Spatial microbiome and synthetic community analyses reveal Trinickia sclerotiorum sp. nov. promotes sclerotia mortality of Sclerotinia sclerotiorum.

Bulk microbiome analysis obscures the spatial heterogeneity of microbial communities, limiting the understanding of bacterial distribution inside fungal sclerotia, which are the survival structures of the plant pathogen Sclerotinia sclerotiorum. Although studies have profiled microbiomes of different fungal structures, sclerotia microbiome remains largely unexplored. We applied bulk microbiome using PacBio full-length 16S rRNA sequencing, synthetic community (SynCom), and 10× Genomics Xenium platform to resolve the spatiotemporal contribution of soil bacteria to sclerotia mortality. In the bulk microbiome, the relative abundance of soil bacteria such as Massilia and Trinickia were enriched when sclerotia mortality increased under flooding conditions. Twelve enriched sclerotia-associated bacteria were assembled into a SynCom to study their causality for sclerotia mortality. Spatial microbiome analysis revealed the temporal dynamics and distribution pattern of each SynCom member. Among them, Trinickia sclerotiorum sp. nov. type strain MC (TsMC) was found to reach the most interior sclerotia and its relative abundance coincided with sclerotia mortality. TsMC exhibited the highest importance in four antagonistic assays and SynCom drop-out test. Whole genome sequencing confirmed TsMC as a novel species with antifungal potential. Collectively, we document a novel bacterium for sclerotia mortality and demonstrate the advantage of spatiotemporal distribution in microbiome research.