## 2 Materials and Methods
### Characterization of Volatile Compounds Emitted during Fungal Interaction  
Among the endophytes studied, _A. pullulans_ showed the greatest antagonistic activity against _C. acutatum_ in either in vitro or in vivo assays (please see Results). Thus, the ability of this endophyte to produce VOCs with antimicrobial activity was further studied, aiming to elucidate its mechanisms of action against _C. acutatum_. Accordingly, a bioassay with olives was established following a similar procedure previously described. Briefly, five previously sterilized healthy olives of cv. _Cobrancosa_ (with a maturation index of 3) were introduced in 50 mL flasks (Duran Gaines Synth, Bioblock), inoculated with 1 mL of cell suspension of _A. pullulans_ (10\({}^{6}\) cells/mL) and, after 3 days, additionally inoculated with 1 mL of _C. acutatum_ spore suspension (10\({}^{6}\) spores/mL). Controls were performed by inoculating olives with 1 mL of sterile solution of 0.025% (\(v/v\)) Tween 80 (mock inoculation), with the endophyte (1 mL at 10\({}^{6}\) cells/mL) or with the pathogen (1 mL at 10\({}^{6}\) spores/mL). After inoculation, the flasks were immediately sealed with a polypropylene cap with polytetrafluoroethylene/silicon septum (Duran) and incubated at 25 \({}^{\circ}\)C \(\pm\) 2 \({}^{\circ}\)C, under a daylight regime. Five replicates for each treatment were performed.  
Based on the results obtained in the in vivo bioassays (please see Results), volatile sampling took place 2, 8 and 16 days after pathogen inoculation. At each sampling time, the volatiles were analyzed by headspace solid phase microextraction gas chromatography coupled to mass spectrometry (HS-SPME-GC/MS), following a similar procedure used by [27]. The vial was placed in a water bath at 30 \({}^{\circ}\)C for 5 min to release volatile compounds. Then, under the same conditions of temperature and agitation, the SPME fiber (divinylbenzene/carbonex/polylimethylsiloxane) (DVB/CAR/PDMS 50/30 \(\upmu\)m) (Supelco, Bellefonte, PA, USA) was exposed for 30 min for adsorption of the volatile compounds in the headspace. Volatile compounds were removed from the fiber by thermal desorption (220 \({}^{\circ}\)C) for 1 min in the chromatograph injection port. The fiber was kept in the injection port for 10 min for cleaning and conditioning for further analysis. The gas chromatograph used was a Shimadzu GC-2010 Plus equipped with a Shimadzu GC/MS-QP2010 SE mass spectrometer detector. A TRB-5 MS column (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m) (Teknokroma, Spain) was used. The injector was set at a temperature of 220 \({}^{\circ}\)C, and the manual injection was performed in splitless mode. The mobile phase consisted of helium 5.0 (Linde, Portugal), at a linear velocity of 30 cm/s and a 24.4 mL/min flow rate. The oven temperature was 40 \({}^{\circ}\)C for 1 min, followed by an increase of 2 \({}^{\circ}\)C/min until reaching 220 \({}^{\circ}\)C. The ionization source was maintained at 250 \({}^{\circ}\)C with an energy of 70 eV and a current of 0.1 kV. All mass spectra were obtained by electronic ionization in the m/z range 35-500. Compounds were identified by comparing the mass spectra and through the Kovats index using databases such as NIST 69, PubChem and ChemSpider. Retention indices were obtained using a commercial n-alkane series, C7-C30 (Sigma-Aldrich, St. Louis, MS, USA), by direct splitless liquid injection (1 \(\mu\)L), while all further conditions of GC and MS were settled for the volatile analysis. Retention indices were calculated according to the Kovats index. The identified volatile compounds were expressed based on the areas determined by TIC (total ion chromatogram) integration.