Grapevine decline affects viticulture worldwide. It is caused by a wide range of individual and combined biotic and abiotic factors. Some declines remain unexplained because they are not associated with known pathological symptoms or mineral dysregulation. Vineyard microbiological quality is an important area of study, since grapevine-associated microbiome primarily originates in vineyard soils and determines host health and development. To understand the decrease of growth and yield, and the high mortality of plants in vineyards affected by these declines, a multisite study investigated soil microbial communities. Spatial (terroir: two distinct geographical locations) and temporal (season: autumn and spring) dimensions were added to the inter-row soil status factor (S for areas with symptomatic vines and AS for those with asymptomatic vines). The microbiomes of AS and S soils were analyzed using high-throughput sequencing based on the bacterial 16S rRNA gene, the fungal ITS1 region (Internal Transcribed Spacer), or the fungal 18S rRNA gene for Glomeromycota family. Geographical location was the strongest driver of bacterial and fungal microbial communities, while the seasonal factor primarily influenced bacterial community. Based on metabarcoding analysis, symptomatic soils presented enriched bacterial taxa that can be potentially beneficial for grapevine growth. In addition, fungal diversity and richness, including Glomeromycota division, were greater in symptomatic soils. Fungal genera associated with grapevine diseases were detected across all conditions, with higher abundances in symptomatic soils. These findings reveal that vineyard soils affected by unexplained decline are a potential source of both fungal pathogens and beneficial microorganisms. © 2022 The AuthorsRead less <