## 4 Discussion  
Triterpenoids are an important class of secondary metabolites composed of six isoprene units and have gained significant attention in recent years due to their pharmacological activity [27; 28]. Fungal elicitors have been shown to promote liquid fermentation by fungi to obtain desired substances. For example, [PERSON] et al. studied the impact of yeast extracts and specific polysaccharide elicitors on secondary metabolites, including anthocyanins and phenolic acids, in the suspension cultivation of _Vitis vinifera_[29]. [PERSON] et al. described the effects of two algal polysaccharide elicitors, laminin and carrageenan, on defense reactions and signaling in tobacco plants [30]. In this study, a plant secondary metabolite database was constructed based on UPLC-MS/MS to identify 48 differential secondary metabolites, including phenolic acids, lignans and coumarins, flavonoids, and terpenoids, in polysaccharide elicitor-induced mycelium of _S. singhuang_. Flavonoids (24.4%), terpenoids (8.8%), and coumarin and lignans (11%) showed significant upregulation. The fungal elicitor promoted the accumulation of triterpene and ketone metabolites in _S. singhuang_. Among the triterpenoids, the levels of betulinic acid and 2-hydroxyoleanolic acid were 2.62-fold and 114.67-fold higher than in the control group, respectively. These compounds have been reported to have various pharmacological effects, such as antimalarial, anti-inflammatory [31; 32], antibacterial [33], and antitumor properties [34; 35]. An analysis of the full-length transcriptome sequencing of _S. singhuang_, which generated 30,704 transcripts, was conducted to investigate the effect of fungal elicitor on metabolites. Using fold change \(\geq\) 2 and FDR \(<\) 0.01 as criteria, 97 DETs were identified between the control and stimulated groups, of which 57 were upregulated and 40 were downregulated. The DETs were annotated into GO, KEGG, COG, and egg NOG databases and categorized. The results of gene annotation and enrichment analysis suggested that the fungal elicitor played a significant role in activating the secondary metabolic pathway and defense genes of _S. singhuang_. Additionally, four stable genes related to secondary  
Figure 9: (**A**) The content of NO in _S. singhuang_ under elicitor treatment. (**B**) The content of total triterpenes in _S. singhuang_ under elicitor treatment. * above the pillars show significant differences. metabolism were identified based on the transcriptome sequencing results, and their differential expression was validated. The results of the KEGG enrichment analysis revealed a significant enrichment of differential metabolites in the biosynthetic pathway of urea and other terpenoids-quinone, which is closely associated with the biosynthetic pathway of the terpene skeleton. Downregulation of 4-hydroxybenzoate in this pathway of urea and other terpenoids-quinone resulted in decreased consumption of polyisopentene diphosphate in the biosynthetic pathway of the terpene skeleton. This, in turn, led to a decrease in the production of branching acids, such as isobranchic acid and 1,4-dihydroxy-2-naphthoic acid, and a reduction in the consumption of polyisopentenyl diphosphate, which accumulates significantly in the biosynthetic pathway of the terpene skeleton. The decrease in the consumption of phytosubstituted monophosphate diammonium salts in the terpene skeleton biosynthetic pathway reduced the synthesis of 1,4-diox-2-naphthoic acid, causing its accumulation. Due to the low consumption, the accumulation in the biosynthetic pathway of the terpene skeleton led to the accumulation of geranylgeranyl pyrophosphate triammonium salt, which can be involved in other pathways that synthesize other terpenoids, such as the biosynthetic pathway of triterpenoids, the biosynthesis of indole diterpenoid alkaloids, and the biosynthesis of monoterrepoids. Based on these findings, it is possible that the accumulation and production of terpenoids are a result of the chain reactions mentioned above.