The bacterial strain used in this study was the patented strain Bacillus XT1 CECT 8661, licenced to Xtrem Biotech S.L., which was isolated from a rhizospheric soil sample in the south of Spain (Béjar et al., 2014). The strain was originally classified as B. methylotrophicus and it was later reclassified as a heterotypic synonym of B. velezensis. It was routinely cultivated on a nutrient broth and nutrient agar at 28°C. The phytopathogenic fungus B. cinerea was kindly provided by the University of Zaragoza (Spain) and was maintained on potato dextrose agar (PDA) and potato dextrose broth (PDB) and incubated at 24°C.

The antifungal activity of XT1 strain against B. cinerea was determined in both solid and liquid mediums. For the solid assay strain XT1 was spread on a 1 cm² area on one side of a PDA agar plate (at 1 cm from the plate wall) and an 8-mm-agar disc of the mycelium of fungi was deposited on the opposite side. The maximum and minimum values of the fungal mycelium radius obtained were measured after a 15-day incubation period at 25°C. The results were expressed as a percentage of the mycelium inhibition rate (IR% = A - B / A × 100) where A was the maximum value of the mycelium radius and B was the minimum value. For the antifungal assay in liquid medium, first, the time in which the antifungal activity was the maximum was determinate cultivating the strain XT1 in MOLP (medium optimal for lipopeptide production) (Ahimou et al., 2000) at different times (24, 48, 72, and 96 h), then the supernatant was tested against B. cinerea following the procedure described below. A 15-day culture of B. cinerea in PDB was crushed in a breaker and filtered with gauze, all under sterile conditions. The spore concentration was adjusted to 5 × 10⁷ conidia mL^-1 and penicillin G (2.5 mg mL^-1) and streptomicin (10 mg mL^-1) was added to the spore suspension. The experiment was carried out on multiwell culture plates (Cellstar^∗) with 48 wells where 900 μl of the spore solution was subjected to 300 μL of different times XT1 supernatant, obtained after centrifugation of the XT1 culture in MOLP at 10000 rpm 20 min. Inoculated medium with cycloheximide 50 μg mL^-1 was used as a positive control for growth inhibition, whilst PDB inoculated with spore suspension without treatment was considered as the negative control. The plates were incubated at 25°C for 7 days. The results were obtained by observing the presence or absence of fungal growth (Frikha-Gargouri et al., 2017).

Three different liquid media have been tested for lipopeptide production: MOLP medium (Ahimou et al., 2000); SG medium (Schaeffer et al., 1965; Leighton and Doi, 1971), and a commercial concentrated beef medium (CM) (ox concentrate 43% and yeast extract 24%). Lipopeptides were extracted according to Yazgan et al. (2001) with slight modifications. Briefly, the culture supernatant of strain XT1 was subjected to an organic extraction with one volume n-butanol three times using a decantation funnel. Then, the organic phase was evaporated with a vacuum concentrator.

Lipopeptide antifungal activity was tested by preparing 20 mL of lipopeptide solution at different concentrations: 20, 10, 8, 6, 4, 2, 1, and 0.5 mg mL^-1 (w/v in distilled water) in 50 mL tubes. Then 0.8 g PDA was added to each lipopeptide solution; the negative control was made with untreated PDA medium. The tubes were then sterilised and the medium were poured into 90 mm Petri plates. Next, a fungal plug of 15 days of mycelial growing B. cinerea was deposited in the middle of each plate and maintained at 25°C for 15 days. After that, the mycelial growth inhibition percentage was calculated after 15 day’s incubation by the comparison between the diameter of mycelial growing in the control plates and the treatments according to the following formula: mycelial growth inhibition = 100-[(diameter of control mycelium diameter of mycelium in lipopeptide medium / diameter of control mycelium) × 100] (Borah et al., 2016).

The minimal inhibitory concentration (MIC), defined as the smallest concentration of lipopetides that inhibits the fungal growth totally and the minimal fungicidal concentration (MFC), defined as the lowest concentration of lipopeptides capable of killing the fungi, was determined. The experiment was carried out in liquid medium according to the protocol described previously in the “Antifungal Activity of XT1 Strain” section. However, in this case 900 μl of the spore solution was exposed to 300 μL of lipopeptide dilutions 20, 10, 8, 6, 4, 2, 1, and 0.5 mg mL^-1. The plates were incubated at 25°C for 7 days. The results were obtained by observing the presence or absence of fungal growth. The whole content from each well, where there was no growth of B. cinerea, was passed to tubes with PDB medium and incubated at 25°C for 7 days for the determination of MFC (Frikha-Gargouri et al., 2017).

The antifungal activity of a lipopeptide solution (10 mg mL^-1) from XT1 was determined after 10, 30, and 60 min of heating at 100°C and after 20 min at 121°C. The stability was also determined at different pH in the range comprised between 3 to 12 (Ghribi et al., 2012). The antifungal activity was tested in liquid medium according to the method described above (Frikha-Gargouri et al., 2017).

Genes encoding NRPS production were amplified by polymerase reaction chain (PCR) from genomic DNA of the XT1 strain. PCR was carried out using the specific and degenerated primers described in Table 1. PCR amplifications were achieved in 50 μL mixtures with PCR buffer, 2 mM MgCl₂, 4 mM of each primer, 5U Taq polymerase, 0.2 mM of each dNTP, and 80–100 ng of genomic DNA. The amplification conditions were: 95°C for 5 min, 40 cycles of 94°C for 1 min, annealing temperature for 1 min, 72°C extension for 1 min; and a final extension at 72°C for 10 min. The annealing temperatures were 45, 43, 50, 53, and 50°C for, Af2/Tf1, As1/Ts2, BmyBF/BmyBR, ItuDF/ItuDR, and SrfA3/LicA3 primers, respectively. The amplification products were analysed by electrophoresis in a 2% (w/v) agarose gel.

The residue obtained from lipopeptide extraccion was dissolved in 10% methanol and analysed by high-pressure liquid chromatography (UPLC) (Acquity UPLC^® BEH300, Waters) coupled to a high definition mass spectrometry (SYNAPT G2 HDMS Q-TOF. Waters). Mass spectrometry was carried out by positive ionisation electrospray (ESI+). The obtained data were processed by the MassLynx^TM software (Waters).

Solid culture medium plates with the negative control treatments and lipopeptides 10 mg⋅mL^-1 from the previously experiment described in the “Antifungal Activity of the Lipopeptides Produced by XT1” section, were used to observe the morphology of B. cinerea. The samples were fixed and observed in a FIB-FESEM (CrossBeam NVision 40^®, Carl Zeiss SMT) Scanning Electron Microscope and Transmission Electron Microscope.

Antifungal activity of lipopeptides produced by XT1 was tested on grapes, strawberries, and tomatoes. B. cinerea was grown for 15 days on a PDA medium at 25°C and conidia were collected with sterile distilled water and filtered through four layers of sterile cheesecloth. The surface of the fruit was sterilised with 5% NaOCl for 5 min and rinsed three times with plenty of sterile water. Wounds of 3 mm were performed with a sterile scalpel on the surface of the grapes, then 15 μl of a 20 mg⋅mL^-1 solutions of lipopeptides were applied on the injury. One hour later, when the fruit was dried at room temperature, 15 μL of a suspension of B. cinerea 10⁸ conidia mL^-1 were inoculated into the wound. Strawberries and tomatoes were cut into slices and treated by spray with the same lipopeptide solution as in the previous case. Then, after 1 h, the fruit was infected by spraying with the conidia suspension of B. cinerea. All the treatments were incubated at 25°C, 70% humidity for 6 days. For each treatment, a total of nine fruits were used and three technical replicates were performed. Effect was measured and expressed as disease incidence (% of infected fruit).

The antioxidant activity was measured extracting the soluble phenols and by FRAP assay (ferric iron reducing antioxidant power assay). Total soluble phenols were extracted from 0.1 g of lyophilized fruits with 10 mL of 80% methanol and 0.1% hydrochloric acid. The mixture was placed in the dark at 4°C for 2 h. The supernatant was filtered and the extract was used for the determination of the phenol content and the FRAP assay. The amount of total phenols was determined according to the Folin-Ciocalteu’s procedure described by Ribereau-Gayon (1968) with slight modifications. The phenol content was estimated from a standard curve of gallic acid (GAE) and the results expressed as mg of gallic acid 100 g^-1 d.w (dry weight). In FRAP assay an extract of fruit (0.2 mL) (prepared as for phenol determination) was added to 2 mL of FRAP solution [0.25 mol L^-1 acetate buffer (pH 3.6) containing 1 mmol L^-1 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) and 20 mmol L^-1 FeCl₃.6H₂O] and incubated 5 min at room temperature measuring the absorbance at 593 nm. A standard of 1 mmol L^-1 L-ascorbic acid in distilled water was prepared. Results were expressed as mmol L^-1 of Fe²⁺ equivalents 100 g^-1 dry weight (Benzie and Strain, 1999).

Data obtained were subjected to ANOVA and multiple pair-wise comparisons were performed by the Duncan’s multiple range test.

Antifungal production in the supernatant was detected after 24 h of aerobic culture and reached its maximum at 72 h. The XT1 strain showed antifungal activity against B. cinerea in both solid and liquid media (inhibition rates of 60 and 100%, respectively).

The production of lipopeptides was tested in different culture media. The data showed that MOLP medium increased the lipopeptide production compared with other media. The best production yield was obtained with this medium (10 g L^-1); however, the production with other media such as SG and the commercial medium (CM) decreased the production of lipopeptide to 2.8 and 2.13 g L^-1, respectively.

The antifungal activity of Bacillus XT1 lipopeptides toward B. cinerea was also analysed. The results demonstrated that lipopeptides produced by XT1 inhibit the growth of B. cinerea. Inhibition rates of 72, 48, 30, and 19% of the mycelium diameter were observed for the concentrations of lipopeptides of 10, 6, 4, and 2 mg mL^-1, respectively, after 15 days of treatment (Figure 1). In general, lipopeptides from XT1 showed antagonistic activity against B. cinerea across a broad spectrum of concentrations in a dose response manner (Figure 1).

Lipopeptides from XT1 were also tested in multiwell culture plates with 48 wells to test the MIC and in culture tubes to test the MFC. As shown in Figure 2A lipopeptides concentrations tested ranging from 20 to 0.5 mg mL^-1 and a significant inhibitory effect of lipopeptides was observed at concentrations as low as 8 mg mL^-1 which corresponds to the MIC. The MFC was also 8 mg mL^-1 (Figure 2B).

The antifungal potency was not affected by any heat treatment. However, the antifungal potency of lipopeptides from XT1 was affected at pH 3 and pH 12. The optimum range of pH for the maximum antifungal efficacy was between 7 and 9.

Genomic analysis of Bacillus XT1 indicates that it contains gene clusters for non-ribosomal lipopeptide synthetases. Amplicons of the expected sizes were obtained for fengycin, surfactin, bacillomycin, and iturin genes srfA-C, fenC, srfA-A, bmyB, and ituD.

Electrospray quadrupole time-of-flight mass spectrometry (Q-TOF MS) analyses were carried out in this study to identify the metabolites produced by XT1. Figure 3 illustrates the total ion chromatogram (TIC) spectrum of the lipopeptide extract from a XT1 culture supernatant.

These analyses showed that the strain XT1 produces several forms of different lipopeptides. Four known surfactins with an acyl chain ranging from C12 to C15 were detected, whereas three known bacillomycins D (C14, C15, and C16) were also detected. Two peaks corresponding to the fengycin A and fengycin B were also observed (Table 2). There were no mass signals for iturin.

Quadrupole time-of-flight mass spectrometry analysis indicated five types of lipopeptides and two predominant compounds. This analysis detected a [M+H] peak at m/z 1022.6729 and afforded the molecular formula C₅₂H₉₁N₇O₁₃ (i-Fit = 20.6 and DBE = 10.5) and corresponding to surfactin and [M+H] peak at m/z 1463.8038 corresponding to fengycin A with the molecular formula C₇₂H₁₁₀N₁₂O₂₀ (i-Fit = 30.2 and DBE = 23.5) (Figures 4C and 4A, respectively).

Quadrupole time-of-flight mass spectrometry analysis indicated five types of lipopeptides and two predominant compounds. This analysis detected a [M+H] peak at m/z 1031.5431 (1053.5276 corresponds to the complementary sodium adduct molecular ion [M+Na]) with the molecular formula C₄₈H₇₅N₁₀O₁₅ (i-Fit = 101.1 and DBE = 16.5) corresponding to bacillomycin D, a [M+H] peak at m/z 1463.8038 with the molecular formula C₇₂H₁₁₀N₁₂O₂₀ (i-Fit = 130.3 and DBE = 23.5) corresponding to fengycin A and a [M+H] peak at m/z 1022.6729 and afforded the molecular formula C₅₂H₉₁N₇O₁₃ (i-Fit = 161.5 and DBE = 10.5) and corresponding to surfactin (Figures 4A, 4B, and 4C, respectively).

Microscopy data of B. cinerea mycelial growth treated with XT1 lipopeptides are shown in Figure 5. Scanning electron microscopy (SEM) analyses of B. cinerea mycelium treated with the MIC/MFC of lipopeptides (8 mg mL^-1) from XT1 showed important alterations of pathogen morphology. Hyphae treated without lipopeptides grew normally with straight appearance and their surfaces were smooth (Figure 5A). However, after exposure to lipopeptides, one of the most striking features was the appearance of structures of resistance (Figure 5B).

Transmission electron microscopy (TEM) images of normal hyphae treated without lipopeptides showed smooth surfaces, intact cells, well defined enclosing cell walls and the cellular organelles in normal arrangements (Figures 5C,E). TEM images of hyphae confirmed that in the treatment of B. cinerea with lipopeptide organelles were degenerated and gathered in clumps (Figures 5D,F).

The MIC/MFC of lipopeptide extract from XT1 (8 mg mL^-1) was applied in order to evaluate the protector effect with different fruit infected with B. cinerea. The lipopeptides were injected in grapes and sprayed onto strawberries and tomatoes. The level of disease infections decreased in all the fruit treated with XT1 lipopeptides.

The disease incidence in grapes, strawberries, and tomatoes treated with B. cinerea was 71, 100, and 100%, respectively. The disease reductions in fruit treated with XT1 lipopeptides were 100, 12, and 50%, respectively (Table 3 and Figures 6, 7). The results show that antifungal lipopeptide treated fruit reduced the disease symptoms significantly compared to non-treated fruits except in strawberries where the effect was not so evident. This reduction is higher for grapes.

The antioxidant activity was tested in grapes where the highest disease reduction was observed. The antioxidant activity of grape extracts, as estimated by the FRAP assay increased significantly with the inoculation of the MIC/MFC of lipopeptides produced by XT1 (Figure 8A). Although an increase in the antioxidant activity was also observed in grapes infected with the pathogen, the highest increases were observed in the treatment with the lipopeptides.

On the other hand, total phenol content also increased with the exposure of fruit to the lipopeptides but the increase is only significant in the uninfected grapes. In this case the increase of total phenol content is of 30% (Figure 8B).