Draft genome sequence of Bacillus amyloliquefaciens subsp. plantarum strain Fito_F321, an endophyte microorganism from Vitis vinifera with biocontrol potential

Bacillus amyloliquefaciens subsp. plantarum strain Fito_F321 is a naturally occurring strain in vineyard, with the ability to colonise grapevine and which unveils a naturally antagonistic potential against phytopathogens of grapevine, including those responsible for the Botryosphaeria dieback, a GTD disease. Herein we report the draft genome sequence of B. amyloliquefaciens subsp. plantarum Fito_F321, isolated from the leaf of Vitis vinifera cv. Merlot at Bairrada appellation (Cantanhede, Portugal). The genome size is 3,856,229 bp, with a GC content of 46.54% that contains 3697 protein-coding genes, 86 tRNA coding genes and 5 rRNA genes. The draft genome of strain Fito_F321 allowed to predict a set of bioactive compounds as bacillaene, difficidin, macrolactin, surfactin and fengycin that due to their antimicrobial activity are hypothesized to be of utmost importance for biocontrol of grapevine diseases.

B. amyloliquefaciens is ubiquitously distributed, Grampositive, rod-shaped, aerobic and endospore-forming bacteria. Together with other different Bacillus species from the Bacillus subtilis group, B. amyloliquefaciens has been reported to develop beneficial relationships with plants by promoting growth, improving resistance to environmental stress or having important biological activities for plant diseases control [12][13][14]. These species produce a variety of antimicrobial compounds, such as bacteriocins, antifungal compounds such as lipopeptides, namely iturins and fengycins, and siderophores [15,16]. Given its biocontrol potential, aligned with its physiological characteristics, such as UV light and heat resistant spores, long shelf life [17] and their advantageous characteristics for formulation, this microorganism is an environmental-friendly alternative to agrochemicals. Indeed, some of B. amyloliquefaciens strains are commercially available as biological control agents or generic plant growth promoters [18,19].
Altogether these characteristics prompted us to explore the B. amyloliquefaciens subsp. plantarum strain Fito_F321, a naturally occurring strain in vineyards that we have isolated from grapevine leaves in the Bairrada appellation -Portugal. In this study, we have obtained the draft genome sequence of B. amyloliquefaciens subsp. plantarum strain Fito_F321, analysed it and compared it with known genome sequences of representative related species, to gain knowledge on the genes involved in plant interaction with grapevine, as well as the genes conferring antimicrobial activity, and thus to evaluate the potential of this strain for further viticulture and agronomic applications.

Classification and features
Strain Fito_F321 was isolated from Vitis vinifera cv. Merlot at Bairrada appellation -Cantanhede, Portugal during the 2012 vine cycle. The samples collection was authorized by the private owner, who is fully acknowledged in this paper, and no specific permissions were required for this activity. Briefly, leaf tissues were homogenised in a sterile saline solution (0.85% NaCl) with a sterile pestle. The bacterial isolates were then obtained after plating the homogenised leaves on PDA medium and incubation for 24 h at 28°C. Sub-cultures were then carried out on the same culture medium until obtaining pure colonies that were further assigned to an isolation code. Microscopic analysis showed that strain Fito_F321 is a Gram-positive and rod shape microorganism (Fig. 1). The classification and general features of strain Fito_F321 are listed in Table 1.
Strain Fito_F321 was taxonomically identified by combining the analysis of the 16S rRNA gene sequence using both SILVA database [20] and EzBioCloud [21], and by genome comparisons. In SILVA the 16S rRNA sequence of strain Fito_F321 showed 99% of similarity to B. amyloliquefaciens subsp. plantarum AS43.3 (CP003838) and to Bacillus amyloliquefaciens subsp. plantarum SQR9 (CP006890), both non-type strains. A last updated available, reclassified these two strains as Bacillus velezensis. In the other hand, results obtained from EzBio-Cloud showed a 99.93% similarity of strain Fito_F321 to B. velezensis CR-502 (type strain). Given these results, the 16S rRNA gene sequence of strain Fito_F321 and  Phylum Firmicutes TAS [71][72][73] Class Bacilli TAS [74,75] Order Bacillales TAS [72,76] Family Bacillaceae TAS [72,77] Genus Bacillus TAS [72,78] Species Bacillus amyloliquefaciens , 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 [80] other representative related and type strains species available on GenBank [22] were then selected for phylogenetic analysis (Fig. 2). The phylogenies were generated using the Neighbor-Joining method [23] and evolutionary distances were computed by the Maximum Composite Likelihood method [24] with 1000 bootstrap replicates. Phylogenetic analysis was conducted in MEGA 7.0 [25].

Extended feature descriptions
The physiological and biochemical features of B. amyloliquefaciens subsp. plantarum strain Fito_F321 were analysed to explore the mechanisms behind its antagonistic potential, namely its ability to produce hydrolytic enzymes, presence of siderophores and phosphate solubilization. The tolerance to pH and salinity conditions were also tested. All tests were performed in triplicate. Given the enzymatic production, the amylase, cellulase, lipase, pectinase, protease and urease activity were screened under in vitro conditions by using specific culture media. Results were expressed as positive activity, when a clear halo around strain colony was observed, and the enzymatic index (EI) was calculated through the ration between the average diameter of the degradation halo (clear zone) and the average diameter of the colony growth. The strain Fito_F321 was able to produce all enzymes under in vitro conditions except ureases. Amongst them, cellulases had the higher enzymatic index (10.50 ± 0.20), followed by pectinases (5.44 ± 0.39). This strain was also able to produce siderophores and to solubilise phosphate under in vitro conditions. Overall, these phenotypic features are of high interest, since they are intimately involved in the biocontrol action. Further, this strain was able to grow between pH 6.0-9.0, with an optimal growth at pH 6.5, and grew under up to 6% NaCl. Interestingly, the morphology of Fito_F321 colonies was altered with salt concentration, and colonies became smaller with increasing NaCl concentration in the culture media. It is recognised that excess of soil salinity reduces both plant growth and yield thus, salt tolerant strains may confer plant tolerance against these abiotic stresses [27].

Genome sequencing information
Genome project history B. amyloliquefaciens subsp. plantarum strain Fito_F321 was selected for sequencing as a part of an ongoing project that focuses on the deep characterization of the grapevine-associated microorganisms and their natural antagonistic potential. Thus, its specific antagonistic activity against important grapevine pathogens, such as grey mould or grapevine trunk diseases, together with its physiological and biochemical unique features such as the ability to growth on a range of pHs and salinity conditions, the production of siderophores, the phosphate solubilisation and the high enzymatic activity, were the drivers for its sequencing. Sequencing of the wild-type B. amyloliquefaciens subsp. plantarum strain Fito_F321 genome was performed at Biocant, Portugal and the draft genome sequencing project has been deposited at DDBJ/ENA/GenBank under the Bioproject PRJNA360208, Biosample ID SAMN06205151 and the accession number MSYT00000000. The version described in this paper is version MSYT01000000. A summary of the project is shown in Table 2. The GenBank accession numbers are shown in parentheses. Sequences were aligned using ClustalW 1.6 [81]. The phylogenetic tree was constructed by using the Neighbor-Joining method [23] and evolutionary distances were computed by the Maximum Composite Likelihood method [24] within MEGA 7.0 [25]. There were a total of 1380 positions in the final dataset. Numbers at the nodes are bootstrap values calculated from 1000 replicates; only bootstrap values > 70 are indicated Growth conditions and genomic DNA preparation B. amyloliquefaciens subsp. plantarum strain Fito_F321 was grown in Luria-Agar medium at 28°C for 24 h. The genomic DNA was extracted by using the Wizard Genomic DNA Purification kit (Promega, Madison, USA), following the standard protocol for Gram-positive bacteria. The DNA integrity was checked by 0.8% agarose gel electrophoresis, the concentration was determined by using Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific) and quality assessed with NanoDrop spectrophotometer (Thermo Scientific, USA). Prior to genome sequencing, the quality of the isolated DNA and the molecular identity was confirmed by the sequencing of the 16S rRNA gene.

Genome sequencing and assembly
A DNA library was built from 1 mg of high-quality genomic DNA. Briefly, genomic DNA was fragmented by nebulization and the sequencing adaptors ligated to create double stranded DNA libraries. After quality assessment by using high sensitivity DNA analysis kit (Agilent Technologies) and library titration with KAPA library quantification kit (Kapa Biosystems), the final genome fragments were pyrosequenced in the GS FLX+ system (Roche, 454 Life Sciences), using GS FLX Titanium Sequencing Kit XL+ at Biocant (Cantanhede, Portugal). The sequencing reads were assembled with the GS Assembler, version 2.9 (Roche, 454 Life Sciences) using the default parameters. The sequencing produced 285,879 reads with an average length of 580 bases. The final assembly yielded − 54 contigs, a genome coverage of 41-fold and generated a genome of 3.86 Mb (Table 2).

Genome annotation
The structural and functional annotations were performed using the Prokaryotic Genome Prediction pipeline [28]. Prediction of non-coding RNA genes and miscellaneous features were performed with the PGP pipeline by using tRNAscan-SE [29], RNAMMer [30] and PILERCR [31]. Coding sequences were predicted with Prodigal [32] and automatically corrected by PGP pipeline based on the Gen-ePRIMP algorithm [33]. Functional annotation of protein coding genes was carried out under Prokaryotic Genome Prediction pipeline in InterProScan [34] against Pfam database [35], TIGRFAM [36], Hamap [37], PIRSF [38], PRINTS [39], SMART [40], SUPERFAMILY [41], ProSite [42] databases and RPS-BLAST against Clusters of Orthologous Groups (COG) database [43]. The product name of the identified coding sequences (CDSs) was assigned by using Pfam database, TIGRFAM and COG annotation [44]. The CDSs that were not assigned to a specific product with these databases were named as hypothetical proteins.

Genome properties
The genome statistics are provided in Table 3, and genome visualisation was performed on Artemis version 16.0.0 [45]. The draft genome sequencing of B. amyloliquefaciens subsp. plantarum strain Fito_F321 was distributed across 54 contigs with an estimated genome size of 3,856,229 bp and an average of GC content of 46.53%. The genome analysis showed that Fito_F321 strain' genome contained 3657 protein coding genes predicted, 95 RNAs and without any CRISP elements. The predicted protein encoding genes showed a total length of 3,424,790 bp which represents 88.81% of the total  The total is based on either the size of genome in base pairs or the total number of genes in the predicted genome genome size. Of these, 2697 proteins were assigned to a COG functional category across 20 categories (Table 4). The majority of protein-coding genes were assigned as function unknown (264 proteins) and general function prediction only (306 proteins), which all together represents 15.59% of the protein encoding genes ( Table 4). The proteins not assigned in COGs (960 proteins) represent 26.25% and the amino acid transport (269 proteins), transcription (227 proteins) and carbohydrate transport and metabolism (191 proteins) were the followed categories with 7.36%, 6.21% and 5.22%, respectively. Interestingly, the defense mechanisms included 43 protein-coding genes, which represent about 1% of the annotated genome, and included β-lactamase (class C), multi-drug efflux pumps as ATP-binding cassette (ABC) transport and the multidrug and toxic compound extrusion (matE), antimicrobial peptides (AMPs) and lanthionine synthetase component C-like protein (LANCL).

Plant-bacteria interactions
The genome of B. amyloliquefaciens subsp. plantarum strain Fito_F321 was also analysed for genes contributing directly or indirectly for plant-growth promotion (PGP) and biocontrol activities (Additional file 1: Table S1):

Colonisation, adhesion, and movement of bacteria across plant root
It is recognized that a crucial feature of a successful plant growth promoter microorganism, as well as of a The total is based on the total number of protein coding genes in the genome biocontrol agent relies on its competence for plant colonisation, notably at roots level [46]. Overall a colonisation process may involve a plant surface adhesion/ attachment and a bacterial biofilm formation [47]. plantarum strain Fito_F321 also displays a swarming motility, which allows a rapid surface colonization [48]. Herein, genes encoding for swrA (BVY13_02415), swrB (BVY13_ 00300) and swrC (BVY13_18860) were predicted. Overall, the swarming motility requires both flagella biosynthesis and surfactant production [48]. Other genes such as hook-associated proteins -flgK (BVY13_02315), fliD (BVY13_02345), or hag flagellin (BVY13_02340) can be expressed in response to root exudates secreted by plant roots. Bacterial flagellins can interact with host and are involved in elicitation of general plant immune response [49]. Furthermore, this strain may also produce biofilms. Indeed, the sporulation transcription factor spo0A (BVY13_05345) was here identified, and has an important role on biofilm formation, by repressing the expression of AbrB [50,51].
Spo0A is essential for surface-adhered cells prior to transition to a three-dimensional biofilm structure [50,51].

Plant-growth promotion
B. amyloliquefaciens subsp. plantarum strain Fito_F321 encodes proteins that enhance the plant growth such as those involved in the biosynthesis of indole-3-acetic acid, a plant auxin. Herein, genes encoding for tryptophan, the main precursor of IAA [52], were identified and include trp genes such as trpA (BVY13_06245), trpB (BVY13_06240) or trpE (BVY13_06220). Going forward, the synthesis of volatile compounds, as 2,3-butanediol and acetoin, released by some Bacillus strains, may also enhance the plant growth promotion and be involved in the eliciting induce systemic resistance [53]. Herein, a set of genes that catalyse the 2,3-butanediol pathway, such as butanediol dehydrogenase bdhA gene (BVY13_ 17360), acetolactate synthase als and alsD (BVY13_ 14285, BVY13_09195) and acetolactate decarboxylase alsD (BVY13_14290) were identified. Regarding nitrogen fixation, several nif genes were not identified among Fito_F321 genome, though other genes involved in nitrate reduction pathways were predicted. Further, a scaffold protein nifU (BVY13_03720) and a cysteine desulfurase nifS, which are involved in the Fe-S cluster assembly and required for the activation of nitrogenase, where identified. Another feature of B. amyloliquefaciens subsp. plantarum strain Fito_F321 is the nirK gene responsible for the nitric oxide synthase, a signalling molecule that protects Gram-positive strains from antibiotics and oxidative stress [54,55]. Regarding the phosphate solubilisation, no pqq genes were predicted for this bacterial strain. These genes encode a pyrroloquinoline quinine, a PGP agent involved in the phosphate solubilisation process [56]. However, Fito_F321 strain displayed a phytase activity (BVY13_15080) that contributes to the subsequent use of phosphorous by the plant. This activity is important for the plant growth under phosphate limitation [57,58]. These predictions are in agreement with the in vitro results obtained by using the Pikovskaya culture medium, which unveiled the ability of Fito_F321 strain to solubilise phosphate. Another important feature is that this strain encodes an inositol 2-dehydrogenase (BVY13_ 11530), important for the inositol catabolism. Inositol or other inositol derivatives are end-products of phytate degradation, abundant in the plant rhizosphere and can be use by microorganisms as carbon sources [57,59]. An indirect PGP effect can also be mediated through the siderophores production. Siderophores are iron (Fe)specific chelating small molecules secreted by bacteria and have high affinity with ferric ionic from soils and surrounding environments [60], thus increasing the bioavailability of Fe for plants, by promoting its solubilisation. On other hand, the siderophores production by BCAs may also confer a clear competition for the available carbon sources, allowing for plant colonisation in detriment with other microorganisms. The B. amyloliquefaciens subsp. plantarum strain Fito_F321 encodes genes for ABC transporters for iron and iron uptake, which was further supported by the genome analysis using antiSMASH 3.0 [61] that also predicted siderophores.

Antimicrobial resistance
In the meantime, the strain Fito_F321 encodes antimicrobial resistance genes (Additional file 1: Table S1) such as bacitracin (bcr -BVY13_11500), fosfomycin (fosB -BVY13_12675) and tetracycline (BVY13_08560) [62]. Regarding bacitracin and fosfomycin resistance genes, these are antibiotics that interfere with peptidoglycan synthesis of the bacterial cell wall [63,64]. Given the bacitracin, herein multiple genes encoding for ABC transporter system were identified, which are associated with bacitracin resistance. Tetracycline antibiotics inhibit the bacterial ribosome, and thus, protein synthesis [65]. In Fito_F321 strain genome, the resistance to tetracycline occurs via active efflux (BVY13_08560).  [68]. This analysis allowed for the calculation of the intergenomic distances between genomes and the probability of belonging to the same species. The general comparison is shown in Table 5 and the intergenomic distances, through the DDH estimate and ANI, are in Table 6

Conclusions
In this study, we have characterized the genome of B. amyloliquefaciens subsp. plantarum strain Fito_F321, a Table 6 Comparative analysis of the in-silico genome distances between B. amyloliquefaciens subsp. plantarum strain Fito_F321 withboth B. amyloliquefaciens and B. velezensis, through the DNA-DNA hybridization (DDH method) and average nucleotide identities (ANI) The nucleotide sequences were download from GenBank and the respective accession numbers are shown in the table. In-silico DNA-DNA hybridization (DDH) was calculated by using the Genome-to-Genome Distance Calculator (GGDC 2.1) [67] and ANI values were computed through pairwise genome comparison by using the MUMmer software [68]. Values with grey colour are of a below cut-off (< 95%) natural grapevine-associated microorganism, which was isolated from grapevine leaves. Given its genomic and physiological characteristics, this microorganism may provide an interesting model to study the plant-microbial interactions and their role in grapevine protection. The intergenomic distances amongst genomes showed that B. amyloliquefaciens subsp. plantarum strain Fito_F321 is highly close to type strain B. amyloliquefaciens subs. plantarum FZB42, with a DDH estimate value of 85.90% and a ANIm value of 94.14%, and more distant to the type strain B. amyloliquefaciens subsp. amyloliquefaciens DSM7.
The predicted gene compounds of B. amyloliquefaciens subsp. plantarum strain Fito_F321 such as bacillaene, difficidin, macrolactin, surfactin, fengycin and siderophore, together with other protein-coding genes herein presented, are of utmost importance for its biocontrol activities and could explain its positive plant-microbial interactions, as well as its role on the natural protection of vineyard. Thus, these gene clusters suggest that the strain Fito_F321 can produce bioactive compounds of biocontrol value, which represents a source of novel bioactive compounds and that may be essential for the grapevine protection in the pursue of a more sustainable viticulture.

Additional files
Additional file 1: Table S1. General overview of genes involved in bacterium-plant interaction in B. amyloliquefaciens subsp.