2006 Annual Report 4d.Progress report. 4d. Progress Report This report serves to document research conducted under a reimbursable agreement between ARS and the University of California, Davis. Additional information on research can be found in the report for inhouse project 5306-22000-013-00D. This continuing research was conducted to develop effective measures to prevent or minimize transmission of soilborne pathogens and nematodes via irrigation water and the survival and reproduction of plant-parasitic nematodes. To assess effective measures to prevent or minimize transmission of pathogenic fungi and nematodes via irrigation water in ornamental production systems, we have investigated the efficacy of ozonation on the nematodes Caenorhabditis elegans and Meloidogyne javanica in recycled irrigation water. Experiments were conducted in the laboratory using a closed-loop ozonation system to circulate test irrigation water while injecting gaseous ozone. Ozone was generated using the corona discharge method and was injected using a Mazzei negative pressure injector. Experiments were conducted to determine dissolved ozone concentrations over time and survival of nematodes when exposed to dissolved ozone in irrigation water. Ozone residuals were determined at time intervals spectrophotometrically. Nematode bioassays were performed by placing vermiform nematodes into recycled irrigation water, and treating with ozone. Samples were taken every fire minutes and worms were scored as active or inactive. Nematode bioassays were conducted using different rates of ozone treatment. The concentrations of dissolved ozone achieved were lower than those achieved in tap water. More than one hour of exposure was required to reach dissolved ozone concentrations similar to those experienced immediately with clean water. Nematodes were inactivated in ozonated irrigation water although longer exposure was required with irrigation water than with clean water. Complete inactivation took from 30 minutes to 120 minutes depending on nematode species and ozone delivery rates. Another approach to disinfesting irrigation water of nematodes and pathogens is the use of ultra-violet (UV) light. Experiments were performed to assay the effects of UV light on plant-parasitic nematodes. Aqueous suspensions of M. javanica juveniles were exposed to UV light with either a mercury lamp or a UV/Vis Xenon (Xe) arc lamp system (200 to 700 nm). The majority of tests were performed with nematodes suspended in artificial tap water (ATW), but preliminary experiments were also conducted using recycled irrigation water obtained from a nursery. Nematodes were exposed to the UV light by placing an uncovered Petri dish with 10 ml of the nematode suspension under the light source. Different treatments were created by differing the duration of exposure for the mercury lamp or by varying the number of pulses in the Xe arc lamp. Worms were scored as either active or inactive after exposure. The rest of the sample was left for 24 hours and then recounted to assess the delayed effects of UV exposure. The effect of visible light on nematodes was also checked using the Xe arc lamp and filtering out the UV spectrum. Experiments were conducted using the mercury lamp with the nematodes in ATW, using the Xe arc lamp with ATW water, and also the Xe arc lamp and recycled irrigation water. The bioassay nematodes in ATW were completely inactivated by exposures of 300-350 mJ/cm2 of emitted UV energy. Nematodes in irrigation water did not show complete inactivation until 2250 mJ/cm2 due to the ability of contaminants to absorb UV light. Visible light did not have any measurable effect on nematode activity. Experiments also were done to test the efficacy of the pulsed Xe UV source against two species of fungi commonly found in irrigation water: Fusarium oxysporum (we used F. oxysporum f.sp. dianthi in our experiments) and Phytophthora (we used P. capsici in our experiments). Cultures were grown on appropriate agar media for 5-7 days prior to experiments. Spores of both species were harvested from the culture plates by adding sterile water and, in the case of Fusarium, dislodging spores from mycelium with a sterile glass rod to create a spore suspension. In the case of Phytophthora, flooded plates were allowed to stand for 30-40 minutes until most sporangia had released zoospores. In both cases, the resulting spore suspensions were then filtered through cheesecloth to separate spores from mycelial fragments. Aliquots of spore suspension then were added to measured volumes of distilled or recycled nursery water in empty petri dishes to yield suspensions of known concentration. Culture plates of mycelium growing in agar media also were used in experiments where they were exposed directly to UV. The light emissions from the Xe lamp were collimated using a 19.5 cm long piece of 9.5 cm dia. PVC pipe, at the bottom of which petri dishes were placed that contained spore suspensions or mycelial cultures. Immediately below the target samples was a joulemeter used to measure the photon energy delivered to the target in real time. The photon energy output from the Xe lamp was measured to be 47.4 mJ/cm2/pulse total energy, and 8.9 mJ/cm2/pulse UV energy. While there were differences between experiments, taking the data in aggregate showed that both Fusarium and Phytophthora could be killed by exposure to 80-120 mJ/cm2 of UV photons. However, the efficacy of UV photons was degraded in experiments wherein spores were suspended in recycled nursery water instead of distilled water. The impurities in recycled irrigation water absorbed UV photons and protected the fungal spores. For this reason, we now are experimenting with electronic systems that utilize pulsed RF energy to aggregate (flocculate) impurities. Our goal over the coming year is to develop nonchemical methods for flocculating impurities so that they can be removed from recycled water by filtration before the water is run through a UV or ozone treatment device. In addition, we are experimenting with surfactant materials that have been shown to have antifungal properties (through the degradation of cell membranes), and which might not be adversely affected by water impurities. We also assessed the influence of the potential nematode biological control agent L. enzymogenes strain C3 against nematodes by conducting laboratory experiments. Experiments included assessment of plants grown in a soil-free system using plant growth pouches and cabbage as the host plant with cyst and root-knot nematodes (Heterodera schachtii and Meloidogyne javanica), and plants growing in sand-filled containers using zinnia as the host plant with M. javanica. Cabbage and zinnia were germinated and then grown in a plant growth chamber. The plants were inoculated with C3 post-germination and then subsequently with infective nematode juveniles. Data collected included nematode reproduction, plant growth, and C3 colonization in cabbage. Experiments were repeated. The root systems were colonized by C3, and nematode reproduction was reduced in the C3-treated plants in all experiments, although the differences as compared to control treatments were not always statistically significant. C3 treated plants showed fewer cysts or galls with fewer eggs per cyst or gall. The C3 did not show a negative influence on root or shoot biomass. Publications: Chen, J., W. H. Moore, G. Y. Yuen, D. Kobayashi, and E. P. Caswell-Chen. 2006. Influence of Lysobacter enzymogenes Strain C3 on Nematodes. Accepted for publication: Journal of Nematology. Yakabe, L.E. and J.D. MacDonald. 2005. The effects of a surfactant on Phytophthora ramorum. Phytopathology 95:S115