The effects of small geographical resolution and age on the phyllosphere microbial diversity of <i>Castanopsis eyrei</i> in subtropical forest

ABSTRACT Our understanding of the mechanisms that maintain phyllosphere microbial diversity in natural systems remains far less developed than our understanding of belowground microbiomes. This knowledge gap hinders our comprehension of growth dynamics in the Fagaceae, a predominant tree family in subtropical regions, and the critical role it plays as a major forest community assembly. Here, we tested leaves from Castanopsis eyrei , a widespread subtropical Fagaceae species in south-eastern China, sampled across multiple ages within a forest dynamics plot on Mt. Huangshan. Using third-generation sequencing of full-length bacterial 16S rRNA genes and fungal ITS regions, we characterized the associated phyllosphere microbiota. We found that phyllosphere fungal diversity was high, dominated by Teratosphaeriaceae, Trimorphomycetaceae, and Bulleribasidiaceae, while bacterial diversity was lower and primarily comprised Beijerinckiaceae, Isosphaeraceae, and Acidobacteriaceae. Habitat, rather than host age, emerged as the principal factor influencing fungal and pathogen diversity. Linear mixed-effects models revealed a negative relationship between C. eyrei biomass and phyllosphere pathogen diversity. Co-occurrence network analysis showed that C. eyrei saplings supported the most complex network structure, with Recuromyces acting as a key pathogenic fungus, serving as both a module hub and a connector across all age classes. Overall, these findings highlight the ecological importance of habitat in shaping phyllosphere microbial diversity and underscore the interplay between host function and the maintenance of microbial diversity. IMPORTANCE Plant surfaces host diverse microbial communities that significantly impact host health and overall forest productivity. However, mechanisms maintaining phyllosphere microbial diversity and their consequences for host plants remain poorly understood. Employing a three-generation high-throughput sequencing approach, we investigated the phyllosphere fungal and bacterial diversity across different microhabitats and ages of Castanopsis eyrei , a common species in subtropical forest, China. Our results underscore the presence of exceptionally high microbial diversity on the plant surface, elucidating the taxonomic composition at the family level of key host microorganisms. Furthermore, our observation of a negative correlation between host performance and phyllosphere pathogens underscores the potential self-limiting ability of plants.