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United States Department of Agriculture

Agricultural Research Service

Title: Mutagenesis of B-1,3-Glucanase Genes in Lysobacter Enzymogenes Strain C3 Results in Reduced Biological Control Activity Towards Bipolaris Leaf Spot of Tall Fescue and Pythium Damping off of Sugarbeet

item Palumbo, Jeffrey
item Jochum, Christine - UNIVERSITY OF NEBRASKA
item Tatum, Kristin - RUTGERS UNIVERSITY
item Kobayashi, Donald - RUTGERS UNIVERSITY

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 3, 2005
Publication Date: June 1, 2005
Citation: Palumbo, J.D., Yuen, G.Y., Jochum, C.C., Tatum, K., Kobayashi, D.Y. 2005. Mutagenesis of b-1,3-glucanase genes in lysobacter enzymogenes strain c3 results in reduced biological control activity towards bipolaris leaf spot of tall fescue and pythium damping off of sugarbeet. Phytopathology.95:701-707.

Interpretive Summary: Lysobacter enzymogenes strain C3 contains three genes encoding extracellular ß-1,3-glucanases. These genes were found by their sequence similarity to the equivalent genes in L. enzymogenes strain N4-7. Mutations introduced into each gene, gluA, gluB, and gluC, resulted in the loss of production of each ß-1,3-glucanase enzyme. Similarly, mutant strains containing introduced mutations in each combination of two or three ß-1,3-glucanase genes did not produce the corresponding ß-1,3-glucanase enzymes. Strain G123, containing mutations in all three genes, showed significantly reduced biocontrol activity against Bipolaris leaf spot of tall fescue and Pythium damping off of sugarbeet relative to the wildtype strain C3. This indicates that the ß-1,3-glucanases produced by L. enzymogenes contribute to the bacterium's total biocontrol activity.

Technical Abstract: Lysobacter enzymogenes produces extracellular lytic enzymes capable of degrading the cell walls of fungi and oomycetes. Many of these enzymes, including ß-1,3-glucanases, are thought to contribute to the biological control activity expressed by several strains of the species. L. enzymogenes strain C3 produces multiple extracellular ß-1,3-glucanases encoded by the gluA, gluB, and gluC genes. Analysis of the genes indicates they are homologous to previously characterized genes in the related strain N4-7, each sharing >95% amino acid sequence identify to their respective counterparts. The gluA and gluC genes encode enzymes belonging to family 16 glycosyl hydrolases, while gluB encodes an enzyme belonging to family 64. Mutational analysis indicated the three genes accounted for the total ß-1,3-glucanase activity detected in culture. Strain G123, mutated in all three glucanase genes, was reduced in its ability to grow in a minimal medium containing laminarin as a sole carbon source. Although strain G123 was not affected in antimicrobial activity towards Bipolaris sorokiniana or Pythium ultimum var. ultimum using in vitro assays, it was significantly reduced in biological control activity against Bipolaris leaf spot of tall fescue and Pythium damping off of sugarbeet. These results provide direct supportive evidence for the role of ß-1,3-glucanases in biocontrol activity of L. enzymogenes strain C3.

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