Plant growth-promoting bacteria (PGPB) are valuable for supporting sustainable food production and may alleviate the negative impacts of chemical fertilizers on human health and the environment. While single-strain inoculations have proven unreliable due to poor survival and colonization in the rhizosphere, application of PGPB in multispecies consortia has the potential to improve these outcomes. Here, we describe a new approach for screening and identifying bacterial consortia that improve the growth of corn relative to plants inoculated with a single strain. The method uses the microwell recovery array (MRA), a microfabricated high-throughput screening device, to rapidly explore the maize ( Zea mays L .) rhizobiome for higher-order combinations of bacteria that promote the growth and colonization of the nitrogen-fixing PGPB, Azospirillum brasilense . The device simultaneously generates thousands of random, unique combinations of bacteria that include A. brasilense and members of the maize rhizobiome, then tracks A. brasilense growth in each combination during co-culture. Bacteria that show the highest levels of A. brasilense growth promotion are then recovered from the device using a patterned light extraction technique and are identified. With this approach, the screen uncovered growth-promoting consortia consisting primarily of bacteria from the Acinetobacter - Enterobacter - Serratia genera, which were then co-inoculated with A. brasilense on axenic maize seedlings that were monitored inside a plant growth chamber. Compared to maize plants inoculated with A. brasilense alone, plants that were co-inoculated with these consortia showed accelerated growth after 15 days. Follow-up root colonization assays revealed that A. brasilense colonized at higher levels on roots from the co-inoculated seedlings. These findings demonstrate a new method for rapid bioprospecting of root and soil communities for complementary PGPB and for developing multispecies consortia with potential use as next-generation biofertilizers.
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This content will become publicly available on February 28, 2025
Photo-addressable microwell devices for rapid functional screening and isolation of pathogen inhibitors from bacterial strain libraries
Discovery of new strains of bacteria that inhibit pathogen growth can facilitate improvements in biocontrol and probiotic strategies. Traditional, plate-based co-culture approaches that probe microbial interactions can impede this discovery as these methods are inherently low-throughput, labor-intensive, and qualitative. We report a second-generation, photo-addressable microwell device, developed to iteratively screen interactions between candidate biocontrol agents existing in bacterial strain libraries and pathogens under increasing pathogen pressure. Microwells (0.6 pl volume) provide unique co-culture sites between library strains and pathogens at controlled cellular ratios. During sequential screening iterations, library strains are challenged against increasing numbers of pathogens to quantitatively identify microwells containing strains inhibiting the highest numbers of pathogens. Ring-patterned 365 nm light is then used to ablate a photodegradable hydrogel membrane and sequentially release inhibitory strains from the device for recovery. Pathogen inhibition with each recovered strain is validated, followed by whole genome sequencing. To demonstrate the rapid nature of this approach, the device was used to screen a 293-membered biovar 1 agrobacterial strain library for strains inhibitory to the plant pathogen Agrobacterium tumefaciens sp. 15955. One iterative screen revealed nine new inhibitory strains. For comparison, plate-based methods did not uncover any inhibitory strains from the library (n = 30 plates). The novel pathogen-challenge screening mode developed here enables rapid selection and recovery of strains that effectively suppress pathogen growth from bacterial strain libraries, expanding this microwell technology platform toward rapid, cost-effective, and scalable screening for probiotics, biocontrol agents, and inhibitory molecules that can protect against known or emerging pathogens.
more » « less- Award ID(s):
- 1650187
- NSF-PAR ID:
- 10493933
- Publisher / Repository:
- American Institute of Physics
- Date Published:
- Journal Name:
- Biomicrofluidics
- Volume:
- 18
- Issue:
- 1
- ISSN:
- 1932-1058
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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