Plant leaves harbor a rich diversity of bacteria and fungi that, through their interactions with host plants, assume an influential role in plant physiological and metabolic processes. The unique phyllosphere environment of different plant species may shape and select distinct phyllosphere microbial communities. While most academic research has focused on the phyllosphere microorganisms within the same plant variety, there is relatively limited research on the phyllosphere microbial communities between different varieties. Populus L. is a typical tree species in temperate monsoon climates, widely distributed in northern China, and it constitutes a crucial component of China’s forestry resources. For the purpose of this study, we investigated the community structure and diversity of phyllosphere fungi and bacteria in different poplar varieties under identical growth conditions to elucidate the main factors contributing to differences in phyllosphere microbial communities among these varieties. Our findings revealed variations in nitrogen, phosphorus, starch, and soluble sugar contents among the three poplar species studied. Additionally, there were considerable disparities in both abundance and α diversity index of phyllosphere fungal and bacterial communities among these species. At the phylum level, Ascomycota and Basidiomycota have been identified as the dominant fungal communities; while Proteobacteria and Actinomycetes were dominant bacterial communities. The correlation analysis pointed out that chemical traits in the leaves, in particular the total phosphorus and the quantity of soluble sugar, had a significant correlation with the structure and diversity of the microbial community residing in the phyllosphere. Overall, our results demonstrate that even under identical site conditions, each poplar species harbors its own unique phyllosphere microbial community composition as well as distinct leaf characteristics—highlighting host plant diversity as a crucial factor driving differences in phyllosphere microbial composition.

Phyllosphere microorganisms interact with host plants to regulate growth, promote nutrient uptake and enhance stress tolerance with host specificity, while arbuscular mycorrhizal fungi facilitate plant nutrient absorption and stress adaptation. Current poplar microbial studies mostly focus on hermaphroditic species, with limited research on dioecious poplars. This study selected four poplar species commonly hybridized with Populuscathayana and Populus deltoides in the Xinmin area of Liaoning Province as research subjects: two female plants, DM-9-18 and DX-08-01, and two male plants, 2111 and Qingshan poplar. We performed MiSeq high-throughput sequencing targeting bacterial 16S rRNA, fungal ITS, and arbuscular mycorrhizal fungal (AMF) marker genes from poplar phyllosphere, coupled with chemical quantification of leaf, root and rhizosphere soil, to disentangle clone- and sex-associated divergence in microbial assemblages and their core environmental drivers. No significant gender differences were observed in leaf and rhizosphere nutrient levels and microbial α diversity, whereas male poplars had higher rhizosphere soil nutrients. Male and female poplars genotypes harbored distinct microbial ASVs. The dominant phyllosphere microbes and arbuscular mycorrhizal fungi exhibited gender-specific abundance variations, and nutrient content was the key factor shaping microbial communities. This study clarifies microbial community differences among the four selected hybrid poplar clones. While the experimental design confounds sex with host genotype, the observed patterns provide insights into potential sex-related variations. Our results advance the mechanistic understanding of how dioecious poplar genotype and sexual phenotype jointly filter leaf and root-associated microbial symbionts, with applied implications for hybrid poplar breeding and shelterbelt microbial regulation.