1. INTRODUCTION Some DAPG-producing isolates of Pseudomonas fluorescens act as biocontrol agents and aid in improving crop yields. DAPG can affect nematodes, increasing egg hatch of Globodera rostochiensis but decreasing J2 mobility (Cronin et al., 1997), and DAPG-producing strains of P. fluorescens can suppress Meloidogyne javanica numbers (Siddiqui and Shaukat, 2003a,b). The primary goal of this research was to determine whether direct application of DAPG to diverse nematode species would result in either toxic or stimulatory effects. Plant-parasitic nematodes and free-living nematodes were selected for the study, to determine whether DAPG would affect target and nontarget nematodes. Nematode Culture and Collection. Nematodes were maintained on greenhouse plants (H. glycines, M. incognita), in corn root explant cultures (P. scribneri ), or in media with or without Escherichia coli (C. elegans, P. pacificus and R. rainai). Nematodes were collected on mesh or from Baermann funnels and surface-rinsed. Assays. Nematodes were incubated in synthetic DAPG (Toronto Research Chemicals, Inc, North York, Ontario) in microwell plates at ca. 24 C. Life stages, numbers and incubation times depended on the life cycle and availability of each species (Table 1). Concentrations Tested. -0, 1, 10, 25, 50, and 75 µg/ml DAPG (all but X. americanum). -0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 50, and 75 µg/ml (X. americanum). -DAPG in methanol at 1, 10 and 100 µg/ml DAPG; 0% (water only), and 0.0045%, 0.045%, and 0.45% diluted methanol controls (M. incognita). There were 4-5 replicate wells per trial, with at least two trials per treatment (1-2 trials for X. americanum in DAPG > 20 µg/ml). Statistical Methods. A 3-factor, nested-effects analysis of covariance model was conducted using SAS ® 9.2 Proc GLIMMIX. Significant differences (α = 0.05) in the model intercept were identified using the Extended Shaffer-Royen 2 (ESR) multiple comparisons method, and significant differences (α = 0.05) in model slope among the nematode species were identified using the PDMIX800 3 SAS macro.. Nematode speciesLife stage# per well Incubation Time H=Hours, D=Days Caenorhabditis elegans Eggs J1 Adults 14 12 19 1, 3, & 6 H; 1 & 2 D 6 H; 1 & 2 D Heterodera glycines Eggs J2 200 50 7 D 2 D Meloidogyne incognita Eggs J2 100 50 7 D 2 D Pratylenchus scribneri Juveniles Females 100 13 2-3 D 2-4 D Pristionchus pacificus Eggs Adults 58 48 6 H; 1 & 2 D Rhabditis rainai Eggs Adults 55 53 6 H; 1 & 2 D Xiphinema americanum Adults151 &2 D Table 1. Nematode species, life stage assayed, number per well, and incubation times in DAPG. RESULTS LITERATURE CITED Meyer, Susan L.F. 1, J.M. Halbrendt 2, L.K. Carta 1, A.M. Skantar 1, T. Liu 3, H.M.E. Abdelnabby 4, and B.T. Vinyard 5. 1 USDA, ARS Nematology Laboratory, Beltsville, MD, USA, 2 Pennsylvania State University, Biglerville, PA, USA, 3 Beijing Academy of Agricultural and Forestry Science, Beijing, China, 4 Benha University, Qaliubia, Egypt, 5 USDA, ARS Biometrical Consulting Service, Beltsville, MD, USA. EXPOSURE OF SOIL-DWELLING NEMATODES TO DIACETYLPHLOROGLUCINOL (DAPG) Fig. 4. Xiphinema americanum adults in DAPG. Perc entage viable J2 Perc entage viable adults DAPG Concentration (µg/mL water) Fig. 1. Meloidogyne incognita eggs and J2 in: diluted methanol, DAPG in diluted methanol, or DAPG in water only. Fig. 2. Caenorhabditis elegans eggs in DAPG. Fig. 3. Caenorhabditis elegans J1 in DAPG. Meloidogyne incognita Eggs (Fig. 1): Hatch in water controls was ca. 99.8%. Egg hatch was 91.2%-93.3% in diluted methanol (0.0045%, 0.045% and 0.45%) without DAPG; methanol alone therefore decreased egg hatch. There was a significant decrease in egg hatch with DAPG in diluted methanol, with the greatest effect at low DAPG concentrations. When water alone was used as a solvent for DAPG, there was a significant difference from all other treatments. DAPG in water was toxic to eggs, resulting in a constant decrease in egg hatch as DAPG concentration increased. Meloidogyne incognita J2 (Fig. 1): There was no significant effect of any treatment on J2 viability. Caenorhabditis elegans Eggs (Fig. 2): Egg hatch was stimulated by DAPG at 1 and 3 hours, but no significant effect was observed at 6 hours, 1 day or 2 days. Caenorhabditis elegans J1 (Fig. 3) and Adults: No significant effect of DAPG was observed on J1 or adults. Xiphinema americanum Adults (Fig. 4): DAPG was toxic to adults of X. americanum. After 24 hours immersion, adult mobility averaged 97.8% in water controls, dropping rapidly to a mean of 73% at 2 µg/ml DAPG, and 0% in 50 µg/ml DAPG. This toxic effect was even more pronounced after 2 days. Heterodera glycines Eggs and J2, Pratylenchus scribneri Juveniles and Females, and Pristionchus pacificus & Rhabditis ranai Eggs and Adults: No significant effect of DAPG was observed on these nematodes (graphs not shown). -Nematode responses to DAPG varied with taxon and life stage. When egg hatch and juvenile/adult viability were observed, three of the seven tested nematode species responded to the compound. Eggs of five nematode species were immersed in DAPG; effects on egg hatch varied from stimulatory to inhibitory to no change, depending on the species. Juveniles of four species were tested; DAPG did not affect juvenile viability of any of those species. Adults from four species were tested, but DAPG was only toxic to X. americanum adults. -Methanol is commonly used as a solvent for DAPG, but it was found to alter effects of DAPG on M. incognita eggs. Consequently, water only was used as a solvent for tests with all other nematode species. -DAPG production by soil-dwelling bacteria would not directly result in suppression of population numbers of every plant-pathogenic or bacterial-feeding nematode species. Augmentation of DAPG-producing P. fluorescens populations for biological control of nematodes should be targeted to pathogenic nematode species that have been shown to be sensitive to the compound, or used for indirect effects, such as induced systemic resistance. Acknowledgements: Thanks are extended to Paula Crowley, Sharon Ochs, Maria Hult and Emily Brinker for assistance in the laboratory and/or with preparation of graphs. MATERIALS AND METHODS CONCLUSIONS Cronin, D., Moënne-Loccoz, Y., Fenton, A., Dunne, C., Dowling, D. N. and O’Gara, F. 1997. Role of 2,4-diacetylphloroglucinol in the interactions of the biocontrol pseudomonad strain F113 with the potato cyst nematode Globodera rostochiensis. Appl. Environ. Micro. 63:1357-1361. Siddiqui, I. A., and Shaukat, S. S. 2003a. Plant species, host age and host genotype effects on Meloidogyne incognita biocontrol by Pseudomonas fluorescens strain CHA0 and its genetically-modified derivatives. J. Phytopathology 151:231-238. Siddiqui, I. A., and Shaukat, S. S. 2003b. Suppression of root-knot disease by Pseudomonas fluorescens CHA0 in tomato: Importance of bacterial secondary metabolite, 2,4-diacetylphloroglucinol. Soil Biol. Biochem. 35:1615-1623. Perc entage viable J2 or hatched eggs