Reproducing plant microbiome research reveals site and time as key drivers of apple tree phyllosphere bacterial communities.

Manipulating plant microbiomes is foreseen as a key biocontrol avenue to tackle the accelerating challenges of global change in agriculture. Several recent studies have identified the spatiotemporal dynamics of phyllosphere microbial communities, stressing the need to understand plant microbiome drivers to design efficient biocontrol interventions. Yet, these works are often performed on small sample counts, rarely provide sufficient information on the relative impact of time or local environment, and are seldom repeated to assess reproducibility. To address these limits, we performed a longitudinal sampling across multiple orchards of contrasting agricultural practices to study the ecological drivers of phyllosphere bacterial communities of apple tree (Malus domestica, Borkh.). We sampled up to eight apple cultivars at six orchards (three conventional, three organic) in the Eastern Townships (Canada) in 2022 and 2023. In contrast with common cross-sectional microbiome studies, our work builds on a two-year sampling design, thus allowing for the evaluation of the reproducibility of previous plant microbiome research. Our results support previous findings indicating that site and time are major drivers of apple tree bacterial community structure, yet their relative influence vary across the two sampling years. In addition, our data showed that leaf and flower bacterial alpha diversity is lower at organic sites compared to conventional sites. Overall, this study provides a comprehensive longitudinal multi-site study design highlighting the value of assessing reproducibility in plant microbiome studies and paving the way for future research in this field.