Contrasting Phyllosphere Mycobiome in two Lycopodiaceae Plant Species: Unraveling Potential HupA-Producing Fungi and Fungal Interactions

Background: Huperzine A (HupA) is a natural lycopodium alkaloid renowned for its efficacy in treating neurodegenerative diseases such as Alzheimer's disease. It specifically occurs in the Huperzioideae subfamily of Lycopodiaceae. Fungi associated with Huperzioideae species are potential contributors to HupA biosynthesis, offering promising prospects for HupA production. However, limited knowledge of fungal diversity in lycophytes, coupled with decreased HupA production over time of fungal strains, has impeded the discovery and applications of HupA-producing fungi. Here, we investigated huperzine concentrations and the mycobiome across various tissues of two Lycopodiaceae species, Huperzia asiatica (a HupA producer) and Diphasiastrum complanatum (a non-HupA producer). Our objectives across the tissues of the two species are to unveil the distribution of potential HupA-producing fungi and elucidate fungal interactions within the mycobiome, aiming to uncover the role of HupA-producing fungi and pinpoint their potential fungal facilitators. Results: Among the tissues, H. asiatica exhibited the highest HupA concentration in apical shoots (360.27 μg/ml) whereas D. complanatum showed no HupA presence in any tissue. We obtained 441 Amplicon Sequence Variants (ASVs) from H. asiatica and 497 ASVs from D. complanatum. The fungal communities in bulbils and apical shoots of H. asiatica were low in diversity and dominated by Sordariomycetes, a fungal class harboring the majority of reported HupA-producing fungi. Integrating bioinformatics with published experimental reports, we identified 27 potential HupA-producing fungal ASVs, primarily in H. asiatica, with 12 ASVs identified as hubs in the fungal interaction network, underscoring their pivotal roles in mycobiome stability. Members of certain fungal genera, such as Penicillium, Trichoderma, Dioszegia, Exobasidium, Lycoperdon and Cladosporium exhibited strong connections with the potential HupA producers in H. asiatica's network rather than in D. complanatum's, implying their prospects as fungal facilitators in enhancing HupA production. Conclusions: This study advances our knowledge of fungal diversity in Lycopodiaceae and provides insights into the search for potential HupA-producing fungi and fungal facilitators. It highlights the importance of exploring young tissues, and emphasizes the ecological interactions that promote the fungi-mediated production of complex bioactive compounds, offering new directions for research in fungal ecology and secondary metabolite production. ### Competing Interest Statement The authors have declared no competing interest.