3.5.2S
MOLECULAR APPROACHES TO IMPROVE THE EFFICACY OF BACTERIAL BIOCONTROL AGENTS


LS THOMASHOW1,2, DV MAVRODI2, Z HUANGl, JM RAAIJMAKERSl and DM WELLER1,2

lUSDA-Agricultural Research Service and 2Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA

Background and objectives
Recent efforts to understand the molecular and biochemical basis of biological control in the rhizosphere have established that antibiotics have a dominant role in the control of certain soilborne pathogens. Genes for the synthesis of antibiotics with known biocontrol activity have been cloned, characterized, and found to be regulated by complex global hierarchies that coordinate antibiotic production with other physiological activities underlying microbial growth and adaptation to environmental stress. Here we show how this knowledge has been applied to understand and overcome the causes of inconsistent field performance that until now have retarded the commercial development and widespread use of bacterial biocontrol agents.

Results and conclusions
When genes that contribute to the suppression of soilborne pathogens are known, screening methods can be tailored to identify effective new agents from native populations adapted to particular crops or locations where biological control is needed. The antibiotic 2,4 diacetylphloroglucinol (Phl) is involved in the biological control of various soilborne pathogens by plant-associated fluorescent pseudomonads of worldwide origin. Phi-specific probes and PCR primers based on the DNA sequence of the phID biosynthetic gene were used to distinguish Phi-producing strains among bacterial isolates from the roots of wheat grown in soils that exhibit the natural biological control of take-all called take-all decline. Such strains control take-all when introduced into nonsuppressive soils, and are thought to have a key role in take-all decline [1].

Antibiotic synthesis is tightly linked to the overall metabolic status of the cell, which in turn is dictated by nutrient availability and other environmental stimuli. Altering or uncoupling this linkage can lessen the influence of the environment on antibiotic synthesis, thus stabilizing or increasing antibiotic production and the biocontrol activity of the modified strain. Strains with increased biocontrol activity have been obtained by manipulating the regulatory genes that modulate expression of antibiotic biosynthetic operons or by substituting constitutive promoters in place of environmentally-responsive ones. Selective use of unmodified biocontrol agents under conditions known to be supportive of antibiotic gene expression is another approach to more consistent strain performance.

Because the genes required for antibiotic synthesis are structurally linked and coordinately regulated, they can be transferred as intact functional units to enhance the performance of recipient strains with other desirable attributes. Genes for the synthesis of phenazine-1 -carboxylic acid (PCA) from Pseudomonas fluorescens 2-79 were cloned under the control of the constitutively expressed tac promoter and inserted into several different sites in the genome of the Phi-producing strain P. fluorescens Q8rl-96, which aggressively colonizes the roots of wheat even when applied at very low doses and is highly effective against take-all. The transgenic derivative strains produced both Phi and PCA, colonized roots equivalently to the parental strain, retained activity against take-all, and controlled Rhizoctonia root rot and Pythium root rot significantly better than the parental strain.

References
1. Raaijmakers JM, Weller DM, 1998. Molecular Plant-Microbe Interactions 11, 144-152.