This study investigated how the phyllosphere endophytic microbiota was shaped by cultivars and local enviroment.

Boxwood is a renowned landscape plant and a major evergreen shrub in the U.S. green industry. Its phyllosphere endophytic microbiota play an important role in the plant's health and productivity. However, it remains unknown whether geographic location, cultivars, and growth conditions may impact the boxwood phyllosphere endophytes. Here, shoot samples were collected from four boxwood cultivars in two distinct climatic regions of the U.S., Oregon and Virginia, and in three sampling months: May, August, and November of 2021. DNA from the surface-sterilized shoots was amplified for bacterial 16S rRNA V5-V7 and fungal ITS1 regions, and the amplicons were sequenced for endophytic metagenomics. We found that cultivar and growing region had a stronger effect on the fungal than on the bacterial community diversity, structure, and composition. Divergent microbial composition was associated with the three levels of boxwood blight resistance among the four cultivars. Particularly, marked distinct microbial signature was observed between the most susceptible and tolerant cultivars, including dissimilarity in community structure, taxa with differential abundance, and microbial indicators. Regionwide, core microbiome analysis identified three bacterial and seven fungal genera that were prevalent and abundant at all three sampling times. Many of the core bacterial genera have members beneficial to plant growth and health, while the core fungal genera contain several saprophytes and plant pathogens. This study advanced our understanding of boxwood-associated microbiomes and are fundamental for leveraging, harnessing, and manipulating beneficial microbial associations for better boxwood production and protection.