# New Detection Method for Fungal Infection in Silver Fir Seeds
## 3 Materials and Methods
### Measurement of Volatile Organic Compounds  
Volatile organic compounds (VOC) emitted from seeds were analyzed by headspace solid-phase microextraction coupled with gas chromatography and mass spectrometry (HS-SPME/GC-MS) following a previously used method [55; 56] briefly described below.  
In preliminary studies of VOCs emitted from asymptomatic silver fire seeds (control), divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS), CAR/PDMS, and PDMS sorption fibres (Supelco, Bellefonte, PA, USA) were compared and DVB/CAR/PDMS fibre was selected for further research based on the highest effectiveness of the extraction-desorption cycle. Seeds for chemical analysis were selected at random, targeting those seeds with visible fungal growth. Seeds (1 \(\pm\) 0.05 g, \(\sim\)8 seeds) were placed in 60 mL glass vial with cap and septum (Buchi 049535) and heated at 40 \({}^{\circ}\)C for 60 min. SPME fiber was then added to the vial with a divinylbenzene/carboxylate/polydimethylsiloxane stationary phase (Supelco, Bellefonte, PA, USA). The fiber was exposed to the headspace gas phase at 40 \({}^{\circ}\)C for 30 min. Immediately after exposure, the SPME fibre was inserted into an injection port of the GC-MS instrument for 10 min. GC-MS analyses were performed using an Agilent 7890A gas chromatograph with an Agilent 5975C mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). The injector was operated at a temperature of 250 \({}^{\circ}\)C in splitless mode. Chromatographic separation was performed on a capillary column HP -5 MS (30 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m) at a helium flow rate of 1 mL/min. The initial temperature of the column was 35 \({}^{\circ}\)C and increased to 250 \({}^{\circ}\)C at a rate of 5 \({}^{\circ}\)C/min. The ion source and quadrupole temperatures were 230 \({}^{\circ}\)C and 150 \({}^{\circ}\)C, respectively. The electron impact mass spectra were obtained at ionization energy of 70 eV. Detection was performed in full scan mode for a range of 29-600 atomic mass units.  
The peaks from the chromatogram were integrated, and the percentage of the components in the total ion current (TIC) was calculated. The mass spectral data and calculated retention indices were used to identify the components. Mass spectrometric identification was performed using the NIST (2020) and Wiley (2020) mass spectral libraries and the collections of [PERSON] (2007) and [PERSON] (2008) as well as a private, unpublished library of mass spectra, which had been created using standard chemical compounds. The retention indices of the analytes were determined considering the retention times of the n-alkanes. In a separate run, the mixture of C5C40 n-alkanes (1 \(\upmu\)L) was injected onto the chromatographic column and separated under the conditions previously described for GC-MS analyses of volatiles. Linear temperature-programmed experimental retention indices (Rexp) were calculated using the following expression \(\mathrm{RI}_{exp}=100[n+(\mathrm{t}_{x}-\mathrm{t}_{n})/(\mathrm{t}_{n+1}- \mathrm{t}_{n})]\), where \(n\) is the number of carbon atoms in the alkane, \(\mathrm{t}_{x}\) is the retention time of the analyte, \(\mathrm{t}_{n}\) is the retention time of the \(n\)-alkane eluting immediately before the analyte, and \(\mathrm{t}_{n+1}\) is the retention time of the \(n\)-alkane eluting directly after the analyte. The experimental retention indices (\(\mathrm{RI}_{exp}\)) were compared with those in the retention indices database (\(\mathrm{RI}_{lit}\)) mentioned above.