Mutagenesis of β-1,3-Glucanase Genes in Lysobacter enzymogenes Strain C3 Results in Reduced Biological Control Activity Toward Bipolaris Leaf Spot of Tall Fescue and Pythium Damping-Off of Sugar Beet

Palumbo, J. D., Yuen, G. Y., Jochum, C. C., Tatum, K., and Kobayashi, D. Y. 2005. Mutagenesis of β-1,3-glucanase genes in Lysobacter enzymogenes strain C3 results in reduced biological control activity toward Bipolaris leaf spot of tall fescue and Pythium damping-off of sugar beet. Phytopathology 95:701-707. Lysobacter enzymogenes produces extracellular lytic enzymes capable of degrading the cell walls of fungi and oomycetes. Many of these enzymes, including β-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 β-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 identity to their respective counterparts. The gluA and gluC gene products encode enzymes belonging to family 16 glycosyl hydrolases, whereas gluB encodes an enzyme belonging to family 64. Mutational analysis indicated that the three genes accounted for the total β-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 toward 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 sugar beet. These results provide direct supportive evidence for the role of β-1,3-glucanases in biocontrol activity of L. enzymogenes strain C3. Lysobacter enzymogenes is a bacterial species characterized for its propensity to lyse fungi and other microorganisms. It is known to produce an abundance of extracellular enzymes such as chitinases and glucanases that are capable of degrading the major cell wall components of fungi and oomycetes. L. enzymogenes also produces other factors, such as antibiotics that are antagonistic to the growth of microbes (6,13,42). L. enzymogenes strain C3 has demonstrated biocontrol efficacy toward a number of plant diseases (15,27,46–48), of which extracellular degradative enzymes are predicted to play an important role. A number of observations provide evidence that extracellular enzymes are involved in biocontrol activity of strain C3. Zhang and Yuen (49,50) demonstrated that purified chitinolytic fractions were active against Bipolaris sorokiniana spore germination and inhibited plant host infection by the pathogen. In a separate study (27), the clp gene, which encodes a global regulator, was found to control lytic enzyme production as well as a number of other factors in strain C3. Mutation of this regulatory gene resulted in complete loss of antimicrobial activity and significant reduction in biological control of Bipolaris leaf spot of tall fescue and Pythium damping-off of sugar beet. Despite these supportive studies, direct genetic evidence for the role of cell wall-degrading enzymes such as chitinases and β-1,3-glucanases in biocontrol activity of strain C3 is lacking. Recently, three β-1,3-glucanases were isolated and characterized from the biocontrol agent L. enzymogenes N4-7 (33). The gluA and gluC genes encode enzymes with catalytic domains belonging to glycosyl hydrolase family 16. The enzymes differ structurally in that the gluC gene product contains the presence of a family IV cellulose-binding domain. In contrast, the gluB gene product is related to glycosyl hydrolase family 64. The molecular and biochemical characterization of these enzymes in strain N4-7 provided a solid foundation with which to assess the role of β1,3-glucanases in biocontrol. However, N4-7 is genetically intractable, and this precluded the ability to further analyze β-1,3glucanase activity by a mutational approach. Taxonomically, strain N4-7 is closely related to strain C3 and these strains share a number of phenotypic traits (42). Unlike strain N4-7, strain C3 is genetically tractable and, moreover, has been more extensively studied as a biocontrol agent using a number of pathosystems, ultimately providing a better system by which to assess biocontrol traits. In this study, our goals were to identify and characterize β-1,3-glucanase genes within strain C3 and create sequential knockout mutations of each gene to assess a role for the enzyme activity in biocontrol. We describe the characterization of three β-1,3-glucanase genes in L. enzymogenes strain C3. We further demonstrate that a strain containing mutations in all three β-1,3-glucanase genes is reduced in biocontrol of Bipolaris leaf spot of tall fescue and Pythium damping-off of sugar beet. Corresponding author: D. Y. Kobayashi E-mail address: [email protected] DOI: 10.1094 / PHYTO-95-0701 © 2005 The American Phytopathological Society