2008 Annual Report
1a.Objectives (from AD-416)
Evaluate the etiology and epidemiology of soilborne diseases of small fruit and nursery crops and the biology of causal pathogens. Develop, evaluate, and enhance strategies for the management of soilborne diseases of horticultural crops. Develop knowledge of mechanisms contributing to biological control of soilborne plant diseases.
1b.Approach (from AD-416)
Dose-response relationships between propagule density in irrigation water and disease. Identification of factors influencing pathogen dispersal and disease spread among containers in a nursery. A systematic survey of blueberry fields throughout the PNW will be conducted to identify nematode pathogens. Greenhouse and microplot experiments will be employed to determine the effects of a range of population densities of nematode species found in the survey on the health of a widely planted blueberry cultivar. Test the Electrocatalytic Oxidation/precipitation (EO) method. Identify factors influencing the efficacy of the EO method. Test the EO method in an experimental nursery setting. Use the recently-described genomisotopic approach to purify the two cryptic metabolites from cultures of Pf-5. Derive mutants of Pf-5. Assess the role of novel metabolites in biological control. Test toxicity of the purified metabolites. Evaluate gene expression by Pf-5 on seed surfaces using RNA isolation, labeling and hybridization. Characterize the nutritional composition of seed exudates. Generate derivatives of Pf-5 with mutations in selected genes. Test mutants for spermosphere colonization and biological control of Pythium damping off. Replaced 5358-12220-001-00D (3/03). FY03 $73,538 Program Increase. FY06 $133,650 Program Increase (memo #44). Replacing 5358-12220-002-00D (3/07).
A plant-parasitic nematode survey of blueberries in Washington was started. Blueberry fields with plant-parasitic nematodes were identified for in depth sampling in subsequent years to determine the temporal-spatial population dynamics of plant-parasitic nematode in blueberry. Secondly, a greenhouse study was established to study the pathogenicity of plant-parasitic nematodes on a range of blueberry genotypes. Six blueberry genotypes were obtained from a local nursery and established in pots.
A survey of Verticillium dahliae in woody ornamental nurseries in Oregon was started. Methods for quantifying soil populations were tested and an optimal procedure was selected. A greenhouse study was begun to evaluate V. dahliae inoculum levels and subsequent disease development. Pots of American elm, red maple, and silver maple were established. Finally, isolates of V. dahliae causing wilt symptoms in a commercial raspberry field were collected. Isolates will be used to screen Rubus selections for resistance or susceptibility.
Field microplots (a pot-in-pot system) were established to study the effects of the ring nematode, Mesocriconema xenoplax, on physiology and growth ‘Pinot noir’ grapevines. Vines were grafted onto 6 rootstocks that were previously demonstrated to have different degrees of resistance to M. xenoplax. Soil in plots was infested with M. xenoplax or maintained as non-nematode controls. Arbuscular mycorrhizal fungi inoculum was incorporated in all plots. Thus far, data have been collected on nematode population dynamics, soil respiration, leaf water potentials, plant growth.
Sampling of 3 forest nurseries was conducted to determine placement of field plots in the Methyl Bromide Alternatives Project. Methods for the isolation of Pythium species from soils were compared and the number of cultures by method was recorded. Results are currently being analyzed for effect of method on recovered Pythium species. Pythium species associated with roots of Dougfir were identified by analysis of the ITS region. A greenhouse study on the pathogenicity of these isolates was established.
A procedure was established for isolation of sufficient quantities of high-quality Pf-5 RNA from bacteria grown on the surface of pea seeds. Sufficient biological replicates have been performed for microarray experiments comparing wild-type Pf-5 with a gacA Pf-5 mutant grown on seed surfaces for 24 hours. Quantitative RT-PCR performed on these samples verified that significant differences in gene expression levels can be observed at this time point between wild-type and gacA samples. In addition, a series of Pf-5 mutants was created, each carrying an unmarked deletion in one of 13 gene clusters encoding for known or putative secondary metabolites or exoenzymes. Five of these mutations have been introduced into a single strain, towards the goal of producing a single strain deficient in production of all known secondary metabolites. We have initiated experiments evaluating the biocontrol activity of Pf-5 mutants having single and multiple mutations. NP 303, Component 4
Discovery of a novel insecticidal toxin from the genomic sequence of Pseudomonas fluorescens Pf-5. Genomic sequencing of microorganisms has revealed a tremendous source of biologically-active compounds of potential value for pest management. In collaboration with colleagues in Switzerland and Denny Bruck of the HCRL, ARS scientists in the Horticultural Crops Research Unit in Corvallis, OR identified a new insect toxin in the genome of the biological control agent Pseudomonas fluorescens Pf-5. This also demonstrated that the toxin is active against tobacco hornworm and waxworm larvae, and they published a manuscript describing this finding. This research, demonstrated the power of a genomic approach for the discovery of valuable natural products and addresses National Program 303 Component 4, Problem Statement 4B: Pathogen, Plant, and Antagonist Interactions.
Impact of plant-parasitic nematodes on winegrapes. The ring nematode, Mesocriconema xenoplax, has been shown to significantly reduce the establishment and growth of young grapevines in the Pacific Northwest. Greenhouse experiments were conducted by ARS scientists in the Horticultural Crops Research Unit in Corvallis, OR to determine the effect of this nematode on the growth and physiology of young ‘Pinot noir’ grapevines and on the colonization of the roots by arbuscular mycorrhizal fungi (AMF). The nematode reduced the density of arbuscules and the accumulation of phosphorus and starch in fine roots. Grape roots are intensely colonized by AMF and dependent on these fungi to sequester phosphorus in the low phosphorus soils found in the PNW. Research is ongoing to evaluate the interaction of the ring nematode, nematode resistant rootstocks, and AMF on the growth and physiology of grapevines. Knowledge of the interaction of mycorrhizal fungi and nematode parasitism can lead to the development of more sustainable vineyard management practices. NP Action Plan Components: 2A: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen, 2C Population Dynamics, Spread, and Epidemiology of Pathogens, 4C Application of Sustainable Disease Management Tools.
5.Significant Activities that Support Special Target Populations
Loper, J.E., Gross, H. 2007. Genomic analysis of antifungal metabolite production by Pseudomonas fluorescens Pf-5. European Journal of Plant Pathology. 119:265-278.
Pechy-Tarr, M., Bruck, D.J., Maurhofer, M., Fischer, E., Vogne, C., Henkels, M.D., Donahue, K.M., Grunder, J., Loper, J.E., Keel, C. 2008. Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens. Environmental Microbiology. 10(9):2368-2386.
Loper, J.E., Henkels, M.D., Shaffer, B.T., Valeriote, F.A., Gross, H. 2008. Isolation and identification of rhizoxin derivatives from Pseudomonas fluorescens Pf-5 using a genomic mining strategy. Applied and Environmental Microbiology. 74(10):3085-3093.