Unravelling the fungal endomicrobiome of Picrorhiza kurrooa for increasing in-planta picroside biosynthesis using endophytic Trichoderma harzianum PKRF1

BACKGROUND: Endophytic fungi form an integral part of plant microbiomes, influencing host physiology, stress resilience, and secondary metabolism. While next-generation sequencing (NGS) has greatly advanced the identification of endophytes, it often falls short of assigning functional roles, necessitating integration with culture-based approaches for downstream applications. Picrorhiza kurrooa, a critically endangered Himalayan medicinal herb valued for its hepatoprotective picrosides, suffers from reduced metabolite content in tissue culture-derived plants, likely due to microbiome loss in the course of aseptic in-vitro practices. Moreover, the diversity and functional role of fungal endomicrobiome in P. kurrooa remain unexplored. METHODS: Internal transcribed spacer (ITS)-based amplicon sequencing was performed to assess and compare the endophytic fungal communities of wild-type (Wt) and in-vitro propagated (Tc) P. kurrooa. Fungal taxa unique to Wt-plants were identified and cross-referenced with culturable isolates. A dominant isolate present only in Wt-plants, Trichoderma harzianum PKRF1, was reintroduced into Tc-plants to evaluate its effect on plant growth and picroside biosynthesis. Whole-genome sequencing and comparative genomics of PKRF1 were also conducted to elucidate its functional capabilities and possible candidates for its endophytic nature. RESULTS: Metagenomic analysis revealed a significant reduction in fungal diversity in Tc plants, with several taxa, including Trichoderma, Cyphellophora, and Preussia, exclusively associated with Wt-plants. Inoculation of Tc-plants with PKRF1 led to successful root colonization, enhanced photosynthetic efficiency, biomass, and significantly higher levels of picrosides. Transcript profiling confirmed upregulation of key biosynthetic genes. Genomic analysis of PKRF1 revealed genes associated with multiple plant-beneficial traits, including nutrient acquisition, phytohormone production, stress tolerance, plant colonization, and competitive interactions, distinguishing it from non-endophytic Trichoderma isolates. CONCLUSIONS: These findings provide the first comprehensive insight into changes in endophytic fungal diversity of P. kurrooa associated with in-vitro cultivation. Furthermore, the application of cultivated endophytes from wild plants demonstrated the potential to restore microbial functions lost during in-vitro propagation and enhance secondary metabolite production in cultivated plants. Overall, this approach offers a promising strategy to integrate metagenomic information into beneficial plant-microbe interactions for practical applications.