Characterization of Boxwood Shoot Bacterial Communities and Potential Impact from Fungicide Treatments

Phyllosphere bacterial communities play important roles in plant fitness and growth. The objective of this study was to characterize the epiphytic and endophytic bacterial communities of boxwood shoots and determine how they may respond to commonly used fungicides. In early summer and early fall, shoot samples were collected immediately before and 1, 7, and 14 days after three fungicides containing chlorothalonil and/or propiconazole were applied to the canopy. Total genomic DNA from shoot surface washings and surface-sterilized shoot tissues was used as the template for 16S rRNA metabarcoding, and the amplicons were sequenced on a Nanopore MinION sequencer to characterize the epiphytic and endophytic communities. The bacterial communities were phylogenetically more diverse on the boxwood shoot surface than in the internal tissue, although the two communities shared 12.7% of the total 1,649 identified genera. The most abundant epiphytes were Methylobacterium and Pantoea, while Stenotrophomonas and Brevundimonas were the dominant endophytes. Fungicide treatments had strong impacts on epiphytic bacterial community structure and composition. Analysis of compositions of microbiomes with bias correction (ANCOM-BC) and analysis of variance (ANOVA)-like differential expression (ALDEx2) together identified 312 and 1,362 epiphytes changed in abundance due to fungicide treatments in early summer and early fall, respectively, and over 50% of these epiphytes were negatively impacted by fungicide. The two chlorothalonil-based contact fungicides demonstrated more marked effects than the propiconazole-based systemic fungicide. These results are foundational for exploring and utilizing the full potential of the microbiome and fungicide applications and developing a systems approach to boxwood health and production.