Production and GC-MS analysis of Trichoderma spent media
For each Trichoderma species, six independent liquid cultures
were prepared (to be sampled after 7, 14, and 21 days, each). After 1
week, 2 and 3 weeks, respectively, cultures were pooled into two
meta-samples (each composed by three independent cultures), centrifuged
to collect the spent media and pellets were dried to measure fungal
biomass. The 1 week (1W) spent media were used both for transcriptomic
analysis and phenotypic tests (see below). Regarding the molecular
composition, the metabolites from 1W spent media were extracted from a
volume of 40 mL using 50 mL of chloroform in a separatory funnel. The
extracts were then concentrated in a nitrogen stream to 2 mL and
esterified according to the manufacturer’s instructions with a
BSTFA-TMCS derivatising mixture (99:1 v/v), (Sigma Aldrich, Germany).
Separation of organic compounds in the esterified samples was performed
by gas chromatography (GC 7890A, Agilent Technologies, USA) coupled with
mass spectrometry (MS 5973c, Agilent Technologies, USA). Samples were
injected using an automatic dispenser (7683 Series Injector Agilent
Technologies, USA) in a volume of 2µL. The standard deviation of the
injection according to the manufacturer’s data was a maximum of 0.3%.
The injected sample was excited into the gaseous state at 280°C and
diluted in a 1:5 v/v helium stream (split). The separation of organic
compounds was then carried out using an HP-5MS column (30 m, 0.25 mm
I.D., 0.25 µm particle size (Agilent Technologies, USA)), using helium
as a carrier gas, at a flow rate of 1 mL/min.
Organic compounds were separated using a temperature gradient. The
chromatography column was heated at 100°C for 5 minutes, followed by a
temperature ramp up to 280°C at a rate of 6°C/min. Once the temperature
reached 280°C, it was maintained for a further 8 minutes.
The mass spectrometer scanned the separated ions in the mass range 40 to
800 Da) at an ionisation of 70eV, a filament temperature of 150°C, and a
temperature of 230°C in the ionisation chamber.