ( Tang et al. 2024 ). Likewise, two B. velezensis strains 5YN8 and DSN012 act as biocontrol agents against Botrytis cinerea , a pathogen that causes severe gray mold disease in crops. These strains inhibit spore formation and the growth of B. cinerea through the production of certain secondary metabolites or volatile organic compounds ( Jiang et al. 2018 ). Several strains of B. velezensis isolated from different sources produce diverse secondary metabolites to control plant pathogens. For example, B. velezensis HNA3 produced several secondary metabolites. Similarly, several antimicrobial metabolites to control plant pathogens were produced by different B. velezensis strains ( Zaid et al. 2022 ; Rabbee et al. 2023 ). Besides anti-microbial activity, another B. velezensis SQR9 strain implicated in promoting plant growth by enhancing root colonization through biofilm formation ( Xu et al. 2019 ). Another strain, B. velezensis Ag75, has been identified as a biocontrol agent, phosphate solubilizer and biofertilizer for maize and soybean crops ( Mosela et al. 2022 ). The plant growth promoting and biocontrol capabilities of several B. velezensis strains were deciphered using the genomic sequencing and analysis. These studies were key to understand organism’s capability to produce antimicrobial compounds, enzyme secretion or secondary metabolites that promotes plant growth. For example, whole genome sequencing of B. velezensis HNA3 led to establish several genes clusters associated in promoting plant growth. Among these genes, major percentage of genes are involved in amino acid metabolism, carbohydrate transport, and secondary metabolite biosynthesis (S. et al. 2022). Similarly, the B. velezensis CH1 strain was isolated from high-quality oats, exhibited antimicrobial properties contributing to oat growth and resistance to infections. Comparative analysis of the CH1 strain revealed 13 gene clusters linked with production of 15 secondary metabolites with antimicrobial properties. Furthermore, the strain harboured numerous putative genes for indole acetic acid (IAA) production, spermidine and polyamine synthesis. These results indicate that the possible applications of B. velezensis CH1 as a biofertilizer ( Cheng et al. 2024 ). In this study, we report genome sequences of B. velezensis B26, which was isolated from soil samples collected from a chicken carcass, Manipal, Udupi, India (Location: 13.325922, 74.804554). In our screening assay to identify chondroitinase producers, B. velezensis B26 was identified. This strain demonstrated potent antifungal capabilities against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae ( http://doi.org/10.7324/JAPS.2025.200474 ). This study aims to provide a comprehensive genomic analysis of B. velezensis B26, with a focus on identifying genetic traits that contribute to its biotechnological potential. Methods Whole genome analysis of bacteria The genome of B. velezensis B26 strain was analysed to explore its microbial genomic characteristics. HiMedia Laboratories Pvt Ltd, Maharashtra, INDIA performed the whole-genome analysis. Sample preparation workflow Genomic DNA (gDNA) from B. velezensis B26 was isolated using DNA Mini Kit (Qiagen). The integrity of the gDNA was assessed spectrophotometrically to measure the A260/280 ratio and its concentration. For library preparation, 250ng of DNA was processed with the QIASeq FX DNA Kit (Qiagen), following manufacturer’s protocol to generate fragmented, adapter-ligated and indexed libraries. The Illumina NextSeq 550 with 300-cycle paired-end sequencing chemistry was employed for sequencing the finalized library. Tapestation analysis of NGS libraries For fragment analysis, 2 μL of high-sensitivity D1000 sample buffer and 2 μL of the final library sample were mixed using vortexer for 1 min. The mixture was then loaded onto the D1000 ScreenTape and analyzed using the Agilent 4200 TapeStation System. This system employs DNA electrophoresis to separate fragments up to 1000 base pairs. The trace analysis revealed an average fragment size of 301 base pairs, which, along with the library concentration, indicates the library’s suitability for next-generation sequencing. Analysis workflow In data analysis all reads are checked for quality and then the quality control (QC’)-quality reads are passed through the process flows simultaneously. The detailed steps involved in each process flow are included within each section below. Reads quality control Briefly, raw fastq files were verified using FastQc v0.11.9.8 The details of parameters checked are added in a MultiQC report ( Ewels et al. 2016 ) (Supplementary file 1). The fastp tool (v0.12.4) was utilized to remove adapter contamination from the sequencing data ( Chen et al. 2018 ). A quality score of Q30 or higher indicates a basecall accuracy of 99.9%, meaning that only 1 in 1000 bases is likely to be incorrect. The resulting files for each read were subjected for genome assembly. Genome assembly and annotations A denovo assembly using a De-Brujin graph was performed to organize the short DNA reads into longer contiguous sequences, referred to as contigs. These contigs served as a starting material used for performing a genome annotation, facilitating the assignment of functional roles to various genomic regions of the organism under investigation. The quality of the genome assemblies produced by the Spades ( Bankevich et al. 2012 ) and Megahit ( Li et al. 2015 ) assemblers was evaluated using the Quast tool ( Gurevich et al. 2013 ). Compared with the megahit assembler, the Spades assembler had longer assembled contigs with better N50 values. Hence, the assembly generated by Spades was selected for further downstream analyses. RAST Server: Rapid annotations using subsystems technology The RAST server is an automated platform developed for the bacterial genome annotation. It detects protein-coding regions, along with genes coding for ribosomal and transfer RNAs, allots functional roles to these elements ( Aziz et al. 2008 ). Additionally, it allows comparative analysis through the SEED environment ( Overbeek et al. 2014 ). The RAST server remains a viable tool for efficient and reliable genome annotation. Results Whole-genome analysis of B. velezensis B26 We performed a genome analysis of B. velezensis B26 to understand the biochemical potential of the organism. Figure. 1 depicts the sample preparation and sequencing workflow. Importantly, the assembled genomes may not be 100% complete. However, the assembly generated here meets the standards acceptable to general bioinformatics repositories as part of the data gathered for a publication. These whole-genome shotgun data have been provided with the accession number JAYKOV000000000 (NCBI database). The master record data is available from our various Entrez servers. The individual sequences are available from a hyperlink at the bottom of the WGS master record JAYKOV000000000. Figure 1. Sample preparation and sequencing workflow. The genome assembly method employed was Megahit v. 2023-07-12 tool, the genome representation was full, the genome coverage was 100.0x and the sequencing technology employed was Illumina. Genome annotation was carried out using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) ( Haft et al. 2018 ; Li et al. 2021 ). The annotation results are summarized in Table 1 , which provides an overview of the genome features and information. Table 2 summarizes key assembly statistics, including the number of contigs, total length of the sequenced genome, and the number of proteins annotated. Table 1. Genome annotation data for Bacillus velezensis B26. ##Genome-Annotation-Data-START## Annotation Provider: NCBI Annotation Date: 01/04/2024 08:32:37 Annotation Pipeline: NCBI Prokaryotic Genome Annotation Pipeline (PGAP) Annotation Method: Best-placed reference protein set; GeneMarkS-2+ Annotation Software revision: 6.6 Features Annotated: Gene; CDS; rRNA; tRNA; ncRNA Genes (total): 3,891 CDSs (total): 3,835 Genes (coding): 3,776 CDSs (with protein): 3,776 Genes (RNA): 56 rRNAs: 1 (5S) complete rRNAs: 1 (5S) tRNAs: 50 ncRNAs: 5 Pseudo Genes (total): 59 CDSs (without protein): 59 Pseudo Genes (ambiguous residues): 0 of 59 Pseudo Genes (frameshifted): 40 of 59 Pseudo Genes (incomplete): 32 of 59 Pseudo Genes (internal stop): 8 of 59 Pseudo Genes (multiple problems): 19 of 59 ##Genome-Annotation-Data-END## ##Genome-Assembly-Data-START## Assembly Method: Megahit v. 2023-07-12 Genome Representation: Full Expected Final Version: Yes Genome Coverage: 100.0x Table 2. Assembly statistics for B. velezensis B26. # of Contigs: 31 # of Proteins: 3,776 Total length: 3,947,698 bp BioProject: PRJNA1060932 BioSample: SAMN39250459 Keywords: WGS Annotation: Contigs Organism: Bacillus velezensis – show lineage Biosource: /collected_by = Manipal College of Pharmaceutical Sciences (MCOPS) /collection_date = 2019-12-01 /country = India: Udupi /isolation_source = environmental /mol_type = genomic /strain = B26 WGS: JAYKOV010000001:JAYKOV010000031 Reference: Evaluation of bioactivities of the bacterial strain Bacillus velezensis B26: Unpublished Submission: Submitted (04-JAN-2024) Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), MAHE, Madhav Nagar, Manipal, Karnataka 576104, India – Kolathur,K.K. Identification of subsystem features by The RAST Server: Rapid Annotations using Subsystems Technology (RAST) In addition to utilizing the NCBI PGAP, we also submitted the sequenced genome data of B. velezensis B26 to the RAST server. This service provides fully automated annotation for bacterial genomes. Using the RAST annotation platform, we recognized genes coding for proteins, ribosomal and transfer RNAs, assigned functional roles to these genes, and predicted the subsystems present in the B. velezensis B26 genome (Supplementary Table 1). Furthermore, the annotated genome is presented on a platform that allows for comparative analysis in the SEED environment ( Figure 2 ). Figure 2. Subsystem statistics of the B. velezensis B26 genome annotations. The genome of B. velezensis B26 was annotated using the RAST server, and the features in subsystem are compared within the SEED environment. The figure highlights key subsystems identified in the genome, including genes associated with carbohydrate metabolism, stress response, virulence, disease and defense, as well as genes involved in metabolism. Our analysis revealed the presence of 216 genes involved in carbohydrate metabolism, 3 genes related to phages, prophages, transposable elements, and plasmids, 43 genes for stress response, and 35 genes linked to virulence, disease and defense. The genes related to the stress response and virulence might be responsible for the biocontrol properties of B. velezensis B26 (Supplementary Table 2). Additionally, several genes involved in metabolic processes were identified, including 59 genes related to DNA metabolism, 10 related to phosphorus metabolism, 20 related to nitrogen metabolism, 6 related to sulfur metabolism, 12 related to metabolism of aromatic compounds, 3 related to potassium metabolism, 10 related to phosphorus metabolism, and 24 related to acquisition and metabolism. These metabolic pathways suggest potential biofertilizer activity (Supplementary Table 2). B. velezensis B26 pangenome analysis Pangenome analysis provides valuable understandings into the genome of prokaryotes. Using Roary software, a large-scale pangenomes are constructed by identifying both core and accessory genes. This method aids in understanding the conserved genes within an organism, as well as accessory genome, to understand the fundamentals linked with natural selection and evolutionary dynamics. Pangenome analysis of B. velezensis B26 identified a total of 5,576 genes. Among these genes, 3,480 genes were considered core genes and were found in 99% to 100% of strains. A total of 1, 143 genes were classified as shell genes, which were present in 15% to 95% of strains, whereas 953 genes were identified as cloud genes, which were found in fewer than 15% of strains ( Table 3 ). These analyses indicate that the major portion of the genome is conserved across species. Table 3. Pangenome analysis of B. velezensis B26. Gene_type Criteria Number_of_genes 1 Core genes (99% <= strains <= 100%) 3480 2 Soft core genes (95% <= strains < 99%) 0 3 Shell genes (15% <= strains < 95%) 1143 4 Cloud genes (0% <= strains < 15%) 953 5 Total genes (0% <= strains <= 100%) 5576 Conclusion The whole-genome analysis, genome annotation, and pangenome analysis of B. velezensis B26 deciphered the key genes responsible for its biotechnological applications. A huge number of protein coding genes (3776) responsible for several cellular functions were identified. Importantly, presence of gene cluster coding for stress response, virulence, and defense indicate that B. velezensis B26 can effectively inhibit plant pathogens. Further, presences of genes associated with nitrogen, phosphorus, and sulfur metabolism might suggest the strain’s ability to act as a biofertilizer, supporting plant growth. Altogether, these findings can pave the way for further exploration of B. velezensis B26 in agricultural applications. Ethics and consent Ethical approval and consent were not required. Data availability NCBI Nucleotide Database: Whole-genome sequencing data of Bacillus velezensis B26. Accession number JAYKOV000000000; https://www.ncbi.nlm.nih.gov/nuccore/JAYKOV000000000 . Contigs view; https://www.ncbi.nlm.nih.gov/Traces/wgs/JAYKOV01?display=contigs . Whole-genome shotgun project; https://www.ncbi.nlm.nih.gov/Traces/wgs/JAYKOV01 . These data support the findings of this study and are publicly accessible. Extended data The complete genome sequence and associated supplementary data for Bacillus velezensis B26 are available on Figshare . Figshare : Supplementary file 1 for the complete genome sequences of Bacillus velezensis B26 study. DOI: 10.6084/m9.figshare.28107950 . The project contains the following underlying data: • Supplementary Table 1 : Complete genome sequences of Bacillus velezensis B26. DOI: 10.6084/m9.figshare.28107941 . • Supplementary Table 2 : Complete genome sequence for Bacillus velezensis B26. DOI: 10.6084/m9.figshare.28107947 . Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). Acknowledgements The authors would like to acknowledge the facilities provided by MCOPS, MAHE, Manipal for helping out in execution of this research work. References Aziz RK, Bartels D, Best AA, et al. : The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008; 9 : 75. PubMed Abstract | Publisher Full Text | Free Full Text Bankevich A, Nurk S, Antipov D, et al. : SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J. Comput. Biol. 2012; 19 : 455–477. PubMed Abstract | Publisher Full Text | Free Full Text Chen S, Zhou Y, Chen Y, et al. : fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018; 34 : i884–i890. 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Publisher Full Text Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 06 Feb 2025 ADD YOUR COMMENT Comment Author details Author details 1 Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India 2 Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India Venkatesh Kamath B Roles: Conceptualization, Data Curation, Formal Analysis, Writing – Original Draft Preparation, Writing – Review & Editing Sandeep Mallya Roles: Writing – Original Draft Preparation, Writing – Review & Editing Subrahmanyam Volety Mallikarjuna Roles: Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Kiran Kumar Kolathur Roles: Conceptualization, Investigation, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing Competing interests No competing interests were disclosed. Grant information The author(s) declared that no grants were involved in supporting this work. Article Versions (2) version 2 Revised Published: 08 Jul 2025, 14:170 https://doi.org/10.12688/f1000research.160546.2 version 1 Published: 06 Feb 2025, 14:170 https://doi.org/10.12688/f1000research.160546.1 Copyright © 2025 Kamath B V et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Kamath B V, Mallya S, Mallikarjuna SV and Kolathur KK. The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.12688/f1000research.160546.1 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 1 VERSION 1 PUBLISHED 06 Feb 2025 Views 0 Cite How to cite this report: Sauka D. Reviewer Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r384406 ) The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-384406 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 09 Jun 2025 Diego Sauka , Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176457.r384406 The article provides valuable insights into Bacillus velezensis B26, a bacterium with demonstrated capabilities in pathogen control and plant growth promotion. However, the introduction requires greater clarity and focus. Although the authors include numerous examples of B. velezensis strains with ... Continue reading READ ALL The article provides valuable insights into Bacillus velezensis B26, a bacterium with demonstrated capabilities in pathogen control and plant growth promotion. However, the introduction requires greater clarity and focus. Although the authors include numerous examples of B. velezensis strains with antimicrobial and growth-promoting properties, they fail to effectively connect these examples to the study’s relevance. Moreover, the broader purpose and scientific significance of the work within the context of previous research remain unclear. The text contains redundancies, such as repeated mentions of the bacterium's ability to produce secondary metabolites, without offering a nuanced or differentiated discussion. Furthermore, some references are poorly integrated, as they are merely listed without elaborating on how they support the article’s hypotheses. The transitions between antimicrobial properties, plant growth promotion, and genomic studies are also disjointed, making the narrative difficult to follow. The introduction would benefit from a clearly stated hypothesis or specific objective emphasizing the biotechnological relevance of B. velezensis B26. Establishing a stronger connection between the genomic findings and their practical applications would significantly enhance the cohesion of the text. Specific comments Materials & Methods (M&M) The authors state that they used two different assemblers for genome assembly. However, the results of the SPAdes assembly are not shown in the Results section. This should be clarified. Additionally, the authors include a paragraph explaining the RAST Server but fail to specify its purpose in the study. Later, in the Results section, they mention using the Prokaryotic Genome Annotation Pipeline (PGAP). This inconsistency requires clarification. Results section The number of tables and figures could be reduced. For example: Figure 1 is unnecessary as it repeats information already described in the M&M section. Tables 1 and 2 could be combined for better conciseness. Figure 2 is not particularly informative, as it presents general information from the RAST Server without any meaningful analysis. A better presentation focusing specifically on genes associated with pathogen control and plant growth promotion would improve the article significantly. Highlighting these capabilities with well-analyzed data would make the study more impactful and engaging. Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Microbiology; genomics; biological control of pests I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Sauka D. Reviewer Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r384406 ) The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-384406 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 12 Sep 2025 KIRAN KOLATHUR , Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India 12 Sep 2025 Author Response Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed ... Continue reading Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. The article provides valuable insights into Bacillus velezensis B26, a bacterium with demonstrated capabilities in pathogen control and plant growth promotion. Response: We thank the reviewer for the comments Comment1: However, the introduction requires greater clarity and focus. Although the authors include numerous examples of B. velezensis strains with antimicrobial and growth-promoting properties, they fail to effectively connect these examples to the study’s relevance. Moreover, the broader purpose and scientific significance of the work within the context of previous research remain unclear. The text contains redundancies, such as repeated mentions of the bacterium's ability to produce secondary metabolites, without offering a nuanced or differentiated discussion. Furthermore, some references are poorly integrated, as they are merely listed without elaborating on how they support the article’s hypotheses. The transitions between antimicrobial properties, plant growth promotion, and genomic studies are also disjointed, making the narrative difficult to follow. The introduction would benefit from a clearly stated hypothesis or specific objective emphasizing the biotechnological relevance of B. velezensis B26. Establishing a stronger connection between the genomic findings and their practical applications would significantly enhance the cohesion of the text. Response 1: We thank the reviewer for the comment, and we have revised the introduction as below Introduction “Bacillus velezensis is a valuable bacterium that is extensively used in various biotechnological applications to promote plant growth. Several strains of B. velezensis have exhibited the ability to suppress plant pathogens and promote plant growth through various mechanisms such as antimicrobial compound synthesis, phosphate solubilization, nitrogen fixation and phytohormone modulation. For example, the B. velezensis LT1 strain displays antifungal activity against the soil-borne fungal pathogen Sclerotium rolfsii LC1, mediated by the secretion of diverse antimicrobial compounds (Tang et al. 2024). Similarly, B. velezensis strains 5YN8 and DSN012 act as biocontrol agents against Botrytis cinerea, a pathogen that causes severe gray mold disease in crops. These strains inhibit fungal spore germination and growth through the production of certain secondary metabolites and volatile organic compounds (Jiang et al. 2018). B. velezensis HNA3 and SQR9 strains have also been reported to produce several antimicrobial metabolites that help to control plant pathogens (S. et al. 2022; Rabbee et al. 2023). Besides antimicrobial activity, the SQR9 strain implicated in promoting plant growth by enhancing biofilm-mediated root colonization (Xu et al. 2019). Another strain, B. velezensis Ag75, has demonstrated as a biofertilizer and phosphate solubilizer for crops like maize and soybean (Mosela et al. 2022). Several studies performed whole genome sequencing to decipher the genetic basis of plant growth promotion and biocontrol capabilities in several B. velezensis strains. These studies provide key insights into the organism’s capability to produce antimicrobial compounds, enzyme secretion, and secondary metabolites that are involved in plant growth. Genome analysis of B. velezensis HNA3 led to establishing several gene clusters associated with promoting plant growth. Among these genes, the major percentage of genes are associated with amino acid metabolism, carbohydrate transport, and secondary metabolite biosynthesis (S. et al. 2022). Similarly, the B. velezensis CH1 strain, isolated from high-quality oats, exhibited antimicrobial properties contributing to oat growth and resistance to infections. Comparative genomic analysis of CH1 strain revealed 13 gene clusters linked with production of 15 secondary metabolites with antimicrobial properties. Furthermore, the strain harboured numerous putative genes for indole-3-acetic acid (IAA) production, spermidine, and polyamine synthesis. These results indicate that the possible applications of B. velezensis CH1 as a biofertilizer (Cheng et al. 2024). Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Specific comments Materials & Methods (M&M) The authors state that they used two different assemblers for genome assembly. However, the results of the SPAdes assembly are not shown in the Results section. This should be clarified. Additionally, the authors include a paragraph explaining the RAST Server but fail to specify its purpose in the study. Later, in the Results section, they mention using the Prokaryotic Genome Annotation Pipeline (PGAP). This inconsistency requires clarification. Response: We thank the reviewer for pointing out the discrepancy in the methods section we have modified as “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. We clarify that PGAP was used for primary genome annotation and reporting, while RAST was used specifically for subsystem-level classification. This has been clearly stated in the revised Methods section. “Genome annotation was primarily carried out using NCBI Prokaryotic Genome Annotation Pipeline (PGAP), which provides standardized gene prediction and functional characterization. Besides PGAP tool, the RAST server was used to perform subsystem-based annotation. This analysis aids in identification of gene sets grouped under biologically relevant functional categories”. Results section The number of tables and figures could be reduced. For example: Figure 1 is unnecessary as it repeats information already described in the M&M section. Tables 1 and 2 could be combined for better conciseness. Figure 2 is not particularly informative, as it presents general information from the RAST Server without any meaningful analysis. Response: We thank the reviewer for this helpful suggestion. In response, we have now removed Figure 1. Figure 2 we have modified as Figure 1, which represents subsystem statistics of the B. velezensis B26 genome annotations. • Tables 1 and 2 are separated for better understanding. Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. The article provides valuable insights into Bacillus velezensis B26, a bacterium with demonstrated capabilities in pathogen control and plant growth promotion. Response: We thank the reviewer for the comments Comment1: However, the introduction requires greater clarity and focus. Although the authors include numerous examples of B. velezensis strains with antimicrobial and growth-promoting properties, they fail to effectively connect these examples to the study’s relevance. Moreover, the broader purpose and scientific significance of the work within the context of previous research remain unclear. The text contains redundancies, such as repeated mentions of the bacterium's ability to produce secondary metabolites, without offering a nuanced or differentiated discussion. Furthermore, some references are poorly integrated, as they are merely listed without elaborating on how they support the article’s hypotheses. The transitions between antimicrobial properties, plant growth promotion, and genomic studies are also disjointed, making the narrative difficult to follow. The introduction would benefit from a clearly stated hypothesis or specific objective emphasizing the biotechnological relevance of B. velezensis B26. Establishing a stronger connection between the genomic findings and their practical applications would significantly enhance the cohesion of the text. Response 1: We thank the reviewer for the comment, and we have revised the introduction as below Introduction “Bacillus velezensis is a valuable bacterium that is extensively used in various biotechnological applications to promote plant growth. Several strains of B. velezensis have exhibited the ability to suppress plant pathogens and promote plant growth through various mechanisms such as antimicrobial compound synthesis, phosphate solubilization, nitrogen fixation and phytohormone modulation. For example, the B. velezensis LT1 strain displays antifungal activity against the soil-borne fungal pathogen Sclerotium rolfsii LC1, mediated by the secretion of diverse antimicrobial compounds (Tang et al. 2024). Similarly, B. velezensis strains 5YN8 and DSN012 act as biocontrol agents against Botrytis cinerea, a pathogen that causes severe gray mold disease in crops. These strains inhibit fungal spore germination and growth through the production of certain secondary metabolites and volatile organic compounds (Jiang et al. 2018). B. velezensis HNA3 and SQR9 strains have also been reported to produce several antimicrobial metabolites that help to control plant pathogens (S. et al. 2022; Rabbee et al. 2023). Besides antimicrobial activity, the SQR9 strain implicated in promoting plant growth by enhancing biofilm-mediated root colonization (Xu et al. 2019). Another strain, B. velezensis Ag75, has demonstrated as a biofertilizer and phosphate solubilizer for crops like maize and soybean (Mosela et al. 2022). Several studies performed whole genome sequencing to decipher the genetic basis of plant growth promotion and biocontrol capabilities in several B. velezensis strains. These studies provide key insights into the organism’s capability to produce antimicrobial compounds, enzyme secretion, and secondary metabolites that are involved in plant growth. Genome analysis of B. velezensis HNA3 led to establishing several gene clusters associated with promoting plant growth. Among these genes, the major percentage of genes are associated with amino acid metabolism, carbohydrate transport, and secondary metabolite biosynthesis (S. et al. 2022). Similarly, the B. velezensis CH1 strain, isolated from high-quality oats, exhibited antimicrobial properties contributing to oat growth and resistance to infections. Comparative genomic analysis of CH1 strain revealed 13 gene clusters linked with production of 15 secondary metabolites with antimicrobial properties. Furthermore, the strain harboured numerous putative genes for indole-3-acetic acid (IAA) production, spermidine, and polyamine synthesis. These results indicate that the possible applications of B. velezensis CH1 as a biofertilizer (Cheng et al. 2024). Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Specific comments Materials & Methods (M&M) The authors state that they used two different assemblers for genome assembly. However, the results of the SPAdes assembly are not shown in the Results section. This should be clarified. Additionally, the authors include a paragraph explaining the RAST Server but fail to specify its purpose in the study. Later, in the Results section, they mention using the Prokaryotic Genome Annotation Pipeline (PGAP). This inconsistency requires clarification. Response: We thank the reviewer for pointing out the discrepancy in the methods section we have modified as “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. We clarify that PGAP was used for primary genome annotation and reporting, while RAST was used specifically for subsystem-level classification. This has been clearly stated in the revised Methods section. “Genome annotation was primarily carried out using NCBI Prokaryotic Genome Annotation Pipeline (PGAP), which provides standardized gene prediction and functional characterization. Besides PGAP tool, the RAST server was used to perform subsystem-based annotation. This analysis aids in identification of gene sets grouped under biologically relevant functional categories”. Results section The number of tables and figures could be reduced. For example: Figure 1 is unnecessary as it repeats information already described in the M&M section. Tables 1 and 2 could be combined for better conciseness. Figure 2 is not particularly informative, as it presents general information from the RAST Server without any meaningful analysis. Response: We thank the reviewer for this helpful suggestion. In response, we have now removed Figure 1. Figure 2 we have modified as Figure 1, which represents subsystem statistics of the B. velezensis B26 genome annotations. • Tables 1 and 2 are separated for better understanding. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 12 Sep 2025 KIRAN KOLATHUR , Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India 12 Sep 2025 Author Response Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed ... Continue reading Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. The article provides valuable insights into Bacillus velezensis B26, a bacterium with demonstrated capabilities in pathogen control and plant growth promotion. Response: We thank the reviewer for the comments Comment1: However, the introduction requires greater clarity and focus. Although the authors include numerous examples of B. velezensis strains with antimicrobial and growth-promoting properties, they fail to effectively connect these examples to the study’s relevance. Moreover, the broader purpose and scientific significance of the work within the context of previous research remain unclear. The text contains redundancies, such as repeated mentions of the bacterium's ability to produce secondary metabolites, without offering a nuanced or differentiated discussion. Furthermore, some references are poorly integrated, as they are merely listed without elaborating on how they support the article’s hypotheses. The transitions between antimicrobial properties, plant growth promotion, and genomic studies are also disjointed, making the narrative difficult to follow. The introduction would benefit from a clearly stated hypothesis or specific objective emphasizing the biotechnological relevance of B. velezensis B26. Establishing a stronger connection between the genomic findings and their practical applications would significantly enhance the cohesion of the text. Response 1: We thank the reviewer for the comment, and we have revised the introduction as below Introduction “Bacillus velezensis is a valuable bacterium that is extensively used in various biotechnological applications to promote plant growth. Several strains of B. velezensis have exhibited the ability to suppress plant pathogens and promote plant growth through various mechanisms such as antimicrobial compound synthesis, phosphate solubilization, nitrogen fixation and phytohormone modulation. For example, the B. velezensis LT1 strain displays antifungal activity against the soil-borne fungal pathogen Sclerotium rolfsii LC1, mediated by the secretion of diverse antimicrobial compounds (Tang et al. 2024). Similarly, B. velezensis strains 5YN8 and DSN012 act as biocontrol agents against Botrytis cinerea, a pathogen that causes severe gray mold disease in crops. These strains inhibit fungal spore germination and growth through the production of certain secondary metabolites and volatile organic compounds (Jiang et al. 2018). B. velezensis HNA3 and SQR9 strains have also been reported to produce several antimicrobial metabolites that help to control plant pathogens (S. et al. 2022; Rabbee et al. 2023). Besides antimicrobial activity, the SQR9 strain implicated in promoting plant growth by enhancing biofilm-mediated root colonization (Xu et al. 2019). Another strain, B. velezensis Ag75, has demonstrated as a biofertilizer and phosphate solubilizer for crops like maize and soybean (Mosela et al. 2022). Several studies performed whole genome sequencing to decipher the genetic basis of plant growth promotion and biocontrol capabilities in several B. velezensis strains. These studies provide key insights into the organism’s capability to produce antimicrobial compounds, enzyme secretion, and secondary metabolites that are involved in plant growth. Genome analysis of B. velezensis HNA3 led to establishing several gene clusters associated with promoting plant growth. Among these genes, the major percentage of genes are associated with amino acid metabolism, carbohydrate transport, and secondary metabolite biosynthesis (S. et al. 2022). Similarly, the B. velezensis CH1 strain, isolated from high-quality oats, exhibited antimicrobial properties contributing to oat growth and resistance to infections. Comparative genomic analysis of CH1 strain revealed 13 gene clusters linked with production of 15 secondary metabolites with antimicrobial properties. Furthermore, the strain harboured numerous putative genes for indole-3-acetic acid (IAA) production, spermidine, and polyamine synthesis. These results indicate that the possible applications of B. velezensis CH1 as a biofertilizer (Cheng et al. 2024). Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Specific comments Materials & Methods (M&M) The authors state that they used two different assemblers for genome assembly. However, the results of the SPAdes assembly are not shown in the Results section. This should be clarified. Additionally, the authors include a paragraph explaining the RAST Server but fail to specify its purpose in the study. Later, in the Results section, they mention using the Prokaryotic Genome Annotation Pipeline (PGAP). This inconsistency requires clarification. Response: We thank the reviewer for pointing out the discrepancy in the methods section we have modified as “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. We clarify that PGAP was used for primary genome annotation and reporting, while RAST was used specifically for subsystem-level classification. This has been clearly stated in the revised Methods section. “Genome annotation was primarily carried out using NCBI Prokaryotic Genome Annotation Pipeline (PGAP), which provides standardized gene prediction and functional characterization. Besides PGAP tool, the RAST server was used to perform subsystem-based annotation. This analysis aids in identification of gene sets grouped under biologically relevant functional categories”. Results section The number of tables and figures could be reduced. For example: Figure 1 is unnecessary as it repeats information already described in the M&M section. Tables 1 and 2 could be combined for better conciseness. Figure 2 is not particularly informative, as it presents general information from the RAST Server without any meaningful analysis. Response: We thank the reviewer for this helpful suggestion. In response, we have now removed Figure 1. Figure 2 we have modified as Figure 1, which represents subsystem statistics of the B. velezensis B26 genome annotations. • Tables 1 and 2 are separated for better understanding. Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. The article provides valuable insights into Bacillus velezensis B26, a bacterium with demonstrated capabilities in pathogen control and plant growth promotion. Response: We thank the reviewer for the comments Comment1: However, the introduction requires greater clarity and focus. Although the authors include numerous examples of B. velezensis strains with antimicrobial and growth-promoting properties, they fail to effectively connect these examples to the study’s relevance. Moreover, the broader purpose and scientific significance of the work within the context of previous research remain unclear. The text contains redundancies, such as repeated mentions of the bacterium's ability to produce secondary metabolites, without offering a nuanced or differentiated discussion. Furthermore, some references are poorly integrated, as they are merely listed without elaborating on how they support the article’s hypotheses. The transitions between antimicrobial properties, plant growth promotion, and genomic studies are also disjointed, making the narrative difficult to follow. The introduction would benefit from a clearly stated hypothesis or specific objective emphasizing the biotechnological relevance of B. velezensis B26. Establishing a stronger connection between the genomic findings and their practical applications would significantly enhance the cohesion of the text. Response 1: We thank the reviewer for the comment, and we have revised the introduction as below Introduction “Bacillus velezensis is a valuable bacterium that is extensively used in various biotechnological applications to promote plant growth. Several strains of B. velezensis have exhibited the ability to suppress plant pathogens and promote plant growth through various mechanisms such as antimicrobial compound synthesis, phosphate solubilization, nitrogen fixation and phytohormone modulation. For example, the B. velezensis LT1 strain displays antifungal activity against the soil-borne fungal pathogen Sclerotium rolfsii LC1, mediated by the secretion of diverse antimicrobial compounds (Tang et al. 2024). Similarly, B. velezensis strains 5YN8 and DSN012 act as biocontrol agents against Botrytis cinerea, a pathogen that causes severe gray mold disease in crops. These strains inhibit fungal spore germination and growth through the production of certain secondary metabolites and volatile organic compounds (Jiang et al. 2018). B. velezensis HNA3 and SQR9 strains have also been reported to produce several antimicrobial metabolites that help to control plant pathogens (S. et al. 2022; Rabbee et al. 2023). Besides antimicrobial activity, the SQR9 strain implicated in promoting plant growth by enhancing biofilm-mediated root colonization (Xu et al. 2019). Another strain, B. velezensis Ag75, has demonstrated as a biofertilizer and phosphate solubilizer for crops like maize and soybean (Mosela et al. 2022). Several studies performed whole genome sequencing to decipher the genetic basis of plant growth promotion and biocontrol capabilities in several B. velezensis strains. These studies provide key insights into the organism’s capability to produce antimicrobial compounds, enzyme secretion, and secondary metabolites that are involved in plant growth. Genome analysis of B. velezensis HNA3 led to establishing several gene clusters associated with promoting plant growth. Among these genes, the major percentage of genes are associated with amino acid metabolism, carbohydrate transport, and secondary metabolite biosynthesis (S. et al. 2022). Similarly, the B. velezensis CH1 strain, isolated from high-quality oats, exhibited antimicrobial properties contributing to oat growth and resistance to infections. Comparative genomic analysis of CH1 strain revealed 13 gene clusters linked with production of 15 secondary metabolites with antimicrobial properties. Furthermore, the strain harboured numerous putative genes for indole-3-acetic acid (IAA) production, spermidine, and polyamine synthesis. These results indicate that the possible applications of B. velezensis CH1 as a biofertilizer (Cheng et al. 2024). Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Specific comments Materials & Methods (M&M) The authors state that they used two different assemblers for genome assembly. However, the results of the SPAdes assembly are not shown in the Results section. This should be clarified. Additionally, the authors include a paragraph explaining the RAST Server but fail to specify its purpose in the study. Later, in the Results section, they mention using the Prokaryotic Genome Annotation Pipeline (PGAP). This inconsistency requires clarification. Response: We thank the reviewer for pointing out the discrepancy in the methods section we have modified as “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. We clarify that PGAP was used for primary genome annotation and reporting, while RAST was used specifically for subsystem-level classification. This has been clearly stated in the revised Methods section. “Genome annotation was primarily carried out using NCBI Prokaryotic Genome Annotation Pipeline (PGAP), which provides standardized gene prediction and functional characterization. Besides PGAP tool, the RAST server was used to perform subsystem-based annotation. This analysis aids in identification of gene sets grouped under biologically relevant functional categories”. Results section The number of tables and figures could be reduced. For example: Figure 1 is unnecessary as it repeats information already described in the M&M section. Tables 1 and 2 could be combined for better conciseness. Figure 2 is not particularly informative, as it presents general information from the RAST Server without any meaningful analysis. Response: We thank the reviewer for this helpful suggestion. In response, we have now removed Figure 1. Figure 2 we have modified as Figure 1, which represents subsystem statistics of the B. velezensis B26 genome annotations. • Tables 1 and 2 are separated for better understanding. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Basak S. Reviewer Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r384408 ) The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-384408 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 09 Jun 2025 Surajit Basak , ICMR - National Institute for Research in Bacterial Infections, Beliaghata Kolkata, India Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.176457.r384408 The study delivers an organized, useful genome report on Bacillus velezensis B26 as it narrates about the possible use as a biocontrol agent and biofertilizer. The use of genome assembly and annotation pipeline pipelines and pangenome analysis ... Continue reading READ ALL The study delivers an organized, useful genome report on Bacillus velezensis B26 as it narrates about the possible use as a biocontrol agent and biofertilizer. The use of genome assembly and annotation pipeline pipelines and pangenome analysis provide more insights into the genome sequences. There are some issues that will help to improve the depth of the manuscript. 1) Add a brief in the introduction by stating importance of giving more emphasis on B26 compared to other Bacillus velezensis strains. 2) Elaborate the methodology section with detailed description of the work done and the tools used for performing them along with proper citations of the tools. 3) No methodology is given for pangenome analysis, give a brief methodology of pangenome analysis with proper citations. 4) Why two assembly pipelines have been used is not clear. Also, there are disparity of information provided in the methodology section “The quality of the genome assemblies produced by the Spades (Bankevich et al. 2012) and Megahit (Li et al. 2015) assemblers was evaluated using the Quast tool (Gurevich et al. 2013). Compared with the megahit assembler, the Spades assembler had longer assembled contigs with better N50 values. Hence, the assembly generated by Spades was selected for further downstream analyses.” and in the results section “The genome assembly method employed was Megahit v. 2023-07-12 tool, the genome representation was full….” - Explain this. 5) Subsystem information is given. Discuss about the gene or gene set responsible for B. velezensis B26. 6) Along with the table of pangenome analysis add plots showing the gene distribution. Is there any unique feature of the Bacillus velezensis B26 strain? Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? No Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Basak S. Reviewer Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r384408 ) The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-384408 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 21 Jul 2025 KIRAN KOLATHUR , Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India 21 Jul 2025 Author Response Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a ... Continue reading Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Reviewer 2: The study delivers an organized, useful genome report on Bacillus velezensis B26 as it narrates about the possible use as a biocontrol agent and biofertilizer. The use of genome assembly and annotation pipeline pipelines and pangenome analysis provide more insights into the genome sequences. Response: We thank the reviewer for the comments There are some issues that will help to improve the depth of the manuscript. 1) Add a brief in the introduction by stating the importance of giving more emphasis on B26 compared to other Bacillus velezensis strains. Response 1: As mentioned, we have revised the introduction to highlight why strain B26 was chosen for genome sequencing. Specifically, we emphasize its unique origin (chicken carcass soil), its antifungal spectrum that includes both agricultural and human pathogens, and its potential for broader biotechnological applications. These features distinguish B26 from the most previously characterized B. velezensis strains and justify its genomic investigation. We have modified the introduction accordingly. 2) Elaborate the methodology section with detailed description of the work done and the tools used for performing them along with proper citations of the tools. Response 2: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 3 & 4). 3) No methodology is given for pangenome analysis, give a brief methodology of pangenome analysis with proper citations. Response 3: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 4). Pangenome Analysis “Genome annotation was carried out using Prokka v1.14.6 (Seemann 2014). It generates standardized GFF3 files, which possess predicted coding sequences (CDS), rRNAs, tRNAs, and functional annotations. Pangenome analysis was subsequently performed using the Roary pipeline v3.13.0 (Page et al. 2015), which aids large-scale analysis of prokaryotic genomes by comparing orthologous genes across different strains. This analysis was carried out using minimum BLASTp identity threshold of 95% for gene clustering. This method facilitates the identification of both core genes (present in ≥99% of genomes), shell genes (present in ≥15% and <95%) and cloud genes (present in <15%), thus provides insights into genome conservation, diversity and evolutionary adaptation.” 4) Why two assembly pipelines have been used is not clear. Also, there are disparity of information provided in the methodology section “The quality of the genome assemblies produced by the Spades (Bankevich et al. 2012) and Megahit (Li et al. 2015) assemblers was evaluated using the Quast tool (Gurevich et al. 2013). Compared with the megahit assembler, the Spades assembler had longer assembled contigs with better N50 values. Hence, the assembly generated by Spades was selected for further downstream analyses.” and in the results section “The genome assembly method employed was Megahit v. 2023-07-12 tool, the genome representation was full….” - Explain this. Response 4: We thank the reviewer for pointing out the discrepancy in the methods section (Page no.4) and results section (Page no.5). We have modified as below “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. 5) Subsystem information is given. Discuss the gene or gene set responsible for B. velezensis B26. Response 5: We thank the reviewer for the comment; we have added detailed as below (Results section, page no 6) Functional implications of subsystem genes “Subsystem-based genome annotation of B. velezensis B26 revealed gene sets that may contribute to its biocontrol and biofertilizer activities. The genome contains 43 genes related to stress response and 35 genes involved in virulence, disease, and defense, suggesting the strain’s ability to tolerate environmental stress and inhibit plant pathogens. Additionally, the presence of genes involved in phosphorus (10 genes), nitrogen (20 genes), and sulfur metabolism (6 genes) indicates the stain’s potential for nutrient acquisition and its tole as a biofertilizer. Altogether, these gene sets provide valuable insights into the biotechnological potential of B. velezensis B26”. 6) Along with the table of pangenome analysis add plots showing the gene distribution. Is there any unique feature of the Bacillus velezensis B26 strain? Response 6: We thank the reviewer for this helpful suggestion. In response, we have now included additional visualizations to support the pangenome analysis: A pie chart (now added as Figure 2) illustrates the distribution of gene categories across the pangenome, including core, shell, cloud, and soft-core genes. “Pangenome analysis of B. velezensis B26 identified a total of 5,576 genes. Among these genes, 3,480 genes were considered core genes, present in 99% to 100% of strains. Additionally,1, 143 genes were classified as shell genes, which were present in 15% to 95% of strains, while 953 genes were identified as cloud genes, found in fewer than 15% of the analysed strains (Figure 2, Table 3). These results indicate that a major portion of the B. velezensis B26 genome is conserved, while significant proportion of cloud genes (953 out of 5,576), may represent strain-specific or rare genes. Although many of the genes remains uncharacterized, these accessory genes might contribute to the distinctive biocontrol and plant growth promoting capabilities of B. velezensis B26”. Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Reviewer 2: The study delivers an organized, useful genome report on Bacillus velezensis B26 as it narrates about the possible use as a biocontrol agent and biofertilizer. The use of genome assembly and annotation pipeline pipelines and pangenome analysis provide more insights into the genome sequences. Response: We thank the reviewer for the comments There are some issues that will help to improve the depth of the manuscript. 1) Add a brief in the introduction by stating the importance of giving more emphasis on B26 compared to other Bacillus velezensis strains. Response 1: As mentioned, we have revised the introduction to highlight why strain B26 was chosen for genome sequencing. Specifically, we emphasize its unique origin (chicken carcass soil), its antifungal spectrum that includes both agricultural and human pathogens, and its potential for broader biotechnological applications. These features distinguish B26 from the most previously characterized B. velezensis strains and justify its genomic investigation. We have modified the introduction accordingly. 2) Elaborate the methodology section with detailed description of the work done and the tools used for performing them along with proper citations of the tools. Response 2: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 3 & 4). 3) No methodology is given for pangenome analysis, give a brief methodology of pangenome analysis with proper citations. Response 3: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 4). Pangenome Analysis “Genome annotation was carried out using Prokka v1.14.6 (Seemann 2014). It generates standardized GFF3 files, which possess predicted coding sequences (CDS), rRNAs, tRNAs, and functional annotations. Pangenome analysis was subsequently performed using the Roary pipeline v3.13.0 (Page et al. 2015), which aids large-scale analysis of prokaryotic genomes by comparing orthologous genes across different strains. This analysis was carried out using minimum BLASTp identity threshold of 95% for gene clustering. This method facilitates the identification of both core genes (present in ≥99% of genomes), shell genes (present in ≥15% and <95%) and cloud genes (present in <15%), thus provides insights into genome conservation, diversity and evolutionary adaptation.” 4) Why two assembly pipelines have been used is not clear. Also, there are disparity of information provided in the methodology section “The quality of the genome assemblies produced by the Spades (Bankevich et al. 2012) and Megahit (Li et al. 2015) assemblers was evaluated using the Quast tool (Gurevich et al. 2013). Compared with the megahit assembler, the Spades assembler had longer assembled contigs with better N50 values. Hence, the assembly generated by Spades was selected for further downstream analyses.” and in the results section “The genome assembly method employed was Megahit v. 2023-07-12 tool, the genome representation was full….” - Explain this. Response 4: We thank the reviewer for pointing out the discrepancy in the methods section (Page no.4) and results section (Page no.5). We have modified as below “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. 5) Subsystem information is given. Discuss the gene or gene set responsible for B. velezensis B26. Response 5: We thank the reviewer for the comment; we have added detailed as below (Results section, page no 6) Functional implications of subsystem genes “Subsystem-based genome annotation of B. velezensis B26 revealed gene sets that may contribute to its biocontrol and biofertilizer activities. The genome contains 43 genes related to stress response and 35 genes involved in virulence, disease, and defense, suggesting the strain’s ability to tolerate environmental stress and inhibit plant pathogens. Additionally, the presence of genes involved in phosphorus (10 genes), nitrogen (20 genes), and sulfur metabolism (6 genes) indicates the stain’s potential for nutrient acquisition and its tole as a biofertilizer. Altogether, these gene sets provide valuable insights into the biotechnological potential of B. velezensis B26”. 6) Along with the table of pangenome analysis add plots showing the gene distribution. Is there any unique feature of the Bacillus velezensis B26 strain? Response 6: We thank the reviewer for this helpful suggestion. In response, we have now included additional visualizations to support the pangenome analysis: A pie chart (now added as Figure 2) illustrates the distribution of gene categories across the pangenome, including core, shell, cloud, and soft-core genes. “Pangenome analysis of B. velezensis B26 identified a total of 5,576 genes. Among these genes, 3,480 genes were considered core genes, present in 99% to 100% of strains. Additionally,1, 143 genes were classified as shell genes, which were present in 15% to 95% of strains, while 953 genes were identified as cloud genes, found in fewer than 15% of the analysed strains (Figure 2, Table 3). These results indicate that a major portion of the B. velezensis B26 genome is conserved, while significant proportion of cloud genes (953 out of 5,576), may represent strain-specific or rare genes. Although many of the genes remains uncharacterized, these accessory genes might contribute to the distinctive biocontrol and plant growth promoting capabilities of B. velezensis B26”. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 21 Jul 2025 KIRAN KOLATHUR , Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India 21 Jul 2025 Author Response Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a ... Continue reading Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Reviewer 2: The study delivers an organized, useful genome report on Bacillus velezensis B26 as it narrates about the possible use as a biocontrol agent and biofertilizer. The use of genome assembly and annotation pipeline pipelines and pangenome analysis provide more insights into the genome sequences. Response: We thank the reviewer for the comments There are some issues that will help to improve the depth of the manuscript. 1) Add a brief in the introduction by stating the importance of giving more emphasis on B26 compared to other Bacillus velezensis strains. Response 1: As mentioned, we have revised the introduction to highlight why strain B26 was chosen for genome sequencing. Specifically, we emphasize its unique origin (chicken carcass soil), its antifungal spectrum that includes both agricultural and human pathogens, and its potential for broader biotechnological applications. These features distinguish B26 from the most previously characterized B. velezensis strains and justify its genomic investigation. We have modified the introduction accordingly. 2) Elaborate the methodology section with detailed description of the work done and the tools used for performing them along with proper citations of the tools. Response 2: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 3 & 4). 3) No methodology is given for pangenome analysis, give a brief methodology of pangenome analysis with proper citations. Response 3: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 4). Pangenome Analysis “Genome annotation was carried out using Prokka v1.14.6 (Seemann 2014). It generates standardized GFF3 files, which possess predicted coding sequences (CDS), rRNAs, tRNAs, and functional annotations. Pangenome analysis was subsequently performed using the Roary pipeline v3.13.0 (Page et al. 2015), which aids large-scale analysis of prokaryotic genomes by comparing orthologous genes across different strains. This analysis was carried out using minimum BLASTp identity threshold of 95% for gene clustering. This method facilitates the identification of both core genes (present in ≥99% of genomes), shell genes (present in ≥15% and <95%) and cloud genes (present in <15%), thus provides insights into genome conservation, diversity and evolutionary adaptation.” 4) Why two assembly pipelines have been used is not clear. Also, there are disparity of information provided in the methodology section “The quality of the genome assemblies produced by the Spades (Bankevich et al. 2012) and Megahit (Li et al. 2015) assemblers was evaluated using the Quast tool (Gurevich et al. 2013). Compared with the megahit assembler, the Spades assembler had longer assembled contigs with better N50 values. Hence, the assembly generated by Spades was selected for further downstream analyses.” and in the results section “The genome assembly method employed was Megahit v. 2023-07-12 tool, the genome representation was full….” - Explain this. Response 4: We thank the reviewer for pointing out the discrepancy in the methods section (Page no.4) and results section (Page no.5). We have modified as below “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. 5) Subsystem information is given. Discuss the gene or gene set responsible for B. velezensis B26. Response 5: We thank the reviewer for the comment; we have added detailed as below (Results section, page no 6) Functional implications of subsystem genes “Subsystem-based genome annotation of B. velezensis B26 revealed gene sets that may contribute to its biocontrol and biofertilizer activities. The genome contains 43 genes related to stress response and 35 genes involved in virulence, disease, and defense, suggesting the strain’s ability to tolerate environmental stress and inhibit plant pathogens. Additionally, the presence of genes involved in phosphorus (10 genes), nitrogen (20 genes), and sulfur metabolism (6 genes) indicates the stain’s potential for nutrient acquisition and its tole as a biofertilizer. Altogether, these gene sets provide valuable insights into the biotechnological potential of B. velezensis B26”. 6) Along with the table of pangenome analysis add plots showing the gene distribution. Is there any unique feature of the Bacillus velezensis B26 strain? Response 6: We thank the reviewer for this helpful suggestion. In response, we have now included additional visualizations to support the pangenome analysis: A pie chart (now added as Figure 2) illustrates the distribution of gene categories across the pangenome, including core, shell, cloud, and soft-core genes. “Pangenome analysis of B. velezensis B26 identified a total of 5,576 genes. Among these genes, 3,480 genes were considered core genes, present in 99% to 100% of strains. Additionally,1, 143 genes were classified as shell genes, which were present in 15% to 95% of strains, while 953 genes were identified as cloud genes, found in fewer than 15% of the analysed strains (Figure 2, Table 3). These results indicate that a major portion of the B. velezensis B26 genome is conserved, while significant proportion of cloud genes (953 out of 5,576), may represent strain-specific or rare genes. Although many of the genes remains uncharacterized, these accessory genes might contribute to the distinctive biocontrol and plant growth promoting capabilities of B. velezensis B26”. Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Reviewer 2: The study delivers an organized, useful genome report on Bacillus velezensis B26 as it narrates about the possible use as a biocontrol agent and biofertilizer. The use of genome assembly and annotation pipeline pipelines and pangenome analysis provide more insights into the genome sequences. Response: We thank the reviewer for the comments There are some issues that will help to improve the depth of the manuscript. 1) Add a brief in the introduction by stating the importance of giving more emphasis on B26 compared to other Bacillus velezensis strains. Response 1: As mentioned, we have revised the introduction to highlight why strain B26 was chosen for genome sequencing. Specifically, we emphasize its unique origin (chicken carcass soil), its antifungal spectrum that includes both agricultural and human pathogens, and its potential for broader biotechnological applications. These features distinguish B26 from the most previously characterized B. velezensis strains and justify its genomic investigation. We have modified the introduction accordingly. 2) Elaborate the methodology section with detailed description of the work done and the tools used for performing them along with proper citations of the tools. Response 2: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 3 & 4). 3) No methodology is given for pangenome analysis, give a brief methodology of pangenome analysis with proper citations. Response 3: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 4). Pangenome Analysis “Genome annotation was carried out using Prokka v1.14.6 (Seemann 2014). It generates standardized GFF3 files, which possess predicted coding sequences (CDS), rRNAs, tRNAs, and functional annotations. Pangenome analysis was subsequently performed using the Roary pipeline v3.13.0 (Page et al. 2015), which aids large-scale analysis of prokaryotic genomes by comparing orthologous genes across different strains. This analysis was carried out using minimum BLASTp identity threshold of 95% for gene clustering. This method facilitates the identification of both core genes (present in ≥99% of genomes), shell genes (present in ≥15% and <95%) and cloud genes (present in <15%), thus provides insights into genome conservation, diversity and evolutionary adaptation.” 4) Why two assembly pipelines have been used is not clear. Also, there are disparity of information provided in the methodology section “The quality of the genome assemblies produced by the Spades (Bankevich et al. 2012) and Megahit (Li et al. 2015) assemblers was evaluated using the Quast tool (Gurevich et al. 2013). Compared with the megahit assembler, the Spades assembler had longer assembled contigs with better N50 values. Hence, the assembly generated by Spades was selected for further downstream analyses.” and in the results section “The genome assembly method employed was Megahit v. 2023-07-12 tool, the genome representation was full….” - Explain this. Response 4: We thank the reviewer for pointing out the discrepancy in the methods section (Page no.4) and results section (Page no.5). We have modified as below “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. 5) Subsystem information is given. Discuss the gene or gene set responsible for B. velezensis B26. Response 5: We thank the reviewer for the comment; we have added detailed as below (Results section, page no 6) Functional implications of subsystem genes “Subsystem-based genome annotation of B. velezensis B26 revealed gene sets that may contribute to its biocontrol and biofertilizer activities. The genome contains 43 genes related to stress response and 35 genes involved in virulence, disease, and defense, suggesting the strain’s ability to tolerate environmental stress and inhibit plant pathogens. Additionally, the presence of genes involved in phosphorus (10 genes), nitrogen (20 genes), and sulfur metabolism (6 genes) indicates the stain’s potential for nutrient acquisition and its tole as a biofertilizer. Altogether, these gene sets provide valuable insights into the biotechnological potential of B. velezensis B26”. 6) Along with the table of pangenome analysis add plots showing the gene distribution. Is there any unique feature of the Bacillus velezensis B26 strain? Response 6: We thank the reviewer for this helpful suggestion. In response, we have now included additional visualizations to support the pangenome analysis: A pie chart (now added as Figure 2) illustrates the distribution of gene categories across the pangenome, including core, shell, cloud, and soft-core genes. “Pangenome analysis of B. velezensis B26 identified a total of 5,576 genes. Among these genes, 3,480 genes were considered core genes, present in 99% to 100% of strains. Additionally,1, 143 genes were classified as shell genes, which were present in 15% to 95% of strains, while 953 genes were identified as cloud genes, found in fewer than 15% of the analysed strains (Figure 2, Table 3). These results indicate that a major portion of the B. velezensis B26 genome is conserved, while significant proportion of cloud genes (953 out of 5,576), may represent strain-specific or rare genes. Although many of the genes remains uncharacterized, these accessory genes might contribute to the distinctive biocontrol and plant growth promoting capabilities of B. velezensis B26”. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Nallusamy S. Reviewer Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r365574 ) The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-365574 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 31 Mar 2025 Saranya Nallusamy , Tamil Nadu Agricultural University, Coimbatore, Tamil nadu, India Approved VIEWS 0 https://doi.org/10.5256/f1000research.176457.r365574 The manuscript titled "The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer" presents the whole-genome sequencing of B. velezensis B26, a strain isolated from chicken carcass soil in Udupi, India. The study highlights the strain’s biocontrol potential and plant ... Continue reading READ ALL The manuscript titled "The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer" presents the whole-genome sequencing of B. velezensis B26, a strain isolated from chicken carcass soil in Udupi, India. The study highlights the strain’s biocontrol potential and plant growth-promoting abilities, emphasizing its antifungal properties and enzymatic activities. Whole-genome sequencing was performed using Illumina technology, and genome annotation identified multiple functional genes linked to metabolic processes, stress response, and biocontrol properties. Bacillus velezensis has multiple applications in agriculture, biotechnology, and medicine. It plays a key role in plant growth promotion, biocontrol activity, and industrial applications. Its genome sequencing helps in understanding its potential for various applications, particularly in sustainable agriculture. This article reports the genome sequence of strain B26 to explore its potential in agriculture and biocontrol. Comparative genomics with other strains highlights unique genetic features that contribute to its biocontrol capabilities. Pan genome analysis reported the core and shell genes which is crucial for multiple applications in microbiology, biotechnology, and evolutionary studies. The study employed appropriate and standard methods like SPAdes for genome assembly and RAST & NCBI PGAP for genome annotation. The genome sequence and annotation data have been deposited in NCBI under accession number JAYKOV000000000. Genome sequencing highlights its potential for secondary metabolite production, making it a strong candidate for synthetic biology and metabolic engineering. Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Plant genomics and transcriptomics, structural bioinformatics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Nallusamy S. Reviewer Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r365574 ) The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-365574 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 12 Sep 2025 KIRAN KOLATHUR , Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India 12 Sep 2025 Author Response Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed ... Continue reading Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Response 1 : We thank the reviewer for the insightful feedback. In the revised manuscript, we have modified the introduction section to more clearly articulate (Introduction, Page no.2) “Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Response 1 : We thank the reviewer for the insightful feedback. In the revised manuscript, we have modified the introduction section to more clearly articulate (Introduction, Page no.2) “Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 12 Sep 2025 KIRAN KOLATHUR , Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India 12 Sep 2025 Author Response Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed ... Continue reading Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Response 1 : We thank the reviewer for the insightful feedback. In the revised manuscript, we have modified the introduction section to more clearly articulate (Introduction, Page no.2) “Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Response 1 : We thank the reviewer for the insightful feedback. In the revised manuscript, we have modified the introduction section to more clearly articulate (Introduction, Page no.2) “Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (0) Version 2 VERSION 2 PUBLISHED 06 Feb 2025 ADD YOUR COMMENT Comment keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 3 Version 2 (revision) 08 Jul 25 read read Version 1 06 Feb 25 read read read Saranya Nallusamy , Tamil Nadu Agricultural University, Coimbatore, India Surajit Basak , ICMR - National Institute for Research in Bacterial Infections, Beliaghata Kolkata, India Diego Sauka , Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina Comments on this article All Comments (0) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Basak S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 23 Aug 2025 | for Version 2 Surajit Basak , ICMR - National Institute for Research in Bacterial Infections, Beliaghata Kolkata, India 0 Views copyright © 2025 Basak S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The revised manuscript may be accepted for indexing. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Basak S. Peer Review Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.184085.r397488) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-170/v2#referee-response-397488 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Sauka D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 23 Aug 2025 | for Version 2 Diego Sauka , Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina 0 Views copyright © 2025 Sauka D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors have successfully addressed all the requests and comments made by this reviewer. Competing Interests No competing interests were disclosed. Reviewer Expertise Microbiology; genomics; biological control of pests I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Sauka D. Peer Review Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.184085.r397487) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-170/v2#referee-response-397487 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Sauka D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 09 Jun 2025 | for Version 1 Diego Sauka , Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina 0 Views copyright © 2025 Sauka D. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The article provides valuable insights into Bacillus velezensis B26, a bacterium with demonstrated capabilities in pathogen control and plant growth promotion. However, the introduction requires greater clarity and focus. Although the authors include numerous examples of B. velezensis strains with antimicrobial and growth-promoting properties, they fail to effectively connect these examples to the study’s relevance. Moreover, the broader purpose and scientific significance of the work within the context of previous research remain unclear. The text contains redundancies, such as repeated mentions of the bacterium's ability to produce secondary metabolites, without offering a nuanced or differentiated discussion. Furthermore, some references are poorly integrated, as they are merely listed without elaborating on how they support the article’s hypotheses. The transitions between antimicrobial properties, plant growth promotion, and genomic studies are also disjointed, making the narrative difficult to follow. The introduction would benefit from a clearly stated hypothesis or specific objective emphasizing the biotechnological relevance of B. velezensis B26. Establishing a stronger connection between the genomic findings and their practical applications would significantly enhance the cohesion of the text. Specific comments Materials & Methods (M&M) The authors state that they used two different assemblers for genome assembly. However, the results of the SPAdes assembly are not shown in the Results section. This should be clarified. Additionally, the authors include a paragraph explaining the RAST Server but fail to specify its purpose in the study. Later, in the Results section, they mention using the Prokaryotic Genome Annotation Pipeline (PGAP). This inconsistency requires clarification. Results section The number of tables and figures could be reduced. For example: Figure 1 is unnecessary as it repeats information already described in the M&M section. Tables 1 and 2 could be combined for better conciseness. Figure 2 is not particularly informative, as it presents general information from the RAST Server without any meaningful analysis. A better presentation focusing specifically on genes associated with pathogen control and plant growth promotion would improve the article significantly. Highlighting these capabilities with well-analyzed data would make the study more impactful and engaging. Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Microbiology; genomics; biological control of pests I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 12 Sep 2025 KIRAN KOLATHUR, Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. The article provides valuable insights into Bacillus velezensis B26, a bacterium with demonstrated capabilities in pathogen control and plant growth promotion. Response: We thank the reviewer for the comments Comment1: However, the introduction requires greater clarity and focus. Although the authors include numerous examples of B. velezensis strains with antimicrobial and growth-promoting properties, they fail to effectively connect these examples to the study’s relevance. Moreover, the broader purpose and scientific significance of the work within the context of previous research remain unclear. The text contains redundancies, such as repeated mentions of the bacterium's ability to produce secondary metabolites, without offering a nuanced or differentiated discussion. Furthermore, some references are poorly integrated, as they are merely listed without elaborating on how they support the article’s hypotheses. The transitions between antimicrobial properties, plant growth promotion, and genomic studies are also disjointed, making the narrative difficult to follow. The introduction would benefit from a clearly stated hypothesis or specific objective emphasizing the biotechnological relevance of B. velezensis B26. Establishing a stronger connection between the genomic findings and their practical applications would significantly enhance the cohesion of the text. Response 1: We thank the reviewer for the comment, and we have revised the introduction as below Introduction “Bacillus velezensis is a valuable bacterium that is extensively used in various biotechnological applications to promote plant growth. Several strains of B. velezensis have exhibited the ability to suppress plant pathogens and promote plant growth through various mechanisms such as antimicrobial compound synthesis, phosphate solubilization, nitrogen fixation and phytohormone modulation. For example, the B. velezensis LT1 strain displays antifungal activity against the soil-borne fungal pathogen Sclerotium rolfsii LC1, mediated by the secretion of diverse antimicrobial compounds (Tang et al. 2024). Similarly, B. velezensis strains 5YN8 and DSN012 act as biocontrol agents against Botrytis cinerea, a pathogen that causes severe gray mold disease in crops. These strains inhibit fungal spore germination and growth through the production of certain secondary metabolites and volatile organic compounds (Jiang et al. 2018). B. velezensis HNA3 and SQR9 strains have also been reported to produce several antimicrobial metabolites that help to control plant pathogens (S. et al. 2022; Rabbee et al. 2023). Besides antimicrobial activity, the SQR9 strain implicated in promoting plant growth by enhancing biofilm-mediated root colonization (Xu et al. 2019). Another strain, B. velezensis Ag75, has demonstrated as a biofertilizer and phosphate solubilizer for crops like maize and soybean (Mosela et al. 2022). Several studies performed whole genome sequencing to decipher the genetic basis of plant growth promotion and biocontrol capabilities in several B. velezensis strains. These studies provide key insights into the organism’s capability to produce antimicrobial compounds, enzyme secretion, and secondary metabolites that are involved in plant growth. Genome analysis of B. velezensis HNA3 led to establishing several gene clusters associated with promoting plant growth. Among these genes, the major percentage of genes are associated with amino acid metabolism, carbohydrate transport, and secondary metabolite biosynthesis (S. et al. 2022). Similarly, the B. velezensis CH1 strain, isolated from high-quality oats, exhibited antimicrobial properties contributing to oat growth and resistance to infections. Comparative genomic analysis of CH1 strain revealed 13 gene clusters linked with production of 15 secondary metabolites with antimicrobial properties. Furthermore, the strain harboured numerous putative genes for indole-3-acetic acid (IAA) production, spermidine, and polyamine synthesis. These results indicate that the possible applications of B. velezensis CH1 as a biofertilizer (Cheng et al. 2024). Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. Specific comments Materials & Methods (M&M) The authors state that they used two different assemblers for genome assembly. However, the results of the SPAdes assembly are not shown in the Results section. This should be clarified. Additionally, the authors include a paragraph explaining the RAST Server but fail to specify its purpose in the study. Later, in the Results section, they mention using the Prokaryotic Genome Annotation Pipeline (PGAP). This inconsistency requires clarification. Response: We thank the reviewer for pointing out the discrepancy in the methods section we have modified as “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. We clarify that PGAP was used for primary genome annotation and reporting, while RAST was used specifically for subsystem-level classification. This has been clearly stated in the revised Methods section. “Genome annotation was primarily carried out using NCBI Prokaryotic Genome Annotation Pipeline (PGAP), which provides standardized gene prediction and functional characterization. Besides PGAP tool, the RAST server was used to perform subsystem-based annotation. This analysis aids in identification of gene sets grouped under biologically relevant functional categories”. Results section The number of tables and figures could be reduced. For example: Figure 1 is unnecessary as it repeats information already described in the M&M section. Tables 1 and 2 could be combined for better conciseness. Figure 2 is not particularly informative, as it presents general information from the RAST Server without any meaningful analysis. Response: We thank the reviewer for this helpful suggestion. In response, we have now removed Figure 1. Figure 2 we have modified as Figure 1, which represents subsystem statistics of the B. velezensis B26 genome annotations. • Tables 1 and 2 are separated for better understanding. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Sauka D. Peer Review Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r384406) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-384406 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Basak S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 09 Jun 2025 | for Version 1 Surajit Basak , ICMR - National Institute for Research in Bacterial Infections, Beliaghata Kolkata, India 0 Views copyright © 2025 Basak S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The study delivers an organized, useful genome report on Bacillus velezensis B26 as it narrates about the possible use as a biocontrol agent and biofertilizer. The use of genome assembly and annotation pipeline pipelines and pangenome analysis provide more insights into the genome sequences. There are some issues that will help to improve the depth of the manuscript. 1) Add a brief in the introduction by stating importance of giving more emphasis on B26 compared to other Bacillus velezensis strains. 2) Elaborate the methodology section with detailed description of the work done and the tools used for performing them along with proper citations of the tools. 3) No methodology is given for pangenome analysis, give a brief methodology of pangenome analysis with proper citations. 4) Why two assembly pipelines have been used is not clear. Also, there are disparity of information provided in the methodology section “The quality of the genome assemblies produced by the Spades (Bankevich et al. 2012) and Megahit (Li et al. 2015) assemblers was evaluated using the Quast tool (Gurevich et al. 2013). Compared with the megahit assembler, the Spades assembler had longer assembled contigs with better N50 values. Hence, the assembly generated by Spades was selected for further downstream analyses.” and in the results section “The genome assembly method employed was Megahit v. 2023-07-12 tool, the genome representation was full….” - Explain this. 5) Subsystem information is given. Discuss about the gene or gene set responsible for B. velezensis B26. 6) Along with the table of pangenome analysis add plots showing the gene distribution. Is there any unique feature of the Bacillus velezensis B26 strain? Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Partly Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? No Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 21 Jul 2025 KIRAN KOLATHUR, Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Reviewer 2: The study delivers an organized, useful genome report on Bacillus velezensis B26 as it narrates about the possible use as a biocontrol agent and biofertilizer. The use of genome assembly and annotation pipeline pipelines and pangenome analysis provide more insights into the genome sequences. Response: We thank the reviewer for the comments There are some issues that will help to improve the depth of the manuscript. 1) Add a brief in the introduction by stating the importance of giving more emphasis on B26 compared to other Bacillus velezensis strains. Response 1: As mentioned, we have revised the introduction to highlight why strain B26 was chosen for genome sequencing. Specifically, we emphasize its unique origin (chicken carcass soil), its antifungal spectrum that includes both agricultural and human pathogens, and its potential for broader biotechnological applications. These features distinguish B26 from the most previously characterized B. velezensis strains and justify its genomic investigation. We have modified the introduction accordingly. 2) Elaborate the methodology section with detailed description of the work done and the tools used for performing them along with proper citations of the tools. Response 2: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 3 & 4). 3) No methodology is given for pangenome analysis, give a brief methodology of pangenome analysis with proper citations. Response 3: We thank the reviewer for the comment; we have added detailed description to the methodology (Page 4). Pangenome Analysis “Genome annotation was carried out using Prokka v1.14.6 (Seemann 2014). It generates standardized GFF3 files, which possess predicted coding sequences (CDS), rRNAs, tRNAs, and functional annotations. Pangenome analysis was subsequently performed using the Roary pipeline v3.13.0 (Page et al. 2015), which aids large-scale analysis of prokaryotic genomes by comparing orthologous genes across different strains. This analysis was carried out using minimum BLASTp identity threshold of 95% for gene clustering. This method facilitates the identification of both core genes (present in ≥99% of genomes), shell genes (present in ≥15% and <95%) and cloud genes (present in <15%), thus provides insights into genome conservation, diversity and evolutionary adaptation.” 4) Why two assembly pipelines have been used is not clear. Also, there are disparity of information provided in the methodology section “The quality of the genome assemblies produced by the Spades (Bankevich et al. 2012) and Megahit (Li et al. 2015) assemblers was evaluated using the Quast tool (Gurevich et al. 2013). Compared with the megahit assembler, the Spades assembler had longer assembled contigs with better N50 values. Hence, the assembly generated by Spades was selected for further downstream analyses.” and in the results section “The genome assembly method employed was Megahit v. 2023-07-12 tool, the genome representation was full….” - Explain this. Response 4: We thank the reviewer for pointing out the discrepancy in the methods section (Page no.4) and results section (Page no.5). We have modified as below “The genome assembly method employed was Megahit v. 2023-07-12 tool, which resulted in a complete genome representation with a coverage of 100.0x using Illumina sequencing technology. Genome assemblies were initially produced using both Spades and Megahit, and their quality was evaluated using Quast tool. Although Spades produced longer assembled contigs and higher N50 values, the assembly generated by Megahit exhibited more completeness and was better suited for downstream tools. Therefore, the Megahit assembly was preferred for genome annotation and comparative analysis”. 5) Subsystem information is given. Discuss the gene or gene set responsible for B. velezensis B26. Response 5: We thank the reviewer for the comment; we have added detailed as below (Results section, page no 6) Functional implications of subsystem genes “Subsystem-based genome annotation of B. velezensis B26 revealed gene sets that may contribute to its biocontrol and biofertilizer activities. The genome contains 43 genes related to stress response and 35 genes involved in virulence, disease, and defense, suggesting the strain’s ability to tolerate environmental stress and inhibit plant pathogens. Additionally, the presence of genes involved in phosphorus (10 genes), nitrogen (20 genes), and sulfur metabolism (6 genes) indicates the stain’s potential for nutrient acquisition and its tole as a biofertilizer. Altogether, these gene sets provide valuable insights into the biotechnological potential of B. velezensis B26”. 6) Along with the table of pangenome analysis add plots showing the gene distribution. Is there any unique feature of the Bacillus velezensis B26 strain? Response 6: We thank the reviewer for this helpful suggestion. In response, we have now included additional visualizations to support the pangenome analysis: A pie chart (now added as Figure 2) illustrates the distribution of gene categories across the pangenome, including core, shell, cloud, and soft-core genes. “Pangenome analysis of B. velezensis B26 identified a total of 5,576 genes. Among these genes, 3,480 genes were considered core genes, present in 99% to 100% of strains. Additionally,1, 143 genes were classified as shell genes, which were present in 15% to 95% of strains, while 953 genes were identified as cloud genes, found in fewer than 15% of the analysed strains (Figure 2, Table 3). These results indicate that a major portion of the B. velezensis B26 genome is conserved, while significant proportion of cloud genes (953 out of 5,576), may represent strain-specific or rare genes. Although many of the genes remains uncharacterized, these accessory genes might contribute to the distinctive biocontrol and plant growth promoting capabilities of B. velezensis B26”. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Basak S. Peer Review Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r384408) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-384408 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2025 Nallusamy S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 31 Mar 2025 | for Version 1 Saranya Nallusamy , Tamil Nadu Agricultural University, Coimbatore, Tamil nadu, India 0 Views copyright © 2025 Nallusamy S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript titled "The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer" presents the whole-genome sequencing of B. velezensis B26, a strain isolated from chicken carcass soil in Udupi, India. The study highlights the strain’s biocontrol potential and plant growth-promoting abilities, emphasizing its antifungal properties and enzymatic activities. Whole-genome sequencing was performed using Illumina technology, and genome annotation identified multiple functional genes linked to metabolic processes, stress response, and biocontrol properties. Bacillus velezensis has multiple applications in agriculture, biotechnology, and medicine. It plays a key role in plant growth promotion, biocontrol activity, and industrial applications. Its genome sequencing helps in understanding its potential for various applications, particularly in sustainable agriculture. This article reports the genome sequence of strain B26 to explore its potential in agriculture and biocontrol. Comparative genomics with other strains highlights unique genetic features that contribute to its biocontrol capabilities. Pan genome analysis reported the core and shell genes which is crucial for multiple applications in microbiology, biotechnology, and evolutionary studies. The study employed appropriate and standard methods like SPAdes for genome assembly and RAST & NCBI PGAP for genome annotation. The genome sequence and annotation data have been deposited in NCBI under accession number JAYKOV000000000. Genome sequencing highlights its potential for secondary metabolite production, making it a strong candidate for synthetic biology and metabolic engineering. Are the rationale for sequencing the genome and the species significance clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of the sequencing and extraction, software used, and materials provided to allow replication by others? Yes Are the datasets clearly presented in a usable and accessible format, and the assembly and annotation available in an appropriate subject-specific repository? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Plant genomics and transcriptomics, structural bioinformatics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (1) Author Response 12 Sep 2025 KIRAN KOLATHUR, Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences (MCOPS), Manipal Academy of Higher Education, Manipal, 576104, India Point-by-point response to Comments We have highlighted all the changes made in the manuscript. We thank the reviewers for the comments and tried to present the data in a detailed manner and the points raised are discussed below. Response 1 : We thank the reviewer for the insightful feedback. In the revised manuscript, we have modified the introduction section to more clearly articulate (Introduction, Page no.2) “Although multiple B. velezensis strains have been reported for plant growth promoting and biocontrol capabilities, most have been isolated from plant-associated environments. Despite these advances, several B. velezensis strains remain unexplored, particularly from non-plant environments. In this context, strain B. velezensis B26 was isolated from a chicken carcass soil, Manipal, Udupi, India (Location: 13.325922, 74.804554). Importantly, this strain demonstrated potent antifungal activity against the opportunistic fungal pathogen Candida albicans and the emerging fungal pathogen Saccharomyces cerevisiae (Ghurye et al. 2025). Therefore, the objective of this study is to perform whole-genome sequencing and comprehensive genomic analysis of B. velezensis B26 to identify genetic traits contributing to its biotechnological potential”. View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Nallusamy S. Peer Review Report For: The complete genome sequences of Bacillus velezensis B26: a promising biocontrol agent and biofertilizer [version 1; peer review: 1 approved, 2 approved with reservations] . F1000Research 2025, 14 :170 ( https://doi.org/10.5256/f1000research.176457.r365574) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/14-170/v1#referee-response-365574 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. 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