Exploring the microbial response as a potential bio-indicator for soil health : insights from a controlled incubator experiment

Soil microorganisms are known to be sensitive to disturbances in their surroundings, and those microbial shifts can reflect the soil health status. Therefore, these shifts can potentially be used as a bio-indicator for soil health. However, measurements taken directly on field samples are often affected by strong spatiotemporal trends that are not related to the soil health status. To address this issue, this study aimed to understand whether the response of the bacterial and fungal soil communities after short-term disturbances in a controlled incubator experiment has the potential to reflect the soil health status. The study was conducted on two arable soils with contrasting health status, which was determined based on extensive background information up to ten years. We studied the response of the soil bacterial and fungal communities after short-term disturbances (drying-rewetting and/or chitin amendment, consecutive disturbances). These disturbances were selected because drying and rewetting alters the moisture dynamics and consequently the dynamics of the bacterial and fungal communities, whereas chitin application releases nitrogen into the soil and boosts the microbial biomass and activity. Three techniques were applied and compared to study the response in the bacterial and fungal communities: metabarcoding (relative changes), PLFA analysis (absolute biomass) and HWC (labile C fraction). Using metabarcoding, the soil microbial communities gave a consistently greater response in the less healthy soil after shortterm disturbances. More specifically, shifts upon drying and rewetting were up to two- to three-fold larger in the less healthy soil compared to the healthy soil. Soils that were exposed to drought for a second time were less responsive, even for the less healthy soil, indicating that a legacy effect is present. Whereas we expected that chitin could at least partially reduce the effects of drought, the combination of both disturbances led to more shifts in the microbial community, but mostly in the less healthy soil. Shortly after disturbances, the response of the bacterial and fungal soil communities measured by metabarcoding, in a pot trial, reflected the soil health status. The magnitude of the response for bacteria and fungi were both valuable for the assessment of soil health. Traditional soil health indicators like HWC and PLFA, but also DNA-related indicators such as diversity and taxonomical indicators were less sensitive to measure this response. This leads us to recommend the magnitude of the response of bacteria and fungi analyzed shortly after disturbances and measured with metabarcoding, being a consistent and sensitive bio-indicator for soil health.Availability of data and materials: The raw demultiplexed sequence data are available in the NCBI Sequence Read Archie under accession number PRJNA735907. Scripts used to run all data analysis can be found at https:// gitlab.com/lljoos/soilmicrobiome_droughtrewettingchitin