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United States Department of Agriculture

Agricultural Research Service

Research Project: GENOMICS AND ENGINEERING OF STRESS TOLERANT MICROBES FOR LOWER COST PRODUCTION OF ETHANOL FROM LIGNOCELLULOSE

Location: Bioenergy Research Unit

Title: High-throughput assay for optimising microbial biological control agent production and delivery

Authors
item Slininger, Patricia
item Schisler, David

Submitted to: Biocontrol Science and Technology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 22, 2013
Publication Date: June 14, 2013
Citation: Slininger, P.J., Schisler, D.A. 2013. High-throughput assay for optimising microbial biological control agent production and delivery. Biocontrol Science and Technology. 23(8):920-943.

Interpretive Summary: Lack of technologies to produce and deliver effective biological control agents (BCAs) is a major barrier to their commercialization. A myriad of variables associated with BCA cultivation, formulation, drying, storage, and reconstitution processes complicates agent quality maximization. An efficient assay using a 96-well microplate format to allow an integrated approach to optimizing these process variables was developed. Relevant variables, such as culture harvest age, cultivation and formulation ingredients, storage atmosphere and temperature, were tested with each step of the assay process to view their individual and combined impact on resultant microbial activity. The utility of this method to evaluate many treatments is demonstrated on seven strains of Pseudomonas fluorescens and Enterobacter cloacae known to suppress fungal diseases of wheat or potatoes. The assay will be useful in the development of many beneficial microbial genera having crop-protective activities by allowing the development of extensive data libraries for trend analysis. This new optimization tool will impact the development of the biological pest control industry and the agricultural community by adding to the technology base needed to successfully manufacture effective new crop protection products in a timely manner. New bio-based products comprised of beneficial bacteria and other microorganisms offer an efficient, environmentally compatible means to control fungal diseases of wheat and potatoes as well as many other agriculturally important pests.

Technical Abstract: Lack of technologies to produce and deliver effective biological control agents (BCAs) is a major barrier to their commercialization. A myriad of variables associated with BCA cultivation, formulation, drying, storage, and reconstitution processes complicates agent quality maximization. An efficient assay using a 96-well microplate format to allow an integrated approach to optimizing these process variables is presented. The assay involves growing the BCA of interest in flasks or fermentors, formulating cells harvested from growth cultures, delivering microliter droplets of formulated cells to microplate wells, air-drying droplets, storing plates, reconstituting dried cells, and monitoring the rate of cell growth to a specified yield using a plate-reading spectrophotometer. Spectrophotometer assessments of cell activity were coupled with micro-dilution plate viable cell enumeration and a wounded potato assay of bioefficacy for potato dry rot suppressive strains. Relevant variables (culture harvest age, cultivation and formulation ingredients, storage atmosphere and temperature) are tested with each step of the assay process to view their individual and combined impact on resultant microbial activity. The utility of this method to evaluate many treatments is demonstrated on seven strains of Pseudomonas fluorescens and Enterobacter cloacae known to suppress fungal diseases of wheat and potatoes.

Last Modified: 4/23/2014