## 2 Results and Discussion
### Characterization of the TaST Mutants  
Through integrative analysis of the fungal genomics and phenotype characters, the roles of the four _TaTS_ genes in the terpene biosynthesis of ACCC 33109 were identified. The _TaTS_ gene disruption mutants _\(\Delta\)Talig4::neo_\(\Delta\)_TaTS::hph_ and chassis strain _\(\Delta\)Talig4::neo_ were cultured on sandwiched PDA plates with _Foc_ for 5 days at 28 \({}^{\circ}\)C for the antagonism assay and qPCR analysis. Considering the antagonism of VOCs is ascribed to the combined action of multiple compounds, the absence of a certain gene cannot cause the complete loss of anti-fungal activity of the VOCs in _Trichoderma_[9; 26]. As shown in Supplementary Figure S5 and Table 2, the VOCs produced by the chassis strain significantly inhibited the _Foc_ growth by 18.56%, 17.16%, and 22.37% at days 3, 4, and 5 respectively. Compared with the chassis strain, deletion of the _TaTS3/4/8/9_ showed significant effects on the biosynthesis of terpenoids, which further changed the composition and contents of the anti-fungal VOCs. Significant inhibition reduction (up to 9.23%) of _Foc_ growth was detected in the _\(\Delta\)Talig4::neo_\(\Delta\)_TaTS9::hph_-3_, \(\Delta\)_Talig4::neo_\(\Delta\)_TaTS9::hph_-130-_, and \(\Delta\)_Talig4::neo_\(\Delta\)_TaTS8::hph_-5_ treated plates on days 3-5 and _\(\Delta\)_Talig4::neo_\(\Delta\)_TaTS3::hph_- and \(\Delta\)_Talig4::neo_\(\Delta\)_TaTS4::hph_-treated plates on day 5 (_p_ < 0.05). The results indicated that the four terpene-synthes-encoding genes are crucial to the biosynthesis of antagonistic VOCs. Deletion of _TaTS3/4/8/9_ also affected the expression levels of other _TaTS_ genes (Figure 5). In comparison to the chassis strain _\(\Delta\)Talig4::neo_, some genes were depressed in the mutant strains, and some genes were overexpressed. In the mutant strains _\(\Delta\)Talig4::neo_\(\Delta\)_TaTS3::hph_  
\begin{table} \begin{tabular}{c c c c c c c} \hline \hline \multirow{2}{*}{**Time**} & \multicolumn{5}{c}{**Inhibitory Rate (\%)**} \\ \cline{2-7}  & \(\Delta\)_Talig4::neo_ & \(\Delta\)_Talig4::neo_ & \(\Delta\)_Talig4::neo_ & \(\Delta\)_Talig4::neo_ & \(\Delta\)_Talig4::neo_ & \(\Delta\)_Talig4::neo_ \\  & \(\Delta\)_TaTS3::hph_ & \(\Delta\)_TaTS4::hph_ & \(\Delta\)_TaTS8::hph_-5_ & \(\Delta\)_TaTS9::hph_-3_ & \(\Delta\)_TaTS9::hph_-130_ \\ \hline Day 3 & 18.56 & 14.90 ** & 17.21 & 14.70 ** & 14.63 ** & 17.83 \\ Day 4 & 17.16 & 14.45 ** & 17.96 & 10.60 *** & 13.78 *** & 18.17 \\ Day 5 & 22.37 & 15.33 ** & 18.88 * & 13.14 *** & 19.40 * & 19.08 ** \\ \hline \hline \end{tabular}   
* * *, **, and *** indicate statistically significant differences of the same row between _\(\Delta\)Talig4::neo_ and _\(\Delta\)Talig4::neo_\(\Delta\)_Talig4::neo_; _\(\Deltaand \(\Delta\)_Talig4::neo\(\Delta\)TaTS4::lph_, the mRNA transcription levels of the _TaTS_ genes were unchanged or depressed significantly (0.05-0.36-fold; \(p\) < 0.05). In the \(\Delta\)_Talig4::neo\(\Delta\)TaTS8::lph_, the mRNA transcription levels of _TaTS3_ and _TaTS6_ increased (2.42- and 2.03-fold; \(p\) < 0.05), while the other _TaTS_ genes had no changes or decreased in terms of the mRNA expression levels (0.09-0.39-fold; \(p\) < 0.05). Only one gene, _TaTS5_, was overexpressed in the \(\Delta\)_Talig4::neo\(\Delta\)TaTS9::lph_, with the highest level of 32.5-fold (_p_ < 0.001), and the other _TaTS_ genes had no changes or decreased in the mRNA expression levels (0.05-0.38-fold; \(p\) < 0.05). The results indicate that the _TaTS_ genes of ACCC 33019 can be classified into the constitutive and inducible types, which form a complex network and function in independent (i.e., _TaTS1_ and _TaTS4_), synchronous (i.e., _TaTS2_ and _TaTS7_) as well as complementary (i.e., _TaTS5_ and _TaTS9_; _TaTS3_, _TaTS6_ and _TaTS8_) ways in the biosynthesis of terpenoids. Moreover, no _TaTS_ genes were detected in the corresponding deletion mutants by qPCR, suggesting the mutant strains lacked genome complementarity to homologs and retained recombination stability.