Phyllosphere Bacterial Communities in Spinach Are Influenced by Soil Nitrogen Availability

Nitrogen is an essential macronutrient for the growth and development of plants. For spinach, as for most crops, nitrogen-based fertilizers are used to improve productivity and quality. Although the significance of the soil microbiome in the inorganic nitrogen cycle is well recognized, soil nitrogen's impact on phyllosphere bacterial communities remains unclear. This study investigated changes in the community composition and diversity of bacteria residing on spinach leaves cultivated in soils with varying nitrogen levels. Sixty-eight spinach samples were processed using culture-dependent and -independent methods to evaluate phyllosphere-associated bacterial communities. In culture-dependent methods, populations of mesophiles, psychrotrophs, coliforms, and lactic acid bacteria were quantified by plate counts using appropriate culture media. In culture-independent methods, bacteria were determined by amplicon sequencing based on the V3-V4 region of the bacterial 16S rRNA gene. Counts of mesophiles, psychrotrophs, coliforms, and lactic acid bacteria on low-nitrogen soil-grown spinach were 7.4, 7.1, 6.9, and 5.5 log CFU/g, respectively, whereas high-nitrogen soil-grown spinach showed counts of 7.5, 7.0, 7.0, and 5.7 log CFU/g. These counts did not differ significantly according to the nitrogen levels used for spinach cultivation ( P > 0.05). Amplicon sequencing, however, revealed differences in relative abundance between different taxa levels and soil nitrogen levels. The relative abundance of Erwiniaceae and Planococcaceae was higher in high-nitrogen soil-grown spinach. The relative abundance of Pseudomonadaceae, Enterobacteriaceae, and Rhizobiaceae was higher in low-nitrogen soil-grown spinach. Soil nitrogen content significantly affected spinach phyllosphere microbiome beta diversity. Our results suggest that the bacterial communities of the spinach phyllosphere are influenced by soil nitrogen content. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .