# New Detection Method for Fungal Infection in Silver Fir Seeds
## 4 Results
### VOC Measurements  
Fungal colonized seeds for each morphotype (green or white) were moved into 60 mL vials for gas measurements. The dominant chemical components identified by GC-MS from the six plates are shown in Figure 6. The source data used in this figure are listed in Table 13 in the Appendix A. This figure shows the average value of all samples and the average and standard deviation for the uninfected and infected seed categories. The limit of 1% of TIC gave 28 components that could be compared with the 130 chemical components identified in the samples by GC-MS, which meant that these represented 21.5% of the components in Table 13, in the Appendix A. These components represented 86% and 88% of TIC for infected and uninfected samples, respectively. The most abundant substance was limonene, whose concentration differed slightly between uninfected and infected samples, but not significantly as shown by the standard deviation whiskers in Figure 6. Among the 28 components, the one that differed most greatly between uninfected and infected samples was \(\alpha\)-terpineol which was almost absent in uninfected samples. For other components whose relative content (% of TIC, Total Ion Current) differed between the categories, \(\gamma\)-cadinene, guaia6-9-diene, selina-4(15)6-diene, \(\alpha\)-selinee, \(\gamma\)-cadinene, \(\beta\)-caryophyllene, and \(\delta\)-elemene showed higher levels in infected samples and these differences were larger than the standard deviation bars between means of infected and uninfected samples. There were several components whose relative contents were significantly higher in uninfected samples including \(\alpha\)-pinene, \(\beta\)-cadinene, himachala-2_4_-diene, garmacene D, longifolene, \(\beta\)-pinene, and \(\alpha\)-longipinene. Such visual data exploration provides interesting insights into the components that might help distinguish between the uninfected and infected samples. However, a statistical analysis using the t-test was also performed, with a goal to determine which of the chemical components identified by GC-MS measurements differed at the \(p\)-value level of 0.05 between uninfected and infected samples. In this case, the analysis was restricted to components with 1% of TIC as in the results mentioned earlier. This analysis of the GC-MS results aimed to assess chemical components that might help discriminate between uninfected and infected samples. In Figure 7, these data are presented in graphical form, where two factors are used. First, the difference in the amount of substance must be signif \begin{table}
\begin{tabular}{l l l l l} \hline \hline \multirow{2}{*}{**Accession Sequences**} & \multirow{2}{*}{**Colony**} & \multicolumn{3}{c}{**Closest Match**} \\ \cline{3-5}  & & **Species** & **Accession** & **Identity [\%]** \\ \hline OK642412 & G1, green & _Xylaria ellisii_ & NR, 172972 & 99.32 \\ OK642413 & G2, green & _Aspergillus baranensis_ & KY980621 & 98.26 \\ OK642414 & G3, green & _Trichoderma koningii_ & MT102397 & 100.00 \\ OK642415 & W1, white & _Trichoderma harzianum_ & MK738148 & 100.00 \\ OK642416 & W2, white & _Trichoderma harzianum_ & MT557111 & 100.00 \\ OK642417 & W3, white & _Trichoderma harzianum_ & MK738148 & 100.00 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Top BLAST match of six isolates to sequences in GenBank The colony name for the samples (G1–G3, W1–W3) are the same as those used in Table 3.  
icant, which could be represented by the factor \(\text{TIC avg\_diff}=\text{TIC}_{Uhin\_{fected}}-\text{TIC}_{Infected}\). However, it is also important that this value is significantly different from the total amount of the component present in the sample, which can be defined as follows: \(\text{TIC pct\_diff}=\text{TIC avg\_diff}/2\text{TIC}_{All}\). The list of chemical elements presented in Figure 7 are included in Table 16 in the Appendix A. The full details of the results of the GC-MS measurements are listed in Table 17 in the Appendix A.  
Figure 6: Dominant chemical components identified by the GC-MS method.