Isolate and growth conditions
We sourced isolate FSP34 of F. circinatum (35) from the Fusarium collection of the Forestry and Agricultural Biotechnology Institute (FABI; University of Pretoria, South Africa). For routine culturing, the fungus was incubated on potato dextrose agar (PDA; 10 g/L; Merck Group) in darkness at 25°C for 7 days. For obtaining conidia, a 7-day-old culture was flooded with 2 mL of 0.2 M phosphate-buffered saline (PBS) consisting of 10 mM NaH2PO4, 10 mM Na2HPO4, and 150 mM NaCl at pH 7.2. Following collection with a sterile pipette, the Countess 3 Automated Cell Counter (ThermoFisher Scientific) was used to quantify the concentration of the spore suspension.
Isolation and quantification of EVs from fungal and plant tissue
Fungal EVs were isolated from 72 hour planktonic and biofilm cultures were prepared in 200 mL potato dextrose broth (PDB; 10 g/L; Merck Group). This involved sequential centrifugation steps (4,000 g for 15 min and 15,000 g for 30 min at 4°C) for pelleting fungal tissue, after which the supernatant was passed through a 0.45 µm membrane filter (Merck Millipore) to eliminate any remaining cells or debris. By making use of the Amicon Ultra-4 Centrifugal system (100-kDa pore size, Millipore), the supernatant was concentrated to approximately 20 mL. The concentrated supernatant was then subjected to ultracentrifugation (100,000× g for 1 hour at 4°C) using Beckman Coulter equipment. The supernatant was discarded, and the EV-enriched pellet was washed twice with PBS at 100,000× g for 1 hour at 4°C. Finally, the EV pellet was stored in PBS at 4°C until further use.
Isolation of EVs from the roots and needles of P. patula seedlings were performed as previously described (36). Briefly, a total of two healthy seedlings per isolation were used, whereby entire leaves and roots were rinsed and weighed, and distal zones of needles and roots from the same plant were collected. After removing excess water, the plant material was ground using a pestle and mortar in the presence of vesicle isolation buffer containing 5.9 g/L NaCl, 0.002 g/L CaCl2, and 4.3 g/L 2-(N-Morpholino)ethanesulfonic acid hydrate (MES hydrate). This homogenate was filtered by passing it through a cheesecloth, and then centrifuged for 20 min at 700 × g at 4°C to separate the extracts. Concentrated EVs obtained using the Amicon Ultra-4 Centrifugal system(100-kDa pore size) were then subjected to size-exclusion chromatography (SEC) as described previously (36, 37). This involved application of a mixture containing EVs with a cell membrane-specific dye (FM4-64; Thermo Fisher Scientific) to a Sepharose CL-2B column. Fractions with fluorescence above 3.0 RFU were pooled as "EV signal" after measurement.
Analyses of the general properties of vesicles
The general features of the extracted EVs were studied using electron microscopy and nanoparticle tracking analysis (NTA). Purified EVs were spotted onto carbon-coated grids for adsorption for 5 min, negatively stained with 1% (w/v) uranyl acetate for 3 min and subjected to transmission electron microscopy (TEM) using the JEM-2100F Field Emission Electron Microscope (JOEL Ltd., Tokyo, Japan). Scanning electron microscopy (SEM) was carried out as previously described (38) for planktonic cells and biofilms in order to observe EV-like structures on the surface of the cells. Briefly, after culturing fungal biofilms on glass slides, the samples were rinsed with PBS and treated with a 2.5% glutaraldehyde/formaldehyde solution (), followed by fixation with 1% osmium tetroxide. Subsequent dehydration steps using graded ethanol concentrations (30%, 50%, 70%, 90%, and absolute ethanol) and a 50:50 mixture of hexamethyldisilazane (HMDS) were performed. The dried samples were mounted on aluminum stubs, carbon-coated (Quorum Q150T ES sputter coater), and observed using a JEOL JSM 6490LV scanning electron microscope (GenTech Scientific Inc., Arcade, NY, USA). For NTA, 5 mL of an EV suspension was vortexed for 1 minute, and diluted with PBS to a total of 10 mL, of which 1.5 mL was immediately analysed with the NanoSight NS500 (Malvern Panalytical, UK) in triplicate (each run = 30 s video). PBS was treated as a blank. The videos were captured and analysed using the NanoSight NTA software (NTA 3.3 Dev Build 3.3.104, Malvern Panalytical, UK). The camera sensitivity and detection threshold were optimized per video, and the temperature was set at 22°C. We further determined the concentrations of EVs derived from planktonic cells (pkcdEVs), biofilms (bdEVs), and pine seedling needles (ndEVs) and roots (rdEVs) based on protein content using the QuantiPro™ BCA Assay Kit (Merck) following manufacturer’s instructions.
Evaluating the effects of EVs on conidial germination and biofilm formation
The analyses of the effects of EV on conidial viability and germination, as well as biofilm formation were conducted using previously published methodologies (39–41). To achieve this, two sets of experiments were performed using PDB. In the one experiment, EVs (pkcdEVs, bdEVs, and pdEVs) were co-incubated with conidia to a final concentration of 5 x 105 spores/mL and 15 µg/ml EVs at room temperature 16 h while shaking (100 rpm, Shake-O-Mat, Labotec South Africa). Following this, cultures treated and not treated with EVs were examined using an automated cell counter (Countess 3 FL, Thermo Fisher Scientific) that incorporates Trypan Blue staining that selectively colours dead cells blue, distinguishing them from viable ones. We also observed the cultures between 2 and 24 h following shaking using a haemocytometer to determine the number of germ tubes emerging from viable conidia. In the second experiment, all types of EVs isolated were co-incubated with conidia as before, but incubated statically to allow biofilm formation, after which biomass and ECM were analysed as described above at 72 h and 7 days. Biofilms formed without EV treatments were treated as a positive control, while a negative control included PDB with EVs only and EVs that were heat-inactivated in PDB at 90°C for 15 minutes.
Biofilm biomass and ECM quantification
Biofilm and planktonic cultures were grown as described previously (38) using either 96-well polystyrene plates (Greiner bio-one, South Africa) or 500 mL pyrex Erlenmeyer flasks (Merck, South Africa). In case of the 96-well plates, conidia were suspended to a final concentration of 2 x 105 spores/mL in 400 µL PDB, followed by incubation at room temperature for 72 h and7days under stationary conditions to allow biofilm formation. Planktonic cell cultures were grown in the same way in these plates, except that they were incubated on a shaker (100 rpm, Shake-O-Mat, Labotec South Africa). In case of the Erlenmeyer flasks, we inoculated 200 mL of PDB with conidia to a final concentration of 1 x 106 spores/mL, followed by incubation at room temperature for 72 hours on an orbital shaker at 100 rpm for the planktonic cultures or statically for the biofilm cultures.
Biomass and ECM analysis of planktonic and biofilm (72 and 7 days) cultures of FSP34 in 96-well polystyrene plates were conducted as before (38). Briefly, biofilms were then rinsed with PBS to remove non-adherent cells, while planktonic cells were collected by centrifugation (10000 g for 10 min) and rinsed the same as the biofilms. Cells were then fixed with methanol and air-dried, followed by respectively crystal violet and fuchsin staining (38). The former allows visualization of fungal cells alongside certain components within the biofilm matrix, while the latter allows for visualization and distribution of polysaccharides in the matrix. We then used a spectrophotometer (SpectraMax paradigm, Multimode detection platform) to quantify biomass (540 nm) and ECM production (530 nm).
Morphological analyses of fungal cells exposed to vesicles
We further aimed to determine whether vesicles have potential long-term effects on F. circinatum. To do this, a conidial suspension (5 x 105 spores/mL) was pre-treated with 5 µg/mL pkcdEVs and bdEVs, as well as 15 µg/mL ndEVs and rdEVs for 24 h. The pre-treated spores were then allowed to grow for 7 days at room temperature on triplicate PDA plates (Merck, Modderfontein GP, South Africa; 4g/L Potato Infusion, 20 g/L D(+)-Glucose), and 15 g/L agar). Following this, mycelial growth was quantified from by measuring the diameter of colonies after 3, 7, and 14 days. In addition, a loopful of cells taken from the periphery of a 7-day-old and 14-day-old culture and analysed using a light microscope at 10x magnification.
Reproducibility and statistical analyses
All experimental data, unless specified otherwise, were subjected to one-way ANOVA or non-parametric Student's t-test for comparison. Statistical analyses were performed using GraphPad statistical software (GraphPad 5 Software, San Diego, CA, USA). The level of significance was determined through the representation of mean ± Standard Deviation (SD) and defined using probability thresholds: *, P ≤ 0.05; **, P ≤ 0.002; ***, P ≤ 0.001; ****, P ≤ 0.0001. In most cases, each experiment was conducted with three technical replicates and five biological replicates, unless stated otherwise. Additionally, all experiments included both positive and negative controls to assess the validity and reliability of the experimental results.