Healthy leaves were randomly collected from O. violaceus at different locations grown in Nanjing University of Science and Technology, Nanjing, China. To isolate the phyllosphere fungi, mixed leaves (10 g) were placed in sterile flasks containing 30 mL of potassium phosphate buffer (PBS, 0.1 M, pH 7.0), and sonicated for 10 min at 40 KHz power level at 20°C using a ultrasonic cleaner (Lv et al., 2013). The mixture was then vortexed for 10 min and the resulting suspension was serially diluted and spread onto Czapek-dox agar plates (0.3% NaNO₃, 0.1% K₂HPO₄, 0.05% MgSO₄·7H₂O, 0.05% KCl, 0.001% FeSO₄, 3% sucrose, and 1.5% agar; Han et al., 2016) supplemented with streptomycin sulfate (250 mg L⁻¹) and ampicillin (250 mg L⁻¹) to inhibit bacterial growth. The plates were incubated at 24°C and the purified isolates were stored at −80°C for further use. Colony morphology was determined after 14 days of incubation on Potato Dextrose Agar (20% potato, 2% dextrose, 2% agar, PDA; Bai and Shaner, 1996) at 24°C in darkness. Fungal internal transcribed spacer (ITS) region (ITS1, 5.8S rRNA and ITS4) (Figueroa et al., 2014; Zhang T. et al., 2016), was amplified using primers ITS-1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS-4 (5′-TCCTCCGCTTATTGATATGC-3′; Zhang T. et al., 2016). PCR were assayed in 25 μL of mixtures with 2.5 μL of 10 × buffer (with Mg²⁺) (Sangon Biotech, China), 1 μL of dNTP (2.5 mM) (Sangon Biotech, China), 0.2 μL of enzymes (Sangon Biotech, China), 1 μL of primers and 0.5 μL of DNA. The applied temperature cycle was: initial denaturation at 95°C for 5 min, 30 cycles of 45 s of denaturation at 95°C, 45 s of annealing at 55°C, 1 min of extension at 72°C, and a final extension for 10 min at 72°C. PCR products were purified employing a SanPrep extraction kit (Sangon Biotech, China). The sequencing reaction was assayed through the Applied Biosystems 3730XL automated sequencer system. The sequences were compared with Genbank databases followed by sequence alignment. Phylogenetic trees were performed by neighbor-joining algorithm, using MEGA version 4.1 (Tempe, AZ, USA).

The bacterial strains such as Agrobacterium tumefaciens KYC55 (pJZ372, pJZ 384, pJZ410) and P. aeruginosa PAO1 (wild type) were used in this study based on their QS dependent phenotypes. P. aeruginosa PAO1 was kindly provided by Prof. Qianhong Gong, Ocean University of China (Qingdao, China). A. tumefaciens KYC55 was kindly presented by Prof. Jun Zhu, University of Pennsylvania (Philadelphia, USA). A. tumefaciens KYC55 was propagated in Luria Bertani medium (LB, Sangon Biotech, China) containing 1% tryptone, 0.5% yeast extract, and 1% NaCl (Wu et al., 2015) without antibiotics at 30°C. P. aeruginosa PAO1 was cultivated in LB at 37°C unless otherwise specified.

P. cucumerina was cultivated in PDA medium for 7 days until the mycelium was widespread over the plate. Two hundred and fifty milliliters Erlenmeyer flasks containing 100 mL of Fungus No. 2 medium (2% sorbitol, 2% maltose, 1% glutamine, 1% glucose, 0.3% yeast extract, 0.05% tryptophan, 0.05% KH₂PO₄, 0.03% MgSO₄, pH 6.4, autoclaved at 121°C for 15 min; Xin et al., 2007) were inoculated with the fungal strain. The flasks were incubated at 28°C at 140 rpm for 15 d. After incubation, the fungal biomass and the fermentation broth were separated by centrifugation at 10,000 rpm for 15 min. The fungal biomass was lyophilized using a lyophilizer (LGJ-25C, Four Ring Science Instrument Plant Beijing Co., Ltd., Beijing, China) at −50°C for 15 h. The fungal biomass and the fermentation broth were extracted with the same volume of ethyl acetate (EtOAc). The solvent was pooled and eliminated under reduced pressure and residues were dissolved in methanol (MeOH) and stored at −20°C.

P. cucumerina extract was tested against the selected bacterial strains to determine their minimum inhibitory concentration (MIC) according to Clinical and Laboratory Standards Institute (CLSI) (2015) with an inoculum of about 1–5 × 10⁵ CFU mL⁻¹. Serial two-fold dilution (0.16–10 mg mL⁻¹) of extract was performed in Müller-Hinton broth (Sangon Biotech, China). The MIC was the lowest concentration of extract that inhibited visible cell growth. For growth measurement, overnight cultures of P. aeruginosa PAO1 were subcultured into LB medium to achieve OD₆₂₀ of 0.05. The cultures were then supplemented with extract with varying concentrations and incubated at 37°C at 150 rpm for 15 h. In this study, the extract was firstly set into a high concentration with an amount of MeOH. The extract was then diluted into different concentrations with MeOH. All test extracts with the varying concentrations were supplemented with the same volume. The same amount of distilled water (0) and MeOH served as negative controls. Growth was evaluated by measuring OD₆₂₀ using a microplate reader (Biotek Elx800, USA) in triplicate.

To assess the inhibitory effect of P. cucumerina extract on the levels of C4-HSL and 3-oxo-C12-HSL produced by P. aeruginosa, the overnight cultures of P. aeruginosa PAO1 were diluted 1:1,000 into 50 mL of LB and incubated at 37°C at 200 rpm for 48 h. The extract was added as treated group while the same amount of water and MeOH were used in control groups. After incubation, cells were separated from culture fluids by 15-min centrifugation (12,000 rpm, 4°C). Cell-free culture fluids were extracted with an equal volume of acidified ethyl acetate (0.5% formic acid) for three times. The solvent was pooled and eliminated under reduced pressure at 35°C and residues were dissolved in MeOH and stored at −20°C prior to analysis. The levels of AHLs were determined using LC-MS/MS as described previously (Morin et al., 2003). AHLs were analyzed using HPLC system (SHIMADZU) equipped with a Inertisl-C18 column (250 × 4.6 mm, 5 μm; Shimadzu, Tokyo, Japan). Mobile phase A was formic acid (0.1%) and ammonium formate (5.0 mM) in water. Mobile phase B was MeOH. The flow rate was set as 0.4 mL min⁻¹. The injection volume was 10.0 μL. The gradient was set as: 1–5 min, 20–50% B; 5–20 min, 50–90% B. The eluates were determined by a ThermoFinnigan LCQ Classic system (San Jose, CA) using the positive mode. The nebulizer was set as 15 psi. The drying gas was set as 7 mL min⁻¹ and the temperature was maintained at 300°C. Full-scan MS was from m/z 100 to 1,000. The peaks corresponding to C4-HSL and 3-oxo-C12-HSL were identified according to their fragment MS ions and the retention time of commercial standards (Sigma-Aldrich, USA) for each AHL. The area of the ion m/z 102 was selected to quantify each AHL due to its specificity and better signal-to-noise ratio. The extracted ion chromatograms were employed for area calculation. This is a relative quantification approach for AHLs without the need of standard curve. Data were normalized to the level of the water control for relative quantification.

P. aeruginosa PAO1 biofilms were quantified based on the tube method (Panda et al., 2016). Overnight, P. aeruginosa cultures were diluted 1:50 into 200 μL of Trypticase Soytone broth (TSB, 1.7% tryptone, 0.3% soy protone, 0.25% glucose, 0.5% NaCl, 0.25% KH₂PO₄, pH 7.2, OD₆₂₀ ≈ 0.1; Cycoń et al., 2016) with varying concentrations of extract. Distilled water and MeOH served as negative controls. The tubes were incubated at 37°C for 24 h without agitation. After incubation, excess broth and the planktonic cells were removed. For quantification of the total biofilm biomass, the tubes were washed with PBS (pH 7.2) and then dried at 60°C for 30 min. The biofilms were stained with 200 μL of 0.1% crystal violet for 15 min and then rinsed off by distilled water and bounded crystal violet was re-solubilized in 100% ethanol. The OD was determined at 570 nm using a microplate reader (Biotek Elx800, USA). The biofilm biomass was normalized to the level of the water control for relative quantification.

Subsequently, the effect of extract on exopolysaccharide (EPS) was determined using the sonication method (Liu et al., 2007; Gong et al., 2009; Kim and Park, 2013). Briefly, P. aeruginosa PAO1 was incubated in AB medium (300 mM NaCl, 50 mM MgSO₄, 10 mM potassium phosphate, 0.2% vitamin-free casamino acids, 1% glucose, 1 mM L-arginine; Kim and Park, 2013) in the absence and presence of extract (0.25–1 mg mL⁻¹) at 37°C for 24 h without agitation. The cultures were then centrifuged at 12,000 rpm for 15 min to remove the cells. The cell pellets were resuspended in 10 mL of KCl (0.01 M) and then sonicated with a sonicator (YJ96-II, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) with four cycles of 5 s on and 5 s pauses at 3.5 Hz power level. The sonicated suspension was centrifuged (12,000 rpm, 4°C) once again for 20 min. The supernatant was filtered through a 0.22-μm filter (Millipore, Fisher Scientific). For carbohydrate quantification, 50 μL of the filtrate coupled with 150 μL of concentrated sulfuric acid were added into a 96-well polystyrene microtiter plate and incubation at room temperature for 30 min. The mixture was added with 5% phenol and incubated at 90°C in the water bath for 5 min. The amount of carbohydrate was quantified by measuring OD₄₉₀ and the relative EPS production was calculated.

The overnight cultures of P. aeruginosa PAO1 were diluted 1:50 into 200 μL of TSB, and incubated statically in 1.5-mL tube at 37°C for 24 h. The biofilm was then treated with varying concentration of extract for 24 h. After incubation, both the culture medium and the planktonic cells were removed. For quantification of the total biofilm biomass, the tubes were washed with PBS for three times and then dried at 60°C for 30 min. The biofilm biomass was determined employing the crystal violet staining method as described above.

For the biofilm inhibition assay, the 96-well, round-bottom, sterile polystyrene microtiter plate (Coming Costar, Ltd., New York, USA) with circular glass slides (diameter of 14 mm) and overnight cultures of P. aeruginosa PAO1 were supplemented with extract and incubated at 37°C for 24 h without agitation. Distilled water and MeOH served as negative controls. For quantification of the total biofilm biomass, the glass slides were washed with PBS for three times and then dried at 60°C. Biofilms were stained with 0.1% crystal violet or 0.01% acridine orange for 15 min. After which, biofilms were observed under light microscope (Nikon 80i, Japan) and fluorescence microscope (Nikon 80i, Japan), respectively.

For the biofilm disruption assay, after biofilms formed, the wells were supplemented with or without extract and incubated for 24 h. The biofilms that adhered to the glass slides were then stained using acridine orange for 15 min and visualized under fluorescence microscope. For SEM analysis, slides coated with biofilms were fixed using 2.5% glutaraldehyde and dehydrated with ethanol (50, 70, 80, 90, and 100%). The slides were subsequently dried, gold coated and observed under SEM (JSM6360, JEOL, Tokyo, Japan).

Protease activity was determined according to Wu et al. (2016) using the azocaserin method. Azocaserin is a suitable substrate for the measurement of protease activity (Hazen et al., 1965). During digestion, the colored components are formed and soluble in trichloroacetic acid. After removal of the undigested substrate, the color, which is proportional to the proteolytic activity of the enzyme, can be measured by a microplate reader (Hazen et al., 1965). Briefly, 150 μL of sterile supernatant of P. aeruginosa PAO1 and 250 μL of 2% azocasein (Sangon Biotech, China) in 50 mM Tris-HCl were incubated at 37°C for 4 h. The undigested substrate was precipitated with trichloroacetic acid (10%, 1.2 mL) for 20 min and then centrifuged at 12,000 rpm for 10 min, after which the supernatant was supplemented with 1.4 mL of 1 M NaOH. The absorbance was then measured at 440 nm.

Elastase activity was measured as described previously (Ohman et al., 1980). Briefly, 900 μL of Elastin congo red (ECR) buffer (1 mM CaCl₂, 100 mM Tris, pH 7.5) with 20 mg ECR was mixed with 100 μL of culture supernatant and incubated for 3 h at 37°C. Absorbance of the supernatant was measured at 495 nm after removing the insoluable ECR by centrifugation at 12,000 rpm for 10 min.

Quantification of pyocyanin was performed as previously described with minor modification (O'Loughlin et al., 2013). P. aeruginosa strains were propagated in a 2-mL Pseudomonas broth containing 2% bacto peptone, 0.14% MgCl₂, 1% K₂SO₄, and 1% glycerol and were incubated at 37°C at 150 rpm for 17 h. Cells were separated from culture fluids by 15-min centrifugation at 12,000 rpm. Cell-free culture fluids were used to quantify pyocyanin by measuring OD₆₉₅.

Rhamnolipids were assayed using the orcinol method with minor modification (Kim et al., 2015). Culture supernatant with the volume of 300 μL was extracted with 600 μL of diethyl ether. The organic phase was collected and evaporated to dryness under reduced pressure at 35°C and then added with 100 μL of deionized water. Orcinol solution with the volume of 900 μL (0.19%, Sigma-Aldrich) in 53% H₂SO₄ was mixed with 100 μL of each sample. The mixture was heated at 80°C for 30 min, and then cooled at room temperature for 15 min. Rhamnolipids concentration was determined at 421 nm.

Quantification of alginate was assayed as described previously (Owlia et al., 2007). Sterile-filtered supernatant (70 μL) of P. aeruginosa PAO1 was mixed with 600 μL of boric acid/H₂SO₄ (4:1, v/v). The mixture was vortexed and supplemented with 20 μL of 0.2% carbazole solution. The mixture was vortexed and incubated at 55°C for 30 min. The OD value was determined at 530 nm.

Swimming and swarming motilities were assayed according to Sheng et al. (2015). For swimming, 3 μL of overnight cultures of P. aeruginosa PAO1 were inoculated in the swimming agar medium (1% tryptone, 0.5% NaCl, 0.3% agar) in the presence or absence of extract. For swarming motility, 5 μL of overnight cultures were inoculated in the swarming agar medium containing 1% tryptone, 0.5% NaCl, 0.3% agar, and 0.5% glucose. Plates were incubated at 37°C for 24 h.

P. cucumerina extract (1 mg mL⁻¹) was exposed to a rising temperature gradient for 10 min (30, 50, 70, and 95°C). The stability of P. cucumerina extract was evaluated by an anti-pyocyanin assay as described above. Distilled water and MeOH served as negative controls. Quantification of pyocyanin was performed by measuring OD₆₉₅.

The P. cucumerina extract was analyzed using HPLC system (SHIMADZU) equipped with a XB-C18 column (100 × 2.1 mm, 3 μm; Welch Ultimate, Shanghai, China). The gradient was set as: 1–5 min, 5–20% B; 5–8 min, 20–40% B; 8–20 min, 40–100% B; 20–25, 100–100% B; 25–30 min, 100–5% B. Detection wavelength was 212 nm. For MS system, the nebulizer was set as 55 psi. The drying gas was set as 15 L min⁻¹ and the temperature was maintained at 500°C. Full-scan MS was from m/z 100 to 1,000. Further verification and quantitation of patulin and emodin was performed by LC-MS/MS using their corresponding standards (Sangon Biotech, China). Electrospray ionization was conducted in the negative mode under the same conditions as mentioned above.

Furthermore, pure compounds of patulin (25 and 50 μg mL⁻¹) and emodin (25 and 50 μg mL⁻¹) were added to the growth medium to analyze their activities on P. aeruginosa virulence factors and biofilms. In this assay, patulin was dissolved in distilled water while emodin was dissolved in MeOH. Biofilms, pyocyanin, protease, elastase, and swimming motility were assayed as mentioned above.

Data were expressed as mean values ± SD. The Levene's test for homogeneity of variance was applied to assess the equality of variances for all the variables before variance analysis. Statistically difference was determined by Levene's test coupled with ANOVA followed by Tukey–Kramer test. Statistics were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). P ≤ 0.05 was considered as significant.

A total of 41 fungi were isolated from the healthy leaves of O. violaceus based on their morphological characteristics. The anti-QS capacity was preliminarily screened using A. tumefaciens KYC55 as reporter strain. The A. tumefaciens KYC55 generates the AHL receptor TraR, which can sense exogenous AHLs. This anti-QS capacity was determined by comparing the competitive binding of the AHL molecules of A. tumefaciens and fungal extract to the AHL receptors. The competition was evaluated colormetrically. Among 41 isolated fungi, 7 kinds of fungi showed anti-QS capacities against A. tumefaciens KYC55 (Figure S1). However, only one fungus coded “B-JW-304” was selected for further study due to its potent antivirulence and anti-biofilm capacities against P. aeruginosa PAO1. No antivirulence and anti-biofilm capacities were observed in the other 6 fungi. Colonies of this fungus on PDA were slimy, flat, appressed, with sparse aerial mycelium (Figure S2a). Aerial mycelium varied from buff to salmon pink. Mycelium branched, septate, with abundant anastomosis, forming hyphal coils (Figure S2b). Conidia hyaline, smooth, ellipsoid tapering to rounded ends (Figures S2c,d). Using molecular studies (based on ITS1-5.8S-ITS4), B-JW-304 was identified as P. cucumerina (Figure 1). The 18S rDNA sequence was deposited in Genbank under the accession number KX822030. P. cucumerina is a well-known pathogen that could cause fruit rot (Carlucci et al., 2012). At present, most reports about P. cucumerina are associated with its pathogenic effect and biological control (Sanchez-Vallet et al., 2010; Gamir et al., 2012). However, literature about the secondary metabolites secreted by P. cucumerina is scarce. Therefore, it is valuable to investigate the metabolites and evaluate their bioactive capacities from P. cucumerina.

The yield of EtOAc extract in terms of dry weight was 314.8 mg L⁻¹ of the pooled extract from cells and supernatant. The MIC of extract against P. aeruginosa PAO1 was 1.25 mg mL⁻¹. The growth profile of P. aeruginosa PAO1 was evaluated using P. cucumerina extract at sub-MIC concentrations for 15 h (Figure 2). Results showed that P. cucumerina extract treatment with the concentration of 0.25, 0.5, 0.75, and 1 mg mL⁻¹ had no inhibitory effect on cell growth comparing with the water control during the stationary growth phase (Table S1). However, the growth was statistically different between the cultures with MeOH and 1 mg mL⁻¹ of extract (P < 0.05) at 12 and 14 h while not statistically different at 13 and 15 h (P > 0.05; Table S2). Therefore, we speculated that the experimental error might lead to such difference. To further validate the data, the growth assay was performed again. The bacterial viable count was performed after incubation for 24 h. The reason for selecting 24 h was that most assays of this study were determined after incubation for 24 h. The results showed that extract with the concentration of 1 mg mL⁻¹ had no inhibitory effect on P. aeruginosa growth comparing with the two negative controls (Figure S3).

In P. aeruginosa, the regulatory mechanism between las and rhl is clear. However, the molecular mechanism of pqs in coordinating virulence is currently unknown (Welsh et al., 2015). Therefore, in this study, the putative anti-QS activity of extract against P. aeruginosa was determined by quantitating the AHLs levels produced by this strain. LC-MS/MS analysis confirmed the presence of C4-HSL and 3-oxo-C12-HSL (Figures 3A,B). Treatment with P. cucumerina extract caused a decrease in both peaks and areas of C4-HSL and 3-oxo-C12-HSL as shown in the chromatograms (Figure 3B). The relative quantification of AHLs was presented in Figure 3C. P. cucumerina extract with the concentration of 0.25–1 mg mL⁻¹ caused reduction of C4-HSL by ~22, 61, 57, and 62%, respectively comparing with the water control (Figure 3C). Further, a significant decrease was also observed in 3-oxo-C12-HSL after treatment with P. cucumerina extract from 0.25 to 1 mg mL⁻¹ (Figure 3C). Overall, the data indicated that the anti-QS activity of P. cucumerina extract may be caused by interfering with the production of AHLs.

As shown in Figure 4A, P. cucumerina extract inhibited biofilms formation in a concentration-dependent manner. Extract with the concentration of 0.5–1 mg mL⁻¹ showed potent effects, reducing the biomass of PAO1 strain by ~80, 84, and 85%, respectively. MeOH showed no effect on the biofilm biomass of PAO1 comparing with the distilled water (0).

Since the formation of biofilm by P. aeruginosa is correlated with EPS (Kim and Park, 2013), the impact of extract on EPS production of P. aeruginosa PAO1 was evaluated. Figure 4B showed that extract exhibited concentration-dependent inhibitory activities against EPS and could reduce EPS production up to 46% at 1 mg mL⁻¹.

Direct microscopic observations are recognized to provide useful information on biofilms. Thus, light microscopy and fluorescence microscope were employed. In the control experiment, where bacteria were not treated with extract, a thick coating of biofilms was detected (Figures 5Aa,b). As shown in Figure 5Ac, treatment with 0.25 mg mL-1 of extract showed no significant reduction in the microbial attachment. However, extract with the concentrations of 0.5, 0.75, and 1 mg mL⁻¹ showed significant reduction in the microbial attachment to the glass surface compared to controls (Figures 5Ad–f). The above anti-biofilm activities were further confirmed by fluorescence microscope (Figure 5B). Fluorescence microscope images showed a scattered appearance of extract treated samples compared with the control. The cell clusters were rarely visible on account of poor cohesiveness.

The biofilm disruption activity of P. cucumerina extract against PAO1 was measured employing a standard quantitative biofilm assay method (Ding et al., 2011). As shown in Figure 6A, the extract exhibited strong ability to disrupt the established biofilms when treated with 0.5–1 mg mL⁻¹ extract. Biofilm biomass was reduced by ~52% in the presence of 1 mg mL⁻¹ of extract.

Furthermore, SEM also showed the efficacy of extract as potent biofilm disrupter. The images showed a scattered appearance after treatment with extract when compared with the control colonization (Figure 6B). The cells associated with the surface were almost scattered and the integrity of the biofilms was limited. SEM, a destructive method, relies on rigid sample preparation and may generate pseudo-positive or unexpected results. To circumvent this problem, samples were assayed employing fluorescence microscope after staining with acridine orange (Figure 6C), and the results were similar to SEM analysis.

Five virulence factors (protease, elastase, pyocyanin, rhamnolipid, and alginate) of P. aeruginosa were analyzed to evaluate the effects of extract on virulence (Figure 7). As shown in Figure 7A, the expression level of protease was significantly suppressed by 0.75 and 1 mg mL⁻¹ of extract. At 1 mg mL⁻¹, 27.3% inhibition of protease activities was observed. Production of elastase by P. aeruginosa is one of the QS-dependent behaviors (Husain et al., 2015). As shown in Figure 7B, the reduction in elastase was concentration-dependent. Approximately 42% inhibition in elastase was detected after treatment with extract at 1 mg mL⁻¹.

Pyocyanin is a vital factor for infection and biofilm formation in P. aeruginosa (Wu et al., 2016). Figure 7C showed a significantly decreased production of pyocyanin treated with extract. At 0.25 mg mL⁻¹, there was a 40% decrease in pyocyanin production and at 1 mg mL⁻¹, nearly 70% inhibition was observed. Furthermore, extract also exhibited concentration-dependent inhibitory activities against rhamnolipid and could reduce rhamnolipid production up to 50% at concentration of 1 mg mL⁻¹ (Figure 7D).

As alginate is a constituent of the extracellular matrix of P. aeruginosa biofilms (Luo et al., 2016), the efficiency of extract to reduce the alginate production was determined. Results demonstrated that the alginate production was significantly reduced with increasing concentrations of extract. Approximately 52% reduction was observed when exposure to extract at 1 mg mL⁻¹ (Figure 7E).

Swarming and swimming motility play important roles in QS-mediated biofilm formation in uropathogens (Jones et al., 2004). Inhibition of swarming and swimming motility was observed at concentrations as low as 0.25 mg mL⁻¹ of extract (Figures 8Ac,Bc). P. aeruginosa PAO1 exhibited swarming motility with a total swarming diameter of 33 mm. When treated with extract at concentrations ranging from 0.5 to 1 mg mL⁻¹, bacteria could grow and form a colony in the center with a diameter not exceeding 12 mm, and the tendril formation was also observed to be significantly reduced (Figures 8Ad–f) when compared with the negative controls (Figures 8Aa,b). Furthermore, similar results were observed in swimming motility as well (Figures 8Ba–f).

In this study, thermal stability was measured by 10 min incubation of the extract at varying temperatures ranging from 30 to 95°C, and then the anti-pyocyanin capacity of P. cucumerina extract was determined. Results shown in Figure S4 demonstrated that the P. cucumerina extract was relatively thermal stable. More than 90% activity of the extract was remained even after being maintained at 95°C for 10 min when compared to heat treatment at 30°C.

LC-MS/MS was employed to identify potential bioactive compounds in P. cucumerina extract (Figure 9A). As shown in Table 1, four compounds were identified. The extract ion chromatogram at m/z 177.0556 showed peak eluting at 5.87 min. This peak displayed the fragment at m/z 159 (M-H-H₂O) and 131 (M-H-CH₂O₂) (Figure 9Ba). The spectrum was indicative of mellein (Zhang X. et al., 2016). Compound 2 yielded a quasi-molecular ion [M-H]⁻ at m/z 277.0718 and fragment MS ions at m/z 219 (M-H-C₂H₂O₂) and 191 (M-H-C₃H₂O₃) (Figure 9Bb), being identified as citreoisocoumarin. These fragments are in accordance with the same profile described by one previous study (Rasmussen et al., 2010). The extract ion chromatogram at m/z 153.0194 in P. cucumerina extract showed a peak at 3.39 min (Table 1). This peak displayed the fragments at m/z 109 (M-H-CO₂) and 81 (M-H-CH₂O₃) (Figure 9Bc). The spectrum data was consistent with that of patulin (Desmarchelier et al., 2011), which was further validated by a commercial standard patulin (Figure 9Be). Quantitation analysis showed that the production of patulin was 17.62 μg mL⁻¹ of fungal extract (1 mg mL⁻¹) and this concentration was comparable with the production of 5.5 μg mL⁻¹ of patulin present in fungal broth (Figure S5A). The extract ion chromatogram at m/z 269.0454 showed a peak at 13.18 min. This peak displayed the fragments at m/z 241 (M-H-CO), 225 (M-H-CO₂), and 197 (M-H-CH₂O₃) (Figure 9Bd). This spectrum was indicative of emodin (Song et al., 2008), which was also further validated by a commercial standard emodin (Figure 9Bf). Further, the production of emodin was quantified by 20.37 μg mL⁻¹ of fungal extract (1 mg mL⁻¹) and this concentration was comparable with the production of 6.4 μg mL⁻¹ of emodin present in fungal broth (Figure S5B).

To screen the bioactive compounds responsible for the anti-biofilm and antivirulence activities of P. cucumerina extract, four identified compounds above were tested. Unfortunately, mellein and citreoisocoumarin showed no anti-biofilm and antivirulence activities (Figure S6). Patulin has been reported to inhibit QS-controlled gene expression in P. aeruginosa PAO1 (Rasmussen et al., 2005). Apart from gene expression inhibition, could patulin inhibit QS regulated phenomena such as biofilms and production of virulence factors or not? In this study, the MIC of patulin was 100 μg mL⁻¹. P. aeruginosa PAO1 treated with 25 and 50 μg mL⁻¹ of patulin showed 32 and 52% reduction in biofilm formation, respectively (Figure 10A). The above anti-biofilm activities were further confirmed by fluorescence microscope (Figures 10Be,f). A scattered appearance was observed when exposure to patulin. Patulin also owned strong ability to disperse the established biofilms. Approximately 44% reduction in biofilm biomass was detected after treatment with patulin at 50 μg mL⁻¹ (Figure 10C). SEM also showed the efficacy of patulin as an excellent biofilm disrupter, and a scattered appearance was observed in treated samples compared with the control colonization (Figures 10De,f). This is the first report of the biofilm disruption activity of patulin. To check the antivirulence effect of patulin on P. aeruginosa, virulence factors including protease, elastase, pyocyanin, and swimming were assayed since production of these factors was recognized as an indicator of active QS. Treatment with 50 μg mL⁻¹ of patulin resulted in 31, 62, and 48% inhibition of protease, elastase, and pyocyanin, respectively (Figures 10E,F,H) without affecting cell growth (Figure 10H). Motility behavior was assessed by the swimming motility assay. Treated cells of P. aeruginosa showed shorter diameters on agar plates compared to the untreated controls (Figure 10I).

Emodin was another component identified in P. cucumerina extract by LC-MS/MS. We presumed that emodin might be another principal component for anti-biofilm and antivirulence in P. cucumerina extract. To validate this hypothesis, emodin with concentrations of 25 and 50 μg mL⁻¹ (MIC was 94 μg mL⁻¹) was analyzed. Treatment with 50 μg mL⁻¹ of emodin showed a 45% reduction in biofilm formation (Figure 10A). The above anti-biofilm activities of emodin were further confirmed by fluorescence microscope (Figures 10Bc,d) comparing with the negative controls (Figures 10Ba,b). Biofilm disruption assay demonstrated that emodin (50 μg mL⁻¹) treatment led to a 32% reduction in biofilm biomass (Figure 10C). SEM also revealed that biofilm formation decreased with the concentration of 50 μg mL⁻¹ of emodin as compared to the controls (Figure 10D). The treated bacteria were in the discrete form rather than in a group. In addition, P. aeruginosa dosed with 50 μg mL⁻¹ of emodin resulted in 23, 44, and 50% inhibition of protease, elastase, and pyocyanin, respectively (Figures 10E–G). Emodin treatment with the concentration of 50 μg mL⁻¹ had no significantly inhibitory effect on cell growth comparing with the MeOH control but significantly with the water control (Figure 10H). To further confirm the data, the growth assay was performed again. The results showed that emodin with the concentration of 50 μg mL⁻¹ had no inhibitory effect on P. aeruginosa growth comparing with both the negative controls (Figure S7). Swimming motility was also significantly inhibited after treatment with 25 and 50 μg mL⁻¹ of emodin (Figures 10Ic,d).

Overall, the observed inhibition of P. aeruginosa biofilm and virulence factors suggested that patulin and emodin might act as the principal active components in P. cucumerina extract.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.