The complete genome sequences of <i>Bacillus velezensis</i> B26: a promising biocontrol agent and biofertilizer

Venkatesh Kamath B; Sandeep Mallya; Subrahmanyam Volety Mallikarjuna; Kiran Kumar Kolathur

doi:10.12688/f1000research.160546.2

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Revised

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

[version 2; peer review: 3 approved]

Venkatesh Kamath B¹, Sandeep Mallya², Subrahmanyam Volety Mallikarjuna¹, Kiran Kumar Kolathur ¹

PUBLISHED 08 Jul 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

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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, 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 defense. The raw sequence data generated in this work has been deposited in the NCBI database and the genome sequence is available under the accession number JAYKOV000000000. This genomic data provides insight into the biocontrol ability and plant-growth promoting capabilities of B. velezensis B26.

Keywords

Genome sequencing, Biocontrol agent, Plant growth promotion, Pangenome analysis

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 2; peer review: 3 approved]. F1000Research 2025, 14:170 (https://doi.org/10.12688/f1000research.160546.2) 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)

Revised Amendments from Version 1

We have modified introduction part, removed Figure 1 and added new Figure 2 (Pangenome analysis pie chart).

See the authors' detailed response to the review by Surajit Basak
See the authors' detailed response to the review by Diego Sauka
See the authors' detailed response to the review by Saranya Nallusamy

Introduction

Bacillus velezensis is a valuable bacterium that is extensively used for several 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 plant 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 the 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 (Zaid 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 plants growth promotion and biocontrol capabilities in several B. velezensis strains. These studies provide key insights into organism’s capability to produce antimicrobial compounds, enzyme secretion, and secondary metabolites that are involved in plant growth. The 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 involved in 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 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 that contributing 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 QIAamp DNA Mini Kit (Qiagen). The integrity of the gDNA was assessed spectrophotometrically to measure the A260/280 ratio and its concentration was measured using Qubit broad range dsDNA quantification kit (Thermo Fisher Scientific). 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 quality control were verified using FastQc v0.11.9 (Simon Andrews et al. 2010). FastQC report provided individual details on sequence counts, sequence quality histograms, per sequence quality scores, per base sequence content, per sequence GC content, per Base N content, sequence length distribution, sequence duplication levels, overrepresented sequences, adapter content and status checks. The details of individual reports were then added using MultiQC v1.12, to understand the quantity of data obtained for each of the paired read files for an individual sample (Ewels et al. 2016) (Supplementary file 1). Adapter trimming and quality filtering were performed using fastp tool (v0.12.4) (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 (high quality, adapter-free reads) were used as input for downstream processing.

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, the Megahit assembly exhibited better genome completeness and compatibility with annotation tools employed in the study. Thus, the Megahit assembly was selected for further downstream analyses.

RAST Server: Rapid annotations using subsystems technology

The RAST server is an automated platform developed for 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.

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.

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.

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. 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 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.

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.

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 annotated proteins.

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 1).

Figure 1. 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).

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 strains 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 stains potential for nutrient acquisition and its role as a biofertilizer. Altogether, these gene sets provide valuable insights into the biotechnological potential of B. velezensis B26.

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, 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 analyzed 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.

Figure 2. Pie chart represents group of genes. The pie chart represents groups of genes that were classified into various groups based on their prevalence in the pangenome analyzed.

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, the presence of gene cluster coding for stress response, virulence, and defense indicates that B. velezensis B26 can effectively inhibit plant pathogens. Further, the presence 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.

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Version 2

VERSION 2 PUBLISHED 06 Feb 2025

Author details Author details

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)

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https://doi.org/10.12688/f1000research.160546.2

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© 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.

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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 2; peer review: 3 approved]. F1000Research 2025, 14:170 (https://doi.org/10.12688/f1000research.160546.2)

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Surajit Basak, ICMR - National Institute for Research in Bacterial Infections, Beliaghata Kolkata, India

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Diego Sauka, Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina

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Diego Sauka, Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina

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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 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.

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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

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Reviewer Report 09 Jun 2025

Surajit Basak, ICMR - National Institute for Research in Bacterial Infections, Beliaghata Kolkata, India

Approved with Reservations

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 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.

CITE

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
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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
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Reviewer Report 31 Mar 2025

Saranya Nallusamy, Tamil Nadu Agricultural University, Coimbatore, Tamil nadu, India

Approved

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 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.

CITE

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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
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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
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VERSION 2 PUBLISHED 06 Feb 2025

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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

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23 Aug 2025 | for Version 2

Surajit Basak, ICMR - National Institute for Research in Bacterial Infections, Beliaghata Kolkata, India

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The revised manuscript may be accepted for indexing.

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23 Aug 2025 | for Version 2

Diego Sauka, Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina

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Approved

The authors have successfully addressed all the requests and comments made by this reviewer.

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No competing interests were disclosed.

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Microbiology; genomics; biological control of pests

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09 Jun 2025 | for Version 1

Diego Sauka, Instituto Nacional de Tecnología Agropecuaria (INTA), Hurlingham, Argentina

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Approved With Reservations

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.

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

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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.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

27 Views

09 Jun 2025 | for Version 1

Surajit Basak, ICMR - National Institute for Research in Bacterial Infections, Beliaghata Kolkata, India

27 Views Cite this report Responses(1)

Approved With Reservations

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.

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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”.

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Competing Interests

No competing interests were disclosed.

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Reviewer Report

20 Views

31 Mar 2025 | for Version 1

Saranya Nallusamy, Tamil Nadu Agricultural University, Coimbatore, Tamil nadu, India

20 Views Cite this report Responses(1)

Approved

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.

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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”.

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Competing Interests

No competing interests were disclosed.

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

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

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Methods

Whole genome analysis of bacteria

Sample preparation workflow

Tapestation analysis of NGS libraries

Analysis workflow

Reads quality control

Genome assembly and annotations

RAST Server: Rapid annotations using subsystems technology

Pangenome analysis

Results

Whole genome analysis of B. velezensis B26

Table 1. Genome annotation data for Bacillus velezensis B26.

Table 2. Assembly statistics for B. velezensis B26.

Identification of subsystem features by The RAST Server: Rapid Annotations using Subsystems Technology (RAST)

Figure 1. Subsystem statistics of the B. velezensis B26 genome annotations.

Functional implications of subsystem genes

B. velezensis B26 pangenome analysis

Figure 2. Pie chart represents group of genes. The pie chart represents groups of genes that were classified into various groups based on their prevalence in the pangenome analyzed.

Table 3. Pangenome analysis of B. velezensis B26.

Conclusion

Ethics and consent

Data availability

Extended data

Acknowledgements

References

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