2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Identify and characterize key pathogens to pinpoint critical pathogen vulnerabilities and develop targeted disease management strategies.
Objective 2: Identify plant germplasm and cultivars of small fruits resistant to economically-important soilborne diseases.
Objective 3: Expand knowledge of genomic features and regulatory mechanisms in biological control strains of Pseudomonas spp. to develop more consistent and effective tools for biologically-based disease management.
1b.Approach (from AD-416):
Determine the prevalence and characterize the population diversity of important soilborne pathogens affecting horticultural crops. Results from this research will identify specific pathogen populations that constrain production of horticultural crops. These populations can be targeted in the future to develop more effective, economical, and environmentally-acceptable disease management systems.
Evaluate germplasm of black raspberry (Rubus occidentalis) for resistance to Verticillium wilt, caused by V. dahliae, and germplasm of red raspberry (Rubus ideaus) for resistance to the root lesion nematode, P. penetrans. Our research will identify Rubus genotypes and raspberry cultivars that are resistant to these soilborne diseases, and can be deployed in horticultural production systems in the future.
Develop improved knowledge of the mechanisms by which the biological control agent Pseudomonas fluorescens Pf-5 suppresses disease. Results from this research will enable pathologists, horticulturists, and growers to develop more effective and reliable biological controls for soilborne diseases of horticultural crops.
Towards our first objective to identify key pathogens limiting the production of nursery crops, we completed population genetic diversity analyses of 50 Pythium irregulare isolates from three forest nurseries. P. irregulare exhibited low genetic diversity and there was evidence for migration of P. irregulare among nurseries. This information is essential to develop strategies to minimize pathogen movement between nurseries. We also surveyed nurseries for the presence of the soil-borne pathogen Verticillium dahliae and detected the pathogen on five plant hosts in 6 of the 9 sampled nurseries. To understand the genetic diversity of the virus transmitting plant-parasitic nematode Xiphinema americanum, populations of the nematode were collected from 12 geographically disparate locations across the U.S. from different crops and in varying association with viruses. Molecular data demonstrated the presence of three distinct lineages of X. americanum. These results will be used to develop a diagnostic tool to distinguish virus-transmitting from non-transmitting nematode populations.
Towards our second objective of identifying small fruit germplasm with resistance to soilborne pathogens, we evaluated 17 Rubus genotypes in greenhouse trials and identified two genotypes that exhibited resistance to V. dahliae. Repeated trials are necessary to confirm results. Information will be used to establish damaging threshold inoculum levels at nurseries for susceptible hosts and to develop resistant Rubus genotypes. On raspberry, a field trial was established in collaboration with Washington State University to evaluate the response of 8 raspberry varieties to Pratylenchus penetrans. This long-term trial will provide valuable data upon which growers can base management decisions in the future.
Towards our objective of developing knowledge of mechanisms of biological control of plant diseases, we continued to build a library of mutants of the biological control strain Pseudomonas protegens Pf-5, which produces at least nine antibiotic compounds. To date, the library includes 179 mutants, each having a deletion in one to nine genes for the biosynthesis of those compounds. We evaluated culture extracts of representative mutants to confirm that they lack production of the antibiotic corresponding to each biosynthetic gene deleted. We also tested the mutants in a series of biological assays to determine which antibiotics produced by Pf-5 are responsible for suppression of selected fungi. Analysis of the genomic sequence of Pf-5 revealed gene clusters that are likely, but not known, to produce antibiotics. In collaboration with a natural product chemist, we identified the product of one of these gene clusters as toxoflavin and quantified the levels of toxoflavin production by Pf-5.
Patterns of gene expression by biocontrol bacteria on seed surfaces. To better understand mechanisms of biological control of seeding diseases by Pseudomonas protegens Pf-5, we used new genomic techniques to identify genes that are expressed by Pf-5 living on seed surfaces. This study identified ca. 900 genes expressed by Pf-5 on seeds, where interactions between a biological control agent and seed-infecting pathogens occur. By comparing the set of genes expressed by wildtype Pf-5 to the set expressed by a mutant that is deficient in biological control, we highlighted new genes that can be investigated in the future for their roles in the biological control of plant disease. Before this study, scientists knew a great deal about what genes are expressed by bacteria grown in laboratory cultures, but very little about the genes expressed by biological control bacteria on plant surfaces. This study will allow scientists to use more directed strategies to improve the reliability of biological control for agriculture.
Pathogenicity of forest nursery Pythium species to Douglas-fir (tree) seedlings. Forest nursery managers need information about the Pythium species that cause tree seedling disease. The main tool for disease control, methyl bromide soil fumigation, is being phased out under the Montreal Protocol. ARS scientists in Corvallis, Oregon, identified eight Pythium species that kill Douglas-fir seedlings and eight other Pythium species that cause root lesions. These findings will help forest nursery managers target disease control programs towards the Pythium species that cause the most damage to the 200 million tree seedlings that are produced annually in forest nurseries of the Western United States.
Plant-parasitic nematodes in semi-arid vineyards of the Pacific Northwest. ARS scientists investigated the spatial distribution of plant-parasitic nematodes in semi-arid vineyards to provide grape growers with information to better manage nematodes as little is known about their distribution and pathogenicity. The horizontal and vertical distributions varied among the plant-parasitic nematodes found in this region. Root-knot and ring nematodes were concentrated at shallow soil depths in close proximity to water and roots demonstrating an intimate association with the host grapevine. The dagger and root lesion nematodes were either randomly distributed in the vineyard or aggregated on the edges of the vine row, respectively, indicating that plants other that grape are also hosts for these nematodes. These results indicate that there is potential to reduce the treated area when targeting root-knot and ring nematodes semi-arid vineyards in the Pacific Northwest.
Gigot, J., Zasada, I.A., Walters, T.W. 2012. Integration of brassicaceous seed meals into red raspberry production systems. Applied Soil Ecology. 64:23-31.
Lim Kent, L., Penesyan, A., Hassan, K.H., Loper, J.E., Paulsen, I. 2013. Effect of tannic acid on the transcriptome of the soil bacterium Pseudomonas protegens Pf-5. Applied and Environmental Microbiology. 79:3141-3145.
Kidarsa, T.A., Shaffer, B.T., Goebel, N.C., Roberts, D.P., Buyer, J.S., Johnson, A., Kobayashi, D.Y., Paulsen, I., Loper, J.E. 2013. Genes expressed by the biological control bacterium Pseudomonas protegens Pf-5 on seed surfaces under the control of the global regulators GacA and RpoS. Environmental Microbiology. 15:716-735.
Hartney, S., Mazurier, S., Girard, M., Mehnaz, S., Davis, E.W., Gross, H., Lemanceau, P., Loper, J.E. 2013. Ferric-pyoverdine recognition by Fpv outer-membrane proteins of Pseudomonas protegens Pf-5. Journal of Bacteriology. 195:765-776.
Cheng, X., De Bruijn, I., Van Der Voort, M., Loper, J.E., Raaijmakers, J.M. 2013. The Gac Regulon of Pseudomonas fluorescens SBW25. Environment Microbiology Reports. 5:608-619.
Lim, C., Hassan, K.A., Penesyan, A., Loper, J.E., Paulsen, I. 2013. The effect of zinc limitation on the transcriptome of Pseudomonas protegens Pf-5. Environmental Microbiology. 15:702-715.
Weiland, G.E., Beck, B.R., Davis, E.A. 2013. Pathogenicity and virulence of Pythium species obtained from forest nursery soils on Douglas-fir seedlings. Plant Disease. 97: 744-748.
Zasada, I.A., Riga, E., Pinkerton, J.N., Wilson, J.H., Schreiner, R.P. 2012. Plant-parasitic nematodes associated with grapevines, Vitis vinifera, in Washington and Idaho. American Journal of Enology and Viticulture. 63:522-528.
Gigot, J., Walters, T., Zasada, I.A. 2013. Impact and occurrence of Phytophthora rubi and Pratylenchus penetrans in commercial red raspberry (Rubus idaeus) fields in northwestern Washington. International Journal of Fruit Science. 13:357-372.