Scalable Agricultural Microbiome Sampling: Operational Definitions, Pooling Strategies, and Preservation Methods

bioRxiv (Cold Spring Harbor Laboratory)

Abstract

Abstract Scalable soil microbiome monitoring requires sampling methods that are reproducible across operators, field sites, and logistical constraints. Here, we evaluated three key methodological choices that commonly limit comparability in agricultural rhizosphere studies: how the rhizosphere sampling unit is operationally defined, sample pooling strategies, and preservation methods. We introduce the RhizoCore, a standardized root-zone soil core defined by core diameter, depth, position relative to the plant, and subsample volume, as a practical proxy for traditional rhizosphere sampling. The RhizoCore method captured more than 92% of the sequencing depth found in traditional rhizosphere samples, with differences limited predominantly to low-abundance taxa. Preservation methods significantly affected bacterial communities, while sample pooling showed greater impact on fungal diversity and substantially reduced within-group variability across all treatments. Despite these effects, differential abundance analysis revealed minimal compositional changes, with only a small fraction of microbial taxa significantly affected by either pooling or preservation method. Our findings demonstrate that the RhizoCore method provides a reproducible, and scalable approach for rhizosphere sampling that balances scientific rigor with practical field implementation, offering a framework for large-scale soil microbiome monitoring programs and for improving comparability among agricultural microbiome studies across diverse environmental conditions.

Authors 7

1. Adam Ossowicki corresponding

   Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" · Universidad de Málaga

   Affiliation as printed

   Departamento de Microbiología, Facultad de Ciencias, Campus Universitario de Teatinos s/n, Universidad de Málaga, 29010 Málaga, Spain

   Departamento de Protección de Cultivos, Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Campus Universitario de Teatinos, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), 29010 Málaga, Spain

2. Thom Griffioen

   Syngenta (Switzerland)

   Affiliation as printed

   Syngenta Crop Protection AG, Schaffhauserstrasse 101, Stein AG, Switzerland

3. Enrichetta Mileti
   Affiliation as printed

   Syngenta Biologicals, Via Cagliari 1 -Zona Industriale, 66041 Atessa (CH), Italy

4. Valentino Attanasi
   Affiliation as printed

   Syngenta Biologicals, Via Cagliari 1 -Zona Industriale, 66041 Atessa (CH), Italy

5. Chris Hames
   Affiliation as printed

   Metagen Pty, Ltd., Gatton, Ǫueensland, Australia

6. Víctor Carrión Aachen Institute of Biology, Leiden University

   Leiden University · Netherlands Institute of Ecology · Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora" · Universidad de Málaga

   Affiliation as printed

   Departamento de Microbiología, Facultad de Ciencias, Campus Universitario de Teatinos s/n, Universidad de Málaga, 29010 Málaga, Spain

   Departamento de Protección de Cultivos, Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Campus Universitario de Teatinos, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), 29010 Málaga, Spain

   Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen, Netherlands

   Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands

7. Ben O. Oyserman

   Syngenta (Switzerland)

   Affiliation as printed

   Syngenta Crop Protection, Rosentalstrasse 67, 4058 Basel, Switzerland