Optimizing nematode management by defining natural chemical bases of behavior

This project was based on the hypothesis that nematodes interacting with plants as either parasites or beneficial saprophytes are attracted to their host by natural products. This concept was supported by numerous observations that parasitic nematodes are attracted to root exudates. Our overall goal was to identify nematode sensory compounds from root exudates and to use that information for reducing nematicide applications. We applied skills of the investigators to achieve three specific objectives: 1) Identify nematode behavioral cues (e.g., attractants or repellents) in root exudates; 2) Identify new natural nematicidal compounds; and 3) Combine a natural attractant and a nematicide into a nematode trap. Because saprophytic nematodes benefit plants by mineralizing organic matter, we sought compounds attractive primarily to parasitic nematodes. The project was constructed on several complementary foundations. First, data from Dr. Spiegel’s lab showed that under aseptic conditions Ditylenchus dipsaci, a parasite on onion, is attracted to certain fractions of onion root exudates. Second, PI Phillips had a sizeable collection of natural plant products he had identified from previous work on Rhizobium-legume interactions, which could be tested “off the shelf”. Third, Dr. Ferris had access to aseptic and natural populations of various saprophytic and parasitic nematodes. The project focused on five nematode species: D.dipsaci, Heterodera avenae, and Tylenchulussemipenetransat ARO, and Meloidogyne javanicand Caenorhabditis elegans at UCD. Ten pure plant compounds, mostly flavonoids, were tested on the various nematode species using six different assay systems. Results obtained with assorted test systems and by various scientists in the same test systems were essentially irreproducible. Many convincing, Many convincing, i.e. statistically significant, results in one system or with one investigator could not be repeated with other assays or different people. A recent report from others found that these compounds, plus another 30, were inactive as attractants in three additional parasitic nematode species (Wuyts et al. Nematology 8:89- 101, 2006). Assays designed to test the hypothesis that several compounds together are required to attract nematodes have thus far failed to find a reproducibly active combination. In contrast to results using pure plant compounds, complex unfractionated exudates from aseptic onion root reproducibly attracted D. dipsaci in both the ARO and UCD labs. Onion root exudate collection, separation into HPLC fractions, assays using D. dipsaci and MS-MS experiments proceeded collaboratively between ARO and UCD without any definitive identification of an active compound. The final active fraction contained two major molecules and traces of several other compounds. In the end, analytical studies were limited by the amount of onion root exudate and the complexity of the purification process. These tests showed that aseptic plant roots release attractant molecules, but whether nematodes influence that release, as insects trigger release of attractants from plants, is unknown. Related experiments showed that the saprophyte C. elegans stimulates its prey, Pseudomonas bacteria, to increase production of 2, 4-diacetylphloroglucinol (DAPG) a compound that promotes amino acid exudation by plant roots. It is thus possible that saprophytic nematodes are attracted primarily to their bacterial or fungal prey and secondarily to effects of those microorganisms on root exudation. These observations offer promising avenues for understanding root-zone interactions, but no direct routes to controlling nematodes in agriculture were evident. Extracts from two plant sources, Chrysanthemum coronarium and Sequoia sempervirens, showed nematicidal activity at ARO and UCD, respectively. Attempts to purify an active compound from S. sempervirens failed, but preliminary results from C. coronarium are judged to form a potential basis for further work at ARO. These results highlight the problems of studying complex movement patterns in sentient organisms like nematodes and the issues associated with natural product isolation from complex mixtures. Those two difficulties combined with complications now associated with obtaining US visas, slowed and ultimately limited progress on this project. As a result, US investigators expended only 65% of the $207,400 originally planned for this project. The Israeli side of the project advanced more directly toward its scientific goals and lists its expenditures in the customary financial report.