Complete genome of Pseudomonas chlororaphis strain UFB2, a soil bacterium with antibacterial activity against bacterial canker pathogen of tomato

Strain UFB2 was isolated from a soybean field soil in Mississippi and identified as a member of Pseudomonas chlororaphis. Strain UFB2 has a broad-spectrum antimicrobial activity against common soil-borne pathogens. Plate assays showed that strain UFB2 was especially efficient in inhibiting the growth of Clavibacter michiganensis 1–07, the causal agent of the devastating bacterial canker of tomato. Here, the complete genome sequence of P. chlororaphis strain UFB2 is reported and described. The strain UFB2 genome consists of a circular chromosome of 6,360,256 bp of which 87.86 % are protein-coding bases. Genome analysis revealed multiple gene islands encoding various secondary metabolites such as 2,4-diacetylphloroglucinol. Further genome analysis will provide more details about strain UFB2 antibacterial activities mechanisms and the use of this strain as a potential biocontrol agent.


Introduction
Bacterial strains of Pseudomonas chlororaphis are aerobic Gram-positive bacteria and many of the strains possess a wide-spectrum antifungal activity against soil-borne plant pathogens [1][2][3][4][5]. P. chlororaphis strains have been reported to be efficient plant-growth-promoting bacteria, which can be used as inoculants for biofertilization, phytostimulation and biocontrol [6]. The use of P. chlororaphis strains as biocontrol agents is promising because they are capable of producing a variety of antimicrobial secondary metabolites including phenazine-1-carboxamide, 2-hydroxyphenazine, pyrrolnitrin, hydrogen cyanide, chitinases and proteases [6][7][8]. Moreover, P. chlororaphis is considered to be nonpathogenic to humans, wildlife, or the environment according to U.S. environmental protection agency (EPA) [9]. Antimicrobial activities and low risks to animals and the environments have made the bacterium P. chlororaphis highly potential biocontrol agents in agriculture [8,10]. A genome-wide research and analysis could provide useful information about the mechanisms of how P. chlororaphis protects plants against soil-borne phytopathogens. Currently, the whole genomes of a few P. chlororaphis strains that exhibit antifungal activity have been sequenced. These include P. chlororaphis strain PA23 that can protect canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum [2,11], and P. chlororaphis PCL1606 that was isolated from avocado rhizosphere and exhibited biocontrol activity against soil-borne phytopathogenic fungi [1]. In addition, another functionally-uncharacterized strain, P. chlororaphis subsp. aurantiaca JD37, was recently sequenced (NCBI reference sequence: NZ_CP009290.1). Genome sequences of P. chlororaphis strains with significant antibacterial activity have not been reported previously.
Strain UFB2 was isolated from a soybean field soil in Mississippi. Preliminary analysis of the 16S rRNA gene indicated that it is a member of P. chlororaphis. Plate assays indicated P. chlororaphis strain UFB2 has a broad spectrum of antimicrobial activities, especially against bacterial canker pathogen of tomato: Clavibacter michiganensis [12,13]. Greenhouse trials demonstrated both living cells and Fig. 1 Image of P. chlororaphis UFB2 cells and plate assay of UFB2 antibacterial activity against Clavibacter michiganensis 1-07. The plate bioassay was conducted as described by Scholz-Schroeder and colleagues [44]  Evidence codes -IDA Inferred from Direct Assay, TAS Traceable Author Statement (i.e., a direct report exists in the literature), NAS Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [67] culture extract of strain UFB2 can be used for disease management of bacterial canker of tomato. In this study, the P. chlororaphis strain UFB2 complete genome sequence and annotation are reported. The gene islands within strain UFB2 genome that encode various secondary metabolites, including antimicrobial compounds, are also described. The detailed description of the strain UFB2 genome will shed light into further studies of biocontrol effectiveness and applications of Pseudomonas species.

Classification and features
Strain UFB2 was isolated from rhizosphere soil sample collected from soybean field near Cleveland, Mississippi, USA, where healthy soybean plants were found growing in charcoal rot disease patch. Phylogenetic analyses based on multilocus sequence typing [14] (gyrB, rpoB, rpoD and 16 s rRNA) revealed that strain UFB2 belongs to Pseudomonas chlororaphis (Fig. 2). Strain UFB2 is rod-shaped, motile, non-spore-forming Gram-negative bacterium in the order Pseudomonadales of the class Gammaproteobacteria. UFB2 cells are approximately 3.0 ± 0.8 μm in width and 0.9 ± 0.3 μm in length (Fig. 1). The strain is relatively fast-growing, forming approximately 1 mm opaque yellowish colonies after overnight incubation at 28°C on nutrient-broth yeast extract agar [15]. Strain UFB2 can also be grown on rich media such as LB [16] and PDA, as well as M9 minimal medium [17]. Phenotypic characterization of strain UFB2 was carried out using the API 50CH system as recommended by manufacturer. According to the result, strain UFB2 could utilize almost all carbon sources in API 50CH tests, including D-glucose, D-galactose, L-rhamnose, Dmannitol, D-raffinose, D-fructose, D-arabinose, D-ribose, L-arabinose, L-xylose and D-xylose, but not potassium gluconate.
Plate bioassays demonstrated that strain UFB2 possesses significant antibacterial activity against a broad array of plant bacterial pathogens. Other than Clavibacter michiganensis 1-07, the tested bacteria sensitive to strain UFB2 also include Erwinia amylovora [18,19], Burkholderia glumae [20], Ralstonia solanacearum Rso [21,22] and Erwinia carotovora WSCH1 [19,23]. Of the tested plant pathogenic bacteria, the Gram-positive bacterium Clavibacter michiganensis 1-07, the pathogen causing bacterial canker of tomato [24], is most sensitive to strain UFB2 with a radius of 28 ± 1 mm clear inhibitory zone (Fig. 1). In addition, the growth of fungal pathogen Geotrichum candidum Km, which causes sour rot of citrus fruits, tomatoes, carrot and some vegetables [25], can also be inhibited by strain UFB2. To test the field biocontrol efficacy of strain UFB2, greenhouse . Phylogenetic analyses were performed using MEGA, version 6.06 [51]. The tree was built using the Neighbor-Joining method [52]. Bootstrap analysis with 1000 replicates was performed to assess the support of the clusters experiments were set up according to the method described by Lu and Ingram [26]. Preliminary data showed the control efficacies of both strain UFB2 culture extract and living cells on bacterial canker of tomato are equivalent to that of streptomycin at the recommended rate for plant disease management. The genome of strain UFB2 was sequenced with the aim to identify the genes associated with the antimicrobial characters. The information about the genome sequence of strain UFB2 is summarized in Table 1, and its phylogenetic position is shown in Fig. 2.

Genome sequencing information
Genome project history P. chlororaphis strain UFB2 was selected for sequencing because of its significant antimicrobial activities and its potential as a biocontrol agent for agricultural use. Genomes of three P chlororaphis strains have been sequenced as of May 2015. Sequencing of the whole genome of strain UFB2 makes more data available for genome comparison and analysis within P. chlororaphis species.
The genome project is deposited in the Genomes On-Line Database [27] and the NCBI BioProject database [28]. The annotated genome is publicly available from the Intergrated Microbial Genomes Database [29] under the accession number Gp0111981 and GenBank under accession number CP011020. A summary of the project information is provided in Table 2.
Growth conditions and genomic DNA preparation P. chlororaphis strain UFB2 was cultured in liquid NBY medium overnight at 28°C in a shaker at 220 rpm. The genomic DNA was extracted from 50 mL of the culture using the Wizard Genomic DNA Purification Kit (Promega Corporation, Madison, WI, USA). Totally approximately 900 μg of DNA were obtained with an OD260/ 280 of 1.9. The DNA sample was used for library

Genome sequencing and assembly
One standard library with an average insert size of 400 bp and three mate pair libraries with an average insert size of 2,000 bp, 5,000 bp and 8,000 bp were prepared and sequenced on the Illumina MiSeq instrument according to the manufacturer's instructions. The genome was de novo assembled using a method as described by Durfee et al. [30] using DNAStar Seqman NGen (Version 12, DNASTAR, Inc. Madison, WI U.S.). The standard library and 2,000 bp mate pair library were selected for the de novo assembly. A total of 30 million short reads were scanned and extracted from the raw data files as input data. The short reads were preprocessed by Seqman NGen to trim adaptors and filter lowquality reads. Automatic Mer size and a minimum match percentage of 98 % were selected. 29 million short reads were assembled into 29 contigs and SeqMan Pro (Version 12, DNASTAR, Inc. Madison, WI U.S.) was used to order the contigs in one scaffold according to the mate pair data. The first round assembled sequence was then used as a template for a complete reassembly. The 2,000 bp and 8,000 bp mate pair data were incorporated to proofread the first assembly and to maximize coverage and quality. Adjacent contigs, if possible, were merged. Remaining gaps were filled by PCR and Sanger sequencing. No contigs that might correspond to plasmids remained unassembled. IslandViewer [31] was used to predict and identify genomic islands.

Genome annotation
Automatic annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline [32], which combines gene calling algorithm with similarity-based BLASTn comparison of genomes was visualized by BRIG [53] and UFB2 genome the image was generated with Circos [54] gene detection approach to predict protein-coding genes, structural RNAs (5S, 16S, 23S), tRNAs and small noncoding RNAs. Additional gene prediction analysis and functional annotation were performed by the Integrated Microbial Genomes platform [29].

Genome properties
The complete genome of P. chlororaphis strain UFB2 consists of one circular chromosome of 6,360,256 bp with a GC content of 62.03 %. 5,556 genes were identified from the genome, of which 5,473 are protein coding genes. 90 of the 5,556 genes were predicted to be pseudogenes or partial genes. The genome encodes 1 noncoding RNA, 5 rRNA operons and 65 tRNAs. Seventy genomic islands ranging from 4 kbp to 43.5 kbp were also identified throughout the strain UFB2 genome, among which majority of the islands encode hypothetical proteins. The genome features of P. chlororaphis strain UFB2 are summarized in Tables 3  and 4, and the circular chromosome of strain UFB2 is shown in Fig. 3.

Insights from the genome sequence
Blast research of P. chlororaphis strain UFB2 genome against P. syringae pv. syringae B728a (NC_007005), P. putida KT2440 (NC_002947), P. chlororaphis strain PA23 (NZ_CP008696), P. aeruginosa PAO1 (NC_002516), P. fluorescens Pf0-1 (NC_007492) and P. sp. UW4 (NC_019670) genome revealed multiple unique gene regions which were only found in the strain UFB2 genome (Fig. 3). The BLASTn atlas showed noticeable genome diversity of strain UFB2 when compared to other Pseudomonas species. Seventy genomic islands ranging from 4 kbp to 30 kbp were also identified throughout the strain UFB2 genome, indicating significant horizontal gene transfers occurred during the evolution of strain UFB2 to better adapt the environment it inhabited. P. chlororaphis strain UFB2 harbors an intact phl gene cluster (VM99_23970-23995), which is responsible for biosynthesis of the antimicrobial compound 2,4-diacetylphloroglucinol [33,34]. 2,4-diacetylphloroglucinol is an especially efficient agent against soil borne fungal plant pathogens [35]. The phl gene cluster is involved in the Pseudomonas antifungal activity against Clavibacter The total is based on the total number of protein coding genes in the genome michiganensis 1-07 [36]. Hydrogen cyanide [37,38] biosynthesis gene homologs were also identified in strain UFB2 genome. The production of hydrogen cyanide by Pseudomonas species helps protect plants from soil-borne fungal pathogens [39,40]. Biosynthetic gene clusters of common Pseudomonas species-produced antibiotics such as phenazine [41], pyrrolnitrin [42] and pyoluteorin [43] were not identified in strain UFB2 genome. Biosynthetic gene clusters of common toxins that contribute to plant and animal pathogenicity and/or virulence of Pseudomonas species were also searched for within strain UFB2 genome. The toxin biosynthetic gene cluster that were not identified in strain UFB2 genome include the phytotoxin lipopeptide syringomycin [44], tobacco wildfire spotting causal agent tabtoxin [45], bacterial canker of kiwifruit causal agent phaseolotoxin [46], plant-hormone-mimic toxin coronatine [47], and cytotoxic agent pederin [48]. Strain UFB2 genome harbors homolog genes to those in the bacterial apical necrosis causal agent mangotoxin [49] biosynthesis gene cluster. However, mboC gene homolog that is required for mangotoxin production [50] was not identified in strain UFB2 genome. Overall, the lack of the key pathogenicity/virulence genes in strain UFB2 further indicates that strain UFB2 has a great potential as a biocontrol agent.

Conclusions
The complete genome sequence of P. chlororaphis strain UFB2 is described in this report. The strain UFB2 was originally isolated from the rhizosphere of a healthy soybean plant growing in a group of plants exhibiting charcoal rot disease in Mississippi. This strain possesses significant antimicrobial activities against a wide range of plant pathogenic bacteria and fungi. It is evident that the genome of P. chlororaphis strain UFB2 harbors the complete gene set for production of the antimicrobial compounds 2,4-DAPG and HCN, which may largely contribute to its antimicrobial activities. However, gene homologs required for biosynthesis of all the known toxins to plants, such as syringomycin, tabtoxin, phaseolotoxin, tolaasin, coronatine, or pederin, were absent from the strain UFB2 genome. The genome sequence of P. chlororaphis strain UFB2 will help in understanding genetic mechanisms of the antimicrobial activity studies that are useful for development of biologically-based disease management in agriculture.