The Effects of Fungal Infection Combined with Insect Boring on the Induction of Agarwood Formation and Transcriptome Analysis of Aquilaria sinensis

This study investigates the main insects and endophytic fungi that promote the formation of agarwood in Aquilaria sinensis (Lour.) Spreng. and elucidates the effects and mechanisms of different ‘insect + fungus’ combinations on agarwood formation. The results showed that 16 strains of endophytic fungi were isolated from A. sinensis. Fusarium solani, Penicillium chrysogenum, Fusarium equiseti, and Phaeoacremonium alvesii were identified as dominant fungi promoting agarwood formation, while Nadezhdiella cantori was recognized as the dominant insect facilitating this process. The optimal ‘insect + fungus’ combination was Nadezhdiella cantori + Fusarium equiseti. The average agarotetrol contents were 0.046% and 0.054% in February and June, respectively, which were significantly higher than those in cold drilling, fungal-only, and insect-only treatments. RNA sequencing revealed 23,801 differentially expressed unigenes in cjYB1Z4 (optimal combination) versus control BMZ. Upregulated unigenes were enriched in isoflavone biosynthesis, flavonoid biosynthesis, and sesquiterpenoid and triterpene biosynthesis. Fifty sesquiterpene-related differential unigenes encoded seven key enzymes in the MVA pathway, seven key enzymes in the MEP pathway, and seven terpene synthases. Co-expression network analysis indicated that transcription factors (e.g., WRKY33, ABF, WRKY2) potentially regulate agarwood sesquiterpene formation. This work elucidates preliminary effects and molecular mechanisms of insect- and fungi-induced agarwood formation in A. sinensis, advancing agarwood induction technology.