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Research Project: Improved Strategies for Management of Soilborne Diseases of Horticultural Crops

Location: Horticultural Crops Research

2014 Annual Report

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.

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.

Progress Report
Towards our first objective to identify key pathogens limiting the production of small fruit or nursery crops, we completed population genetic diversity analyses of 40 isolates of Pythium sylvaticum and P. ultimum from commercial nurseries. P. sylvaticum exhibit high genetic diversity whereas P. ultimum exhibited low genetic diversity. There was evidence for movement that the both of these Pythium species have been moved among nurseries. We completed the first trial of the mefenoxam sensitivity assay and identified two P. ultimum isolates that were resistant to the fungicide. Information is essential to develop strategies to minimize pathogen movement, particularly of fungicide-resistant isolates, between nurseries. In addition, soilborne pathogens were isolated from Rhododendron and Ribes plants at five Pacific Northwest nurseries as part of an agreement aimed at improving plant health for nursery production. A field trial was initiated to determine the pathogenicity of Phytophthora isolates and the effect of pathogen inoculum level and irrigation treatment on disease development and plant growth. Verticillium dahliae was found at 8 sampled ornamental nurseries on 8 plant hosts. To continue to explore the genetic diversity of the virus transmitting plant-parasitic nematode Xiphinema americanum, primer sets were developed based upon mitogenomic data to distinguish three distinct lineages of X. americanum. Additional geographically disparate populations from across the U.S. from different crops and in varying association with viruses were characterized using these primers. These results will be used to develop a diagnostic tool to distinguish virus-transmitting from non-transmitting nematode populations. Pseudomonas syringae was isolated from blueberry plants exhibiting symptoms of bacterial canker or blight from plantings in Oregon and Washington. Multilocus sequence analysis showed that the isolates represent many of the major lineages of Pseudomonas syringae, and do not fit into a given pathovar. Many isolates were found to be resistant to copper, the major chemical used to manage bacterial canker in commercial plantings. Towards our second objective of identifying small fruit germplasm with resistance to soilborne pathogens, we evaluated 50 Rubus genotypes in greenhouse trials and confirmed results from the previous year indicating that two genotypes may exhibit resistance to V. dahliae. Information will be used to establish damaging threshold inoculum levels for susceptible hosts and to develop resistant Rubus genotypes. On raspberry, a field trial established the previous year to evaluate the response of 8 raspberry varieties to Pratylenchus penetrans continued to be followed with measurements on plant health and nematode population dynamics being collected. This long-term trial will provide valuable data for growers on which to base future management decisions. Towards our objective of developing knowledge of mechanisms of biological control of plant diseases, we showed that, under microaerophilic conditions, the biological control strain Pseudomonas protegens Pf-5 produces toxoflavin, a compound with broad toxicity against bacteria, fungi and plants. The toxoflavin biosynthesis gene clusters of Pf-5 and the plant pathogen Burkholderia glumae are similar, but the cluster of Pf-5 includes an additional gene that has the capacity to degrade toxoflavin. Tests showed that toxoflavin production by Pf-5 was not associated with phytotoxicity against rice, probably because Pf-5 produces very low levels of the compound. We completed tests evaluating ten well-characterized biological control strains of Pseudomonas spp. for toxicity to insects. Many strains exhibited injectable lethality to the tobacco hornworm and injestable toxicity to Drosophila melanogaster and the tobacco hornmworm. Significant progress was also made towards reaching the objectives of pest management alternatives and risk avoidance. Five on-farm, multi-year field trials evaluating alternatives to broadcast fumigation for the suppression of P. penetrans and P. rubi were completed. It was discovered that bed fumigation performed as good as, and in some cases better than, broadcast fumigation for suppression of these pathogens. We evaluated the pathogenicity of Globodera ellingtonae, a new cyst nematode species, to potato in replicated field trials by inoculating potatoes with varying initial densities of G. ellingatone. At the end of the season, top weights and tuber weights of plants as well as final population densities of G. ellingtonae, were determined. Globodera ellingtonae only cause a reduction in tuber yield, not above-ground plant weight, at the highest initial population density. From a molecular perspective, the genome of G. ellingtonae was sequenced with collaborators. In addition, the transcriptome generated from multiple life stages of G. ellingtonae was also sequenced. With collaborators, a project evaluating the host status of grapes to the root-knot nematodes as well as the spatial distribution of plant-parasitic nematodes in established vineyards was completed. We completed three RNAseq experiments evaluating the roles of antibiotics as signaling molecules in Pseudomonas protegens. These experiments revealed that phloroglucinol, an intermediate in the biosynthesis of the antibiotic 2,4-diacetylphloroglucinol, influences the transcription of hundreds of genes in the biological control bacterium Pseudomonas protegens Pf-5. Of the genes influenced by phloroglucinol, transcripts of the pyoluteorin biosynthesis genes exhibited the greatest fold changes, suggesting that specific mechanisms exist for the coregulation of the two antibiotics, 2,4-diacetylphloroglucinol and pyoluteorin. Other genes regulated by phloroglucinol fall in functional role categories for amino acid biosynthesis, energy metabolism, transport, regulatory, and unknown functions. These results support our hypothesis that phloroglucinol serves as a chemical messenger having broad effects on the Pf-5 transcriptome. We also demonstrated that phloroglucinol produced by cells of Pf-5 could induce pyoluteorin production by a co-inoculated strain in culture, thereby causing that strain to suppress a target plant pathogen. These results demonstrate that phloroglucinol can function as an intercellular chemical messenger influencing both secondary metabolism and antibiosis of Pseudomonas. To our knowledge, this is the first example showing a function of intermediates in antibiotic biosynthesis in bacterial cell to cell communication.

1. Understanding the genetic diversity of Xiphinema americanum. X. americanum is an economically important group of plant-parasitic nematodes that transmit viruses to a variety of agricultural crops. ARS scientists at Corvallis, Oregon, used nuclear and mitochondrial DNA sequence to molecularly characterize populations from the U.S. This study identified three distinct groups of X. americanum based upon mitochondrial sequence data. Two of the groups contained nematodes found in association with virus and the third group contained populations from the Western U.S. where virus was absent. These results suggest that mitochondria DNA may be useful in sorting out the identity of X. americanum nematodes. This information will be used to develop a diagnostic tool to distinguish virus-transmitting from non-transmitting nematode populations.

2. Meadowfoam seed meal as an organic soil amendment to control plant-parasitic nematodes and soilborne pathogens. ARS scientists at Corvallis, Oregon, investigated the ability of meadowfoam seed meal, a waste product of the meadowfoam oilseed industry, to provide organic crop growers with an alternative means to control diseases caused by plant-parasitic nematodes and soilborne pathogens. Amendment rates were identified that were able to successfully control root knot nematodes and Pythium irregulare, both of which affect a wide variety of vegetable, fruit, and ornamental crop species. Methods were further refined to prevent unintentional phytotoxicity to crop plants by delaying planting into amended soils. The active compounds released by meadowfoam seedmeal were tracked over time, indicating that most toxicity occurred within the first 48 hours after soil amendment. This research indicates the potential of meadowfoam seed meal as an organic amendment to treat soilborne pathogens and nematodes that negatively affect crop health.

3. Bacteria strain causes mushroom losses. Spoilage of the commercial button mushroom can result from infection by some bacteria in the Pseudomonas fluorescens group, which cause significant economic losses to the mushroom industry and postharvest losses to consumers. ARS scientists at Corvallis, Oregon, and Pullman, Washington, with collaborators at Oxford, United Kingdom, evaluated a set of well-characterized strains of these bacteria for the capacity to cause discoloration of mushroom caps. They discovered that strain Pf-5 of Pseudomonas protegens, a soil bacterium known for the capacity to suppress plant diseases, causes brown blotch symptoms on mushroom caps. Their results showed that Pseudomonas protegens Pf-5 produces two antifungal metabolites (2,4-diacetylphloroglucinol and pyoluteorin) in mushroom caps that are partially responsible for the brown blotch symptoms. These results were surprising because brown blotch has been attributed in the past solely to production of the lipopeptide tolaasin by Pseudomonas tolaasii and other strains of Pseudomonas. This study highlights a new mechanism for mushroom toxicity in Pseudomonas spp. that is dependent on the production of antifungal metabolites other than lipopeptides and provides a new avenue to pursue in minimizing spoilage to mushrooms.

Review Publications
Maul, J.E., Buyer, J.S., Lehman, R.M., Culman, S., Blackwood, C., Roberts, D.P., Zasada, I.A., Teasdale, J.R. 2014. Microbial community structure and abundance in the rhizosphere and bulk soil of a tomato cropping system that includes cover crops. Applied Soil Ecology. 77:42-50. DOI: 10.1016/j.apsoil.2014.01.002.
Chronis, D.N., Chen, S., Skantar, A.M., Zasada, I.A., Wang, X. 2014. A new chorismate mutase gene identified from Globodera ellingtonae and its utility as a molecular diagnostic marker. European Journal of Plant Pathology. 139(2):239-246. DOI:10.1007/s10658-014-0385-x.
Stockwell, V.O., Davis, E.W., Carey, A.B., Shaffer, B.T., Mavrodi, D.V., Hassan, K.A., Hockett, K.L., Thomashow, L.S., Paulsen, I.T., Loper, J.E. 2013. pA506: A conjugative plasmid of the plant epiphyte Pseudomonas fluorescens A506. Applied and Environmental Microbiology. 79:5272-5282.
Henkels, M.D., Kidarsa, T.A., Shaffer, B.T., Goebel, N.C., Burlinson, P., Mavrodi, D.V., Bentley, M.A., Rangel, L.I., Davis Ii, E.W., Thomashow, L.S., Zabriskie, T., Preston, G.M., Loper, J.E. 2014. Pseudomonas protegens Pf-5 causes discoloration and pitting of mushroom caps due to the production of antifungal metabolites. Molecular Plant-Microbe Interactions. 27(7):733-746.
Creason, A.L., Vandeputte, O.M., Savory, E.A., Davis, E.W., Putnam, M.L., Hu, E., Swader-Hines, D., Mol, A., Baucher, M., Prinsen, E., Zdanowska, M., Givan, S.A., El Jaziri, M., Loper, J.E., Mahmud, T., Chang, J.H. 2014. Analysis of genome sequences from plant pathogenic Rhodococcus reveals genetic novelties in virulence loci. PLoS One. 9(7):e101996.
Weiland, G.E., Santamaria, L., Grunwald, N.J. 2014. Sensitivity of Pythium irregulare, P. sylvaticum, and P. ultimum from forest nurseries to mefenoxam and fosetyl-al, and control of Pythium damping-off. Plant Disease. 98:937-942.
Dung, J., Weiland, G.E., Pscheidt, J.W. 2014. Verticillium wilt in the Pacific Northwest. Pacific Northwest Plant Disease Control Handbook. p. 3-22 to 3-28.
Zasada, I.A., Peetz, A.B., Howe, D.K., Wilhelm, L.J., Cheam, D., Denver, D.R., Smythe, A.B. 2014. Using mitogenomic and nuclear ribosomal sequence data to investigate the phylogeny of the Xiphinema americanum species complex. PLoS One. 9:e90035.
Weiland, G.E., Littke, W.R., Haase, D.L. 2013. Forest nurseries face critical choices with the loss of methyl bromide fumigation. California Agriculture. 67(3):153-161.
Weiland, G.E. 2014. Pythium species and isolate diversity influence inhibition by the biological control agent Streptomyces lydicus. Plant Disease. 98(5):653-659.