Biohacking agarwood: the impact of fire drills and brine on endophytes and metabolites of Aquilaria sinensis

Agarwood is a valuable resinous aromatic substance known to have healing properties. Only plants in the Thymelaeace family, specifically the genera Aquilaria and Gyrinops, produce it to defend themselves from insect, bacterial, and fungal attacks. Over-harvesting of natural agarwood has given rise to different artificial agarwood induction techniques. However, the contribution of endophytic microorganisms in this process remains largely unknown. Herein, we employed fire drills and fire drill + brine treatments and investigated their impact on metabolite constituents and endophytes over time. Gas chromatography-mass spectrometry was used to examine the metabolites, and 16 S and ITS amplicon sequencing were applied to check for endophytes. Metabolites from each treatment were related to 16 S and ITS amplicon sequencing results from different times to find out what effect the treatments had and what role endophytes played in making agarwood. Fire drills with 50-80 ml of 0.4 mmol brine treatment resulted in the highest production of essential oil from agarwood, whereas fire drills with 50-80 ml of 4.0 mmol brine treatment produced the highest number of metabolites. Agarwood fragrant compounds such as chromones, sesquiterpenes, and agarotetrol were prominent among the differentially expressed metabolites and were positively associated with the abundance of bacterial endophytes Acidobacteriota, Chlamydiae, Bacteroidota, and Actinobacteria; and the fungal endophytes Rozellomycota, Basidiomycota, Aphelidiomycota, and Mortierellomycota. Saprotrophs and pathogens were prevalent in the treatment groups; however, with time, their proportion declined as Symbiotroph's proportion inclined, indicating successive roles of these fungi in agarwood induction and propagation. Among them, Acidobacteriota, Chlamydiae, Bacteroidota, and Actinobacteria bacterial; and Rozellomycota, Basidiomycota, Aphelidiomycota, and Mortierellomycota fungal endophytes are correlated with enhanced levels of essential agarwood fragrances such as Chromones, Sesquiterpenes, and Agarospirol. Our results show how important bacterial and fungal endophytes are for making agarwood. They also show how these endophytes change when interacting with the host plant after a fire drill and a brine treatment. A moderate brine concentration (0.4 mmol) following a fire drill can thus be employed as a sustainable agarwood production practice.