Plant species, metabolites, and environmental factors shape the phyllosphere microbiota of grazed grasslands

The phyllosphere of permanent grasslands is a reservoir of microbial diversity, yet its composition and the factors influencing its fluctuations are still poorly understood. The aim of this study was to identify the factors driving the structure of the phyllosphere microbiota. Three plant species of two adjacent grasslands, one of the two receiving nitrogen (N) fertilization, were selected: two fructan-accumulating grasses (perennial ryegrass, <i>Lolium perenne</i> and Yorkshire fog, <i>Holcus lanatus</i>) and a non-fructan species (white clover, <i>Trifolium repens</i>). Samples were collected at three periods-two in summer and autumn-and the weather parameters were recorded. Bacterial enumerations and metabarcoding analysis of the plant microbiota were performed. Carbon and N) contents of the leaves and stomatal conductance regulation were assessed. The composition of leaf microbiota was determined mainly by the plant host species<i>-T. repens</i> grouping separately from the other plant species-, the period of the year (early summer versus late summer and autumn), and, to a lesser extent, fertilization. <i>Methylobacterium</i> was particularly dominant in <i>T. repens</i>. A significant effect of pinitol and fructans levels on the composition of the phyllosphere microbiota was observed in <i>T. repens</i> and in the <i>Poaceae</i> species, respectively. The daily temperature range, the rainfall amount, the daily average radiation, and evapotranspiration were significant drivers of the phyllosphere microbiota composition. This study provides insights into plant and environmental factors that shape grassland phyllosphere microbiota.IMPORTANCEThe phyllosphere is estimated to cover more than 10⁸ km² over the globe, and grasslands cover about 30%-40% of the world land area. Grasslands host a vast microbial reservoir, characterized by both the abundance and diversity of microorganisms. Evidence of connections between phyllosphere and raw milk microbiota exists. The significance of our research is in highlighting grasslands as potential reservoirs of beneficial microorganisms for plants and dairy-derived products. The phyllosphere microbiota should be considered when evaluating the ecosystem services of grasslands as it contributes to supporting services by enhancing biodiversity and to provisioning services through microorganisms that can benefit agriculture and the food industry.