Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera - BMC Microbiology

14 Sep.,2022


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Bacterial strain and growth conditions

The strain GL174 was previously isolated from surface-sterilized tissues of Vitis vinifera cv. Glera and identified as Bacillus licheniformis [19]. This strain was cultivated routinely in Nutrient Broth (NB) or Nutrient Agar (NA) at 28 °C.

Grapevine cutting re-inoculation for endophytic proficiency validation

Sterile stem cuttings of grapevine cultivar Glera, approximately 20 mm long, each bearing an axillary bud, were obtained from in vitro plants grown in Murashige and Skoog (MS) solid medium. The GL174 strain was grown overnight in NB medium at 28 °C under shaking. Cells were harvested by centrifugation at 1500×g and resuspended in sterile 10 mM MgSO4 with a final optical density of 0.1. The lower extremity of each cutting was dipped for 1 min into the bacteria suspension. Negative controls were performed by dipping cuttings into sterile MgSO4. Inoculated cuttings were cultivated in MS/2 solid medium, without sugar, at 24 °C, with 25 μmol m − 2 s − 1 light intensity and a photoperiod of 8 h of light and 16 h of dark. Four weeks after cutting inoculation, the presence of GL174 bacteria inside the plants was tested. Sections of plant stems of each cutting were treated to extract endophytes, as previously described. Three different dilutions of the ground plant material were plated onto NA medium and incubated at 28 °C for 48 h [19]. After this preliminary indication, the endophytic colonization of Glera cuttings by GL174 was assessed using a GFP-tagged strain, GL174::gfp, and laser scanning confocal microscopy.

Transformation of Bacillus licheniformis GL174

Plasmid pUTgfp2X contains a mini-Tn5 transposon delivery system, a PpsbA-RBS-gfp2X cassette: two gfp genes, repeated in tandem, plus the additional 35 bp region containing the Ribosome Binding Site, located downstream the constitutive psbA promoter [15].

Bacteria were grown in NA plates added with 50 mg/L Kanamycin for 1 week at 28 °C. In order to transform the strain, electroporation was performed following a modified protocol from Xue et al., [21]. A colony was inoculated in 5 mL of NB 0.5 M sorbitol and grown until it reached 0.9 OD600. Cells were cooled in ice and centrifuged at 4000 g for 10 min at 4 °C. Bacteria were resuspended in 1 mL and washed 3 times with 500 μL of cold electroporation medium (10% glycerol, 0.5 M sorbitol and 0.5 M mannitol). After washing, cells were resuspended in 60 μL of electroporation medium and mixed with 2 μL of pUTgfp2X plasmid (389 ng/μL). Bacteria were incubated 10 min in ice and transferred in pre-chilled electroporation cuvettes. They were electroporated using a Gene-Pulser (Bio–Rad Laboratories, Richmond, CA) set at 2.5 kV, 200 Ω with a resulting time constant of 4.5–5.4 ms. Immediately after electroporation, 1 mL of NB was added to the transformed cells; then bacteria were incubated at 28 °C under shaking for 3 h. After incubation, 100, 200 and 300 μL of bacteria were plated on NA solid medium supplemented with 30 mg/L Kanamycin and incubated at 28 °C. The resulting colonies were analyzed under a fluorescence stereomicroscope (excitation at 488 nm) to check bacteria fluorescence.

Inoculation of Glera cuttings with Bacillus licheniformis GL174::gfp2x

The ability of these strains to colonize and survive within Glera tissues was investigated inoculating cuttings and analyzing the plants by Laser Scanning Confocal Microscopy (LSCM). We used apical cuttings with 2 leaves obtained from 1 month-old plants grown in vitro. One fluorescent colony was inoculated in NB with 30 μg/μL of kanamycin and grown overnight. Bacteria was centrifuged and resuspended in 5 mL of MgSO4 10 mM. The optical density of the suspension was measured and bacteria were diluted with the same buffer to a cell density of 106 cells/mL. One drop (5 μL) of the suspension was placed on the surface of the solid medium MS half-concentrated [22] contained in a vented Magenta box. In correspondence of the drop, one apical cutting was planted in the medium. As negative control, 3 cuttings were inoculated only with MgSO4 buffer. In order to check colonization, we inoculated 6 plants for each strain: 3 cuttings were sampled 30 days after inoculation. Stem samples were 3/4 cm long, inclusive from the inoculation point and the first node. One control plant was also analyzed to control the absence of any fluorescence bacteria inside the tissues.

Laser scanning confocal microscopy of inoculated Glera tissues

Stem explants were first surface-sterilized for 2 min with sodium hypochlorite 1%, rinsed with 70% ethanol and then washed 3 times for 10 min with sterile deionized water. Stems and roots were sliced longitudinally with a blade, and leaf fragments were observed directly. Plant explants were mounted on a slide with a solution of 50% glycerol and covered with a coverslip. Confocal laser scanning microscopy was performed with a Leica SP5 system using an excitation laser of 488 nm (Argon laser) and collecting the emission band of 515–560 nm for GFP fluorescence and of 695–765 nm for chlorophyll fluorescence.

In vitro antifungal effects of endophytic bacteria

The evaluation of in vitro antifungal effects was performed testing strain GL174 against some grapevine-pathogenic fungi like Phaeoacremonium aleophilum, Phaeomoniella spp, Botryosphaeria spp, Botrytis cinerea and more generic plant pathogens Sclerotinia sclerotiorum and Phytophthora infestans. The endophyte was streaked horizontally in the middle of a Petri dish with PCA medium; plates were incubated at 28 °C for 48 h to obtain bacterial growth. After bacterial growth, two inocula of fungal mycelium were placed on the same plate, one on the right and one on the left of the endophyte. Plates with both bacteria and fungi were incubated for one week at 28 °C. High-resolution pictures (600 dpi) of the plates were obtained and the antifungal effect of the bacterium was evaluated comparing the inhibition of mycelium expansion in the presence of the endophyte strain, and measuring the mycelium radius in the direction of the bacterium using photo-editing software. For each plate we calculated the average radius of the mycelia using the following equation: Rm = ((R1-Rin) + (R2-Rin))/2, where Rin is the fungal inoculum radius. An inhibition index was calculated as percentage of reduction of fungal growth comparing Rm and the mycelium radius of control plates containing fungi without bacteria.

In vivo antagonism assay

The biocontrol activity against Botrytis cinerea was tested using two sets of plants: the first set was represented by 60 day-old plants in soil pots and the second one by 60 day-old plants inoculated with GL174 when propagated as cuttings (see above the described inoculation protocol); to mime the endophyte colonization and check their direct effect, the abaxial sides of leaves were infiltrated with a 103 cells/mL bacteria suspension (in 10 mM MgSO4 buffer) by means of a syringe without needle; as negative control some leaves were injected with sterile MgSO4 buffer. On these two groups of plants, the antagonism tests were performed on detached leaves [23] and on leaves in planta.

In the first test, detached fully expanded leaves from plants, both those infiltrated with GL174 and those from previously GL174-inoculated plants, were placed on wet paper and were challenged by placing a mycelial plug (diameter 10 mm) of Botrytis cinerea, collected from a 7-day-old PDA plate, on the middle of the leaves. The trays with the challenged leaves were covered to keep a high relative humidity for the fungus development.

In the second test, leaves of the plants, both those infiltrated with GL174 and those from previously GL174-inoculated plants, were challenged in planta with mycelial plugs as described above. Plants were kept in plastic bags in a growth chamber.

Non-treated leaves were included in all the experimental conditions: not infiltrated leaves and leaves from not inoculated plants were challenged with the fungus. Negative controls were also set providing sterile medium plugs to the leaves to check any detrimental effect of the inoculation method independently from the fungus.

The effects of the fungus infection on every set of treated leaves were evaluated after 1 week of infection collecting pictures of the leaves and measuring the surface of the brown lesions by means of the software ImageJ. The mean of the values recorded on bacterized leaves was compared with the mean of the surface values obtained by non-treated leaves. Five plants were used in each treatment: for each treatment three/four leaves were used and data are expressed as damaged area (cm2) and asterisks indicate statistically significant differences among treatments (T student test; p = 0.05).

Liquid chromatographic-mass spectrometric analysis

Strain GL174 was analyzed in liquid culture for production of lipopeptides. To obtain the crude extract of LPs, bacteria were inoculated in 500 mL of NB, and grown for 96 h at 28 °C under shaking. Bacteria were removed by centrifugation at 4000 g for 20 min. The supernatant was acidified to pH 2 with 6 N HCl. A white precipitate was obtained and collected by centrifugation at 8000 g. The supernatant was discarded and the solid precipitate extracted with 10 mL of methanol using the volume ratio solid-solvent 1:2. Suspensions were stored at 4 °C for 1 h and the clear methanol phase was transferred into a vial for the electrospray ionization mass spectrometry (ESI-MS) analysis [24, 25].

Genome sequencing and sequence analysis

Genomic DNA of Bacillus licheniformis GL174 was extracted using UltraClean® Microbial DNA Isolation Kit (MoBio, Solana Beach CA, USA) from 5 mL of an overnight culture. Afterwards, Genomic DNA was fragmented and sequenced using ION Proton (Life technologies©) sequencing technology. Genome assembly was performed with the Newbler program.

Sequencing data, assembly and gene prediction were submitted to a public database and are available at BioProject database ( with accession number PRJNA274883. The gene annotation process was performed using the annotation pipeline implemented in the BASys bacterial annotation system ( so that all the coding sequences were assigned to a COG (Cluster of Orthologs) functional class. In addition, the identified coding sequences were compared with the InterPro database ( for double annotation of the protein functions. Among all the identified protein functions, we isolated the sequences related to chemotaxis and motility, plant wall degrading enzymes and plant colonization, iron nutrition and metabolism, phosphate nutrition and metabolism, nitrogen uptake and metabolism, lipopeptides and other secondary metabolites biosynthesis and oxidative stress response.