The Biochemical Defense Mechanisms research pursues the long-term goal of identifying natural plant products that increase the defense system of forage perennial plants. The purpose of this research is to improve the longevity of nutritional pasture plants that lack broad disease resistance.

This research focuses on chitinase enzymes in birdsfoot trefoil and endophyte-free tall fescue, two highly nutritional forage plants known to be susceptible to a wide range of pathogens. Chitinases are natural enzymes that occur in many organisms. In plants, most chitinases are classified as pathogenesis-related, although some are related to development. The initial research attempted to discover chitinases in tall fescue; subsequent work attempted to determine the roles of these chitinases–whether pathogenesis-related or developmental. In 1992, this program reported chitinases in tall fescue, which was the first report of such in a perennial grass. Follow-up work showed that tall fescue chitinases are related to plant defense. Total chitinase activity increased six-fold when roots were infected with Meloidogyne marylandi, which is a parasitic nematode. The program also developed spectroscopic techniques that enabled analysis of large plant populations; using these new techniques, the program found that chitinase activity in tall fescue increased in response to a stress elicitor; this finding further supports the view that tall fescue chitinases are not merely related to plant development. The program also found that constitutive activity, that is, the activity in unchallenged plants, differed among cultivars; cultivars known to be persistent in the field expressed high levels of activity, while those known to lack persistence expressed low levels.

A rewarding research project in this area, which began in 1995, involves chitinases in birdsfoot trefoil. This research investigates individual chitinase proteins, called “isoforms.” In 1995, the program group modified a procedure commonly used to separate isoforms; this procedure, known as gel electrophoresis, was altered to embed substrate in the resolving gel. It also used silver nitrate to stain the gel background. This modification not only permitted chitinase isoforms to separate, it also permitted active isoforms to be visualized in a permanently stable medium.

Future publications will publish this procedure and serve to summarize the above trefoil chitinase research as conducted over the past five years. The publication should show that rhizomatous birdsfoot trefoil, which is the most disease-resistant type of trefoil, contains two chitinase isoforms. (These isoforms are nearly absent in birdsfoot trefoil that is susceptible to pathogens.) When they are applied to a petri dish inoculated with a fungus, these two isoforms inhibit fungal growth. Recent work has partially separated these isoforms and found that the light isoform, which migrates quickly to the bottom of the gel, is antifungal. The final step in this phase of the research involves the separation and testing of the heavy isoform.

These findings not only confirm that at least one chitinase isoform in birdsfoot trefoil is pathogenesis-related, they also support observations from grazed pastures at the University of Missouri, in which persistent plants contained these two chitinase isoforms. These findings provide markers that assist plant breeders in the selection of disease resistant plants, which in turn provide livestock producers with highly nutritional plants that survive in the field.

Key Publications

• Marek, S.M., C.A. Roberts, A.L. Karr, and D.A. Sleper. 2000. Chitinase activity in tall fescue as affected by ethephon and cultivar. Crop Sci.40:713-716.
• Marek, S.M., C.A. Roberts, and A.L. Karr. 1995. Silver stain detection of chitinolytic isozymes after polyacrylamide gel electrophoresis. Anal. Biochem. 230:184-185.
• Roberts, C.A., S.M. Marek, W. Lei, and A.L. Karr. 1994. Quantification of chitinase activity by near infrared reflectance spectroscopy. Crop Sci. 34:1070-1073.
• Roberts, C.A., S.M. Marek, T.L. Niblack, and A.L. Karr. 1992. Parasitic Meloidogyne and mutualistic Acremonium elicit chitinase in tall fescue. J. Chem. Ecol. 18:1107-1116.