Draft genome sequence of Bacillus velezensis 2A-2B strain: a rhizospheric inhabitant of Sporobolus airoides (Torr.) Torr., with antifungal activity against root rot causing phytopathogens
© The Author(s). 2017
Received: 24 March 2017
Accepted: 24 November 2017
Published: 5 December 2017
A Bacillus velezensis strain from the rhizosphere of Sporobolus airoides (Torr.) Torr., a grass in central-north México, was isolated during a biocontrol of phytopathogens scrutiny study. The 2A-2B strain exhibited at least 60% of growth inhibition of virulent isolates of phytopathogens causing root rot. These phytopathogens include Phytophthora capsici, Fusarium solani, Fusarium oxysporum and Rhizoctonia solani. Furthermore, the 2A-2B strain is an indolacetic acid producer, and a plant inducer of PR1, which is an induced systemic resistance related gene in chili pepper plantlets. Whole genome sequencing was performed to generate a draft genome assembly of 3.953 MB with 46.36% of GC content, and a N50 of 294,737. The genome contains 3713 protein coding genes and 89 RNA genes. Moreover, comparative genome analysis revealed that the 2A-2B strain had the greatest identity (98.4%) with Bacillus velezensis.
Root rot causing microorganisms are among the most devastating phytopathogens of many horticultural crops resulting in considerable financial loss worldwide. Some of these pathogens include the oomycete Phytophthora capsici , and the fungi Fusarium solani , Fusarium oxysporum , and Rhizoctonia solani . Biocontrol strategies are important alternatives to keep some plant pathogens at low levels in affected crops, particularly when evaluating the risk of the use of pesticides on human health and the environment, and the social pressure to have innocuous horticultural food products. Biocontrol agents including bacterial strains that possess biocide activity against phytopathogens can also have the ability to invoke a systemic resistance (induced systemic resistance) in the host plant [1, 2]. In some cases, these bacterial strains are also able to promote the plant growth by inducing the biosynthesis of phytohormones [3, 4]. The rhizosphere is the area around the plant root that is inhabited by a unique population of microorganisms. The rhizospheric space is characterized by plant root exudates and usually by a high density and diversity of microorganisms. The root exudates have positive and negative effects in the interactions in the rhizosphere [5–7]. Colonizers of the rhizosphere are a great variety of microorganisms including bacteria that commonly have a friendly interaction with the plant host, suppressing at the same time some phytopathogens, and in some cases promoting plant growth [2, 7].
In seeking new options for biocontrol alternatives against phytopathogens, the genomic and biotechnological advances allows the deciphering of the molecular processes that regulate and induce the expression of many genes of plant-associated microorganisms. This will increase the possibilities for newer options of biocontrol agents with improved efficacy to deal with specific pathological problems of important crops.
In the present study we sampled soil and roots of cultivated and wild plants from Zacatecas state in the central-north region of Mexico, and isolated the bacterial strains from rhizosphere of Sporobolus airoides (Torr.) Torr. The bacteria reported in this study has the capacity to inhibit the growth of each of the four virulent isolates of the pathogens P. capsici , R. solani , F. solani and F. oxysporum , which are the causal agents of root rot in chili pepper crops. Levels of indoleacetic acid and biosynthesis of siderophores were analyzed, along with the induction of NPR1, a key gene controlling local resistance and systemic acquired resistance with multiple roles in plant immunity [8, 9]. In addition, the expression of the sesquiterpene cyclase gene involved in the isoprenoids pathway for the biosynthesis of phytoalexin capsidiol  was analyzed. The bacterial strain 2A-2B from this rhizosphere was selected for genome sequencing. A draft genome assembly of 3.953 MB was obtained and deposited in the NCBI GenBank (biosample SAMN05772828 and accession MLCV00000000), and in the Genomes OnLine Database (GOLD) with the accession Gp0177877.
Classification and features
Bacillus velezensis strain 2A-2B is a Gram-positive bacterium, with rapid growth rate in LB liquid medium reaching the stationary phase after 13 h at 28 °C. In contrast, the growth rate of this strain was much slower in LB solid medium with the stationary phase attained at 24 h. The colonies in solid LB medium were observed as circular with pulvinated elevation and had filiform beige-opaque margins.
Classification and general features of Bacillus velezensis strain 2A-2B 
Species Bacillus velezensis
pH range; Optimum
16% (w/v) NaCl
N 22° 49′ 12.354′´
W 102° 41′ 51.59299′´
Extended feature descriptions
Genome sequencing information
Genome project history
The Bacillus velezensis 2A-2B strain was selected due to its capacity to inhibit the growth of four pathogens, the causal agents of root rot in chili pepper. These include: P. capsici , R. solani , F. solani , and F. oxysporum . The 2A-2B strain was not pathogenic in “mirasol” chili pepper when inoculated in the roots.
illumina paired-end library
SPAdes Genome Assembler 3.8.1
Gene calling method
NCBI Prokaryotic Genome Annotation Pipeline
GenBank Date of Release
Source Material Identifier
Biotechnological, Agricultural, Biocontrol
Growth conditions and genomic DNA preparation
A sample taken from a colony of the 2A-2B strain was inoculated in 5 ml of LB liquid medium and cultured for 24 h at 150 rpm at 28 °C. The bacterial culture was centrifuged at 3500 g and the bacterial pellet was subjected to DNA extraction and purification based on the bacterial DNA isolation CTAB protocol . The purity and concentration of the DNA was analyzed by agarose gel electrophoresis and in a Qubit 2.0 Fluorometer (Invitrogen). One nanogram of DNA in 5 ul of water was used to construct the genome libraries tagmented by PCR for Illumina sequencing. The quality and size of fragments in the libraries was verified in a Bioanalyzer (BioAnalizer 2010, Agilent Technologies). The libraries were subjected to standard normalization and 15 pM were used in the sequencing process.
Genome sequencing and assembly
Purified genomic bacterial DNA was used to prepare libraries following Nextera Kit instructions (Illumina, San Diego Ca.). High-throughput sequencing was done under sequencing by synthesis protocol (MiSeq, Illumina) with a 2 × 75 paired-end run at the Sequencing Laboratory at the Unidad de Ciencias Biológicas, Universidad Autónoma de Zacatecas, México. SPAdes Genome Assembler 3.8.1  was used to assemble the genome and the quality of the assembly was evaluated using QUAST 4.1 .
The protein-coding genes, structural RNAs and tRNAs in the draft genome were predicted using the NCBI Prokaryotic Genome Annotation Pipeline  and the GENIX Automated Bacterial Genome Annotation Pipeline .
% of Total
Genome size (bp)
DNA coding (bp)
DNA G + C (bp)
Protein coding genes
Genes in internal clusters
Genes with function prediction
Genes assigned to COGs
Genes with Pfam domains
Genes with signal peptides
Genes with transmembrane helices
Number of genes associated with general COG functional categories
Translation, ribosomal structure and biogenesis
RNA processing and modification
Replication, recombination and repair
Chromatin structure and dynamics
Cell cycle control, Cell division, chromosome partitioning
Signal transduction mechanisms
Cell wall/membrane biogenesis
Intracellular trafficking and secretion
Posttranslational modification, protein turnover, chaperones
Energy production and conversion
Carbohydrate transport and metabolism
Amino acid transport and metabolism
Nucleotide transport and metabolism
Coenzyme transport and metabolism
Lipid transport and metabolism
Inorganic ion transport and metabolism
Secondary metabolites biosynthesis, transport and catabolism
Mobilome: prophages, transposons
General function prediction only
Not in COGs
Insights from the genome sequence
Regarding the exhibited antifungal activity of this B. velezensis strain, we found genes in the genome that code for proteins of the Bac operon and the oligopeptide permease OppA. The Bac proteins are involved in the biosynthesis of bacilysin, a non-ribosomally synthesized dipeptide that is active against a range of bacteria and some fungi. The proteolysis of this dipeptide releases the non-proteinogenic amino acid L-anticapsin, which functions as a competitive inhibitor of glucosamine synthase and can result in the lysis of fungal cells [27, 28]. Also, a beta-glucanase and an endoglucanase are present in the genome of this bacterium. Similarly, surfactin synthetase gene which is present in the 2A-2B strain genome, adds to the capacity of this bacterium to contribute in the antifungal activity against the root rot causal agents. Furthermore, the surfactin lipopeptide of Bacillus subtilis is well documented as elicitor of induced systemic resistance in plants [29–31]. In the genome of the 2A-2B strain of B. velezensis , with a total of 3713 predicted-protein coding genes, the 1.98% corresponds to defense genes; and the 2.5% of genes corresponds to secondary metabolites biosynthesis. In these two functional categories of genes, a possible role in fungal inhibition may be important. In addition, the sesquiterpene cyclase and NPR1 genes induced in chili pepper plantlets, during the 2A-2B strain root inoculation experiments, suggests that this lipopeptide is sensed by the signaling pathway in the plant’s defense system.
In other hand, in relation to the root bacterial colonization, the CheA and CheY genes are present in the genome of 2A-2B strain. These genes encode proteins that act as a two component system of bacterial chemotaxis, which is a response to chemical signals for controlling the direction of flagellar rotation [32–34]. With this two-component chemotaxis system and other plant exudate chemoreceptors, this bacterium could effectively reach the root tissue and proceeds with the plant tissue colonization.
In the carbohydrate transport and metabolism category, 250 genes (6.3% of total genes) were predicted in the 2A-2B strain genome including the PTS trehalose-specific enzyme IIBC component, xylose isomerase and a number of genes related to glucose metabolism. This suggests that the 2A-2B bacterium possesses a broad battery of genes coding for enzymes required to release a variety of carbon sources including some from plant exudates in the rhizosphere.
The B.velezensis 2A-2B strain has a standard genome size compared to others Bacillus species where the mean size fluctuate around 3.7 MB; the 2A-2B strain contains a genome of 3.96 MB. It is remarkable that the 2A-2B strain has only one 16S and one 23S rRNAs, whereas other Bacillus species possesses seven, nine or ten of each.
In this study, we obtained and characterized a draft genome of Bacillus velezensis , the 2A-2B strain isolated from Sporobolus airoides (Torr.) Torr. rhizosphere. The assembled genome contains a total of 3891 genes of which a high number of genes correspond to amino acid and carbohydrate transport and metabolism categories, and transcription and translation functions; whereas the 5.44% of genes have unknown functions. The antagonist characteristics of this bacterium against several fungal phytopathogens, could be explained in part in a number of described genes in the genome that are involved in the biosynthesis of compounds with toxic effects on fungal cell structures.
In comparative analysis with bacterial genomes in the NCBI DataBank using the draft genome and using the16S rRNA sequence, the 2A-2B strain was classified as Bacillus velezensis . The genome of the 2A-2B strain contains genes related to antifungal activity and systemic induced resistance in plants. A battery of genes is present that are involved in carbohydrate transport and metabolism, habilitating the bacterium for the assimilation of a diversity of carbon sources in the rhizosphere. Moreover, two genes, one with five copies, related to a transport system could be involved in the accumulation of glycine betaine with function of osmoprotection. Further analysis of the genome and specific genes of the 2A-2B strain will offer a better understanding of its biology, and the development of novel strategies in the biotechnological use of this bacterium.
The authors thank Miguel Adame for help in the taxonomic identification of the grass plant from which the 2A-2B B. velezensis strain was isolated.
This study was supported with partial funding by CONACYT, grant INFR-2014-226,183, and Redes Temáticas de Colaboración Académica-PROMEP-SEP, 2013 to Saul Fraire; and partial funding by DGAPA-UNAM, grant IA203313 and IN214917 to Julio Vega.
SFV conceived and designed the study, the annotation, and the NCBI and GOLD GenBanks submission. MAR, VBH and SFV the plant and soil sampling in field. VBH, IMR, and YDCR performed the microorganism isolation, microscopic studies and characterization in lab. IMR, YDCR and AAG performed the DNA and genome library preparation and genome sequencing. JVA and IMR accomplished the genome assembly. AFM and SFV performed the COGs and phylogenetic studies. SFV and JVA in drafting the manuscript and fulfilled the final manuscript. All authors read and approved the final manuscript.
The authors declare they have no competing interests.
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