(312d) Rapid Screening and Identification of Microbial Isolate Collections Using Microwell Recovery Arrays (Faculty/Industry Candidate)

Barua, N., Kansas State University
Hansen, R., Kansas State University
Herken, A., Kansas State University
Platt, T., Kansas State University
Screening soil and root microbiomes for discovery of microorganisms that can inhibit the survival and growth of pathogenetic bacteria is important for developing improved probiotic and biocontrol agents. With the aid of the microwell recovery array (MRA), a novel high throughput screening tool, libraries containing hundreds to thousands of unique isolates are rapidly screened for pathogen suppression. The MRA is designed to generate random combinations between a fluorescently-labeled bacterial pathogen and a controlled number of microbiome isolates. Isolates that display the highest levels of pathogen antagonism can be extracted, validated with off-chip assays, then sequenced and identified. Here, MRAs were used to investigate Agrobacterium tumefaciens interactions with other members of plant microbiomes. A. tumefaciens is a key plant biotechnology tool and also the causative agent of Crown Gall disease. While A. tumefaciens pathogenesis and intraspecific interactions have been studied extensively, far less is known about A. tumefaciens interactions with other members of plant microbiomes. Fluorescently labeled A. tumefaciens sp.15955 was combined with non-pathogenic Agrobacterium isolates collected from native plant roots at the Konza Prairie Biological Station (Manhattan, KS) to isolate species capable of suppressing A. tumefaciens sp.15955. First, an inoculum of A.tumefaciens sp. 15955 expressing gfp and root isolates were seeded in microwells at various cellular ratios. After culture, selective recovery of cells from individual wells displaying highest levels of 15955 inhibition was achieved using a high-resolution, light-based extraction system. The interactions are then validated and the isolates are characterized. In our primary data, we have uncovered several candidates for inhibiting A.tumefaciens sp. 15955 growth. Discovery of such growth inhibiting isolates will help improve plant productivity by using them as reliable biocontrol agents that prevent Crown Gall disease.