Jun 04, 2025
Isolation of bacteria from the roots of maize
- Ashley Paulsen1,2,3,
- Dua X Vang1,2,4,
- Larry Halverson1,2,4,3
- 1Department of Plant Pathology, Entomology, and Microbiology, Iowa State University, Ames, IA, 50011;
- 2Ames National Laboratory, Ames, IA, 50011;
- 3Interdepartmental Genetics and Genomics Graduate Program;
- 4Interdepartmental Microbiology Graduate Program
Protocol Citation: Ashley Paulsen, Dua X Vang, Larry Halverson 2025. Isolation of bacteria from the roots of maize. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge8z3og47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 04, 2025
Last Modified: June 04, 2025
Protocol Integer ID: 219578
Keywords: bacteria for the maize rhizosphere, maize rhizosphere, isolation of bacteria, roots of maize protocol, maize protocol, bacteria, maize, bulk soil, marsc, synthetic community
Funders Acknowledgements:
National Science Foundation
Grant ID: DGE-1545453
National Science Foundation
Grant ID: DRL-1759152
The Iowa Agriculture and Home Economics Experiment Station, and the U.S. Department of Energy (DOE) Office of Biological and Environmental Research, Biological Systems Division
Grant ID: FWP No AL-18-380-055
Disclaimer
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do
not necessarily reflect the views of the National Science Foundation.
Abstract
Protocol used to isolate bacteria for the MAize Rhizosphere Synthetic community (MARSc) from the bulk soil, rhizosphere, and endosphere of maize.
Rhizotrons were filled with raw field soil with low inorganic nitrogen inputs, collected from diversified plots at the Marsden long-term cropping system experimental site, as described previously (1)
Two surface-sterilized, pre-germinated Phz51 or BGEM-0111-S/Phz51 (2) maize seedlings were planted in each rhizotron
Plants were grown in a growth chamber with a 16-h/8-h light/dark photoperiod at 24 °C until the V4/V5 developmental stage
Soil, rhizosphere (root surfaces and adherent soil), and endosphere samples were obtained as described previously (1), summarized with modifications below
Note
1. Bulk soil samples were aseptically collected (∼1 g; >1 cm from a root) and then the root system was extracted
2. The rhizosphere was obtained by vortexing and sonicating the roots in 1 mM phosphate buffer (pH 7.2)
3. Washed roots were pulverized in a ball mill with 5 mL of phosphate buffer to collect endosphere samples
Aliquots of all samples were serial diluted in 1mM phosphate buffer and plated onto R2A or 1/10th strength TSA
Plates were incubated at 27˚C until colonies appeared, then colonies were passaged until pure
Genomic DNA of each pure isolate was extracted using the Wizard gDNA Isolation Kit (Promega)
The full 16S rRNA gene (V1-V9) was Sanger sequenced for each isolate using the primers 8F and 1492R and identities confirmed via BLASTn (3)
Protocol references
1. Bay G, Lee C, Chen C, Mahal NK, Castellano MJ, Hofmockel KS, Halverson LJ. 2021. Agricultural Management Affects the Active Rhizosphere Bacterial Community Composition and Nitrification. mSystems 6:10.1128/msystems.00651-21.
2. Sanchez DL, Santana AS, Morais PIC, Peterlini E, De La Fuente G, Castellano MJ, Blanco M, Lübberstedt T. 2023. Phenotypic and genome-wide association analyses for nitrogen use efficiency related traits in maize (Zea mays L.) exotic introgression lines. Front Plant Sci 14.
3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. Journal of Molecular Biology 215:403–410.