Horticultural Crops Research Laboratory

Corvallis, United States

Horticultural Crops Research Laboratory

Corvallis, United States

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Wallace P.K.,Oregon State University | Arey B.,Pacific Northwest National Laboratory | Mahaffee W.F.,Oregon State University | Mahaffee W.F.,Horticultural Crops Research Laboratory
Micron | Year: 2011

The dual beam scanning electron microscope, equipped with both a focused ion- and scanning electron-beam (FIB SEM) is a novel tool for the exploration of the subsurface structure of biological tissues. The FIB can remove a predetermined amount of material from a selected site to allow for subsurface exploration and when coupled with SEM or scanning ion-beam microscopy (SIM) could be suitable to examine the subsurface structure of bacterial biofilms on the leaf surface. The suitability of chemical and cryofixation was examined for use with the FIB SEM to examine bacterial biofilms on leaf surfaces. The biological control agent, Burkholderia pyroccinia FP62, that rapidly colonizes the leaf surface and forms biofilms, was inoculated onto geranium leaves and incubated in a greenhouse for 7 or 14 days. Cryofixation was not suitable for examination of leaf biofilms because it created a frozen layer over the leaf surface that cracked when exposed to the electron beam and the protective cap required for FIB milling could not be accurately deposited. With chemically fixed samples, it was possible to precisely FIB mill a single cross section (5 μm) or sequential cross sections from a single site without any damage to the surrounding surface. Biofilms, 7 days post-inoculation (DPI), were composed of 2-5 bacterial cell layers while biofilms 14 DPI ranged from 5 to greater than 30 cell layers. Empty spaces between bacteria cells in the subsurface structure were observed in biofilms 7- and 14-DPI. Sequential cross sections inferred that the empty spaces were often continuous between FP62 cells and could possibly make up a network of channels throughout the biofilm. FIB SEM was a useful tool to observe the subsurface composition of a foliar biofilm. © 2011.


Weiland J.E.,Horticultural Crops Research Laboratory | Weiland J.E.,Oregon State University | Nelson A.H.,Cornell University | Hudler G.W.,Cornell University
Plant Disease | Year: 2010

Phytophthora cactorum, P. citricola I, and P. plurivora cause bleeding cankers on mature European beech (Fagus sylvatica) trees in the northeastern United States. Inoculation experiments were conducted to compare the aggressiveness of the three Phytophthora spp. on stems, leaf disks, and roots of European beech and common lilac (Syringa vulgaris) seedlings. Isolates were obtained from bleeding cankers on European beech from five cities in New York (Albany, Ithaca, Oyster Bay, Plainview, and Rochester) and from a bleeding canker on sugar maple in Ithaca, NY. Stems were inoculated with colonized agar plugs, leaf disks with a zoospore suspension, and roots via infested soil at three inoculum levels. All organs of inoculated beech and lilac developed disease except for lilac roots inoculated with zoospores of P. cactorum. Disease incidence, severity, and plant survival were dependent on isolate and were also influenced by the tissue inoculated and host. Isolates of P. cactorum were the least aggressive and caused less necrosis than isolates of P. citricola I and P. plurivora. Results emphasize the utility of stem and root inoculation for evaluation of this canker disease and underscore critical differences in species aggressiveness.


Goss E.M.,Horticultural Crops Research Laboratory | Goss E.M.,University of Florida | Press C.M.,Horticultural Crops Research Laboratory | Grunwald N.J.,Horticultural Crops Research Laboratory
PLoS ONE | Year: 2013

Phytophthora plant pathogens contain many hundreds of effectors potentially involved in infection of host plants. Comparative genomic analyses have shown that these effectors evolve rapidly and have been subject to recent expansions. We examined the recent sequence evolution of RXLR-class effector gene families in the sudden oak death pathogen, P. ramorum. We found that P. ramorum RXLR effectors have taken multiple evolutionary paths, including loss or gain of repeated domains, recombination or gene conversion among paralogs, and selection on point mutations. Sequencing of homologs from two subfamilies in P. ramorum's closest known relatives revealed repeated gene duplication and divergence since speciation with P. lateralis. One family showed strong signatures of recombination while the other family has evolved primarily by point mutation. Comparison of a small number of the hundreds of RXLR-class effectors across three clonal lineages of P. ramorum shows striking divergence in alleles among lineages, suggesting the potential for functional differences between lineages. Our results suggest future avenues for examination of rapidly evolving effectors in P. ramorum, including investigation of the functional and coevolutionary significance of the patterns of sequence evolution that we observed.


Takeda F.,U.S. Department of Agriculture | Tworkoski T.,U.S. Department of Agriculture | Finn C.E.,Horticultural Crops Research Laboratory | Boyd C.C.,Cedar Valley Nursery Inc
HortTechnology | Year: 2011

One- or two-node hardwood cuttings were taken from 9-year-old 'Triple Crown' and 'Siskiyou' blackberry (Rubus) plants on 5 Nov. 2009, 3 Dec. 2009, and 21 Jan. 2010. The response of cuttings with and without partially excised axillary buds to an application of cytokinin was compared with control cuttings with intact axillary buds and no cytokinin. Differences in root development were evident in the two cultivars tested. The cuttings of 'Siskiyou' and 'Triple Crown' callused on cut ends, but many of the adventitious roots developed from the base of the axillary buds. Shoots emerged from the bud in %90% of 'Siskiyou' cuttings stuck in November, December, and January. Rooting occurred in more than 90% of cuttings stuck in November and December but declined in cuttings stuck in January. In 'Siskiyou', bud excision had no effect on shoot and root emergence, but cytokinin treatment suppressed rooting in cuttings collected in November and January. Shoot emergence and rooting were poorer in 'Triple Crown' cuttings than in 'Siskiyou'. In 'Triple Crown' cuttings, partial excision of buds reduced shoot emergence only in January but had no effect on rooting at three sticking dates. Cytokinin treatment improved shoot emergence in November and December but reduced rooting in January. The enclosed system is a viable method for propagating 'Siskiyou' blackberry by non-leafy floricane cuttings.


Seguin J.,University of Basel | Seguin J.,Fasteris SA | Rajeswaran R.,University of Basel | Malpica-Lopez N.,University of Basel | And 7 more authors.
PLoS ONE | Year: 2014

Virus-infected plants accumulate abundant, 21-24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this 'siRNA omics' approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.


Johnson K.B.,Oregon State University | Mahaffee W.F.,Horticultural Crops Research Laboratory
Plant Disease | Year: 2010

The blackberry rust pathogen Phragmidium violaceum was first observed in Oregon in spring 2005 on both commercially cultivated Rubus laciniatus (Evergreen blackberry) and naturalized R. armeniacus (Himalayan blackberry). Several commercial plantings suffered severe economic losses. In 2006 to 2008, all five spore stages of this autoecious, macrocyclic rust pathogen were observed annually, and asexual perennation of the pathogen on old leaves or in leaf buds was not evident in the disease cycle. In field experiments, teliospore germination and infection by basidiospores occurred mostly during April. On potted "trap" plants exposed for periods of 1 week under dense collections of dead leaves bearing teliospores, basidiospore infection was associated with wetness durations of >16 h with mean temperatures <8°C. Trap plants placed under the bundles of collected leaves frequently developed spermagonia, whereas only 1 of 630 trap plants placed in a production field of R. laciniatus became diseased, an indication that the effective dispersal distance of basidiospores may be limited. In growth chambers programmed for constant temperatures of 5, 10, 15, 20, 25, and 30°C, a minimum of six continuous hours of leaf wetness was required for infection by urediniospores, with >9 h required for moderate infection (>4 pustules/cm 2) at 15 and 20°C. With diurnal temperature regimes averaging 5, 10, 15, 20, or 25°C, urediniospore germination and infection was highest in the range of 5 to 15°C; similarly, in the diurnal environment, >9 h of leaf wetness was required to attain moderate infection. In the field, lime sulfur applied as a delayed dormant treatment significantly suppressed telio-spore germination and basidiospore infection. Over two seasons, one application of myclobu-tanil, a demethylation-inhibitor fungicide, applied in early May near the time of spermagonial appearance provided effective suppression of the summer epidemic.


Stockwell V.O.,Oregon State University | Johnson K.B.,Oregon State University | Sugar D.,Oregon State University | Loper J.E.,Oregon State University | Loper J.E.,Horticultural Crops Research Laboratory
Phytopathology | Year: 2010

The biological control agents Pseudomonas fluorescens A506 and Pantoea vagans C9-1 were evaluated individually and in combination for the suppression of fire blight of pear or apple in 10 field trials inoculated with the pathogen Erwinia amylovora. The formulation of pathogen inoculum applied to blossoms influenced establishment of the pathogen and the efficacy of biological control. Pantoea vagans C9-1 suppressed fire blight in all five trials in which the pathogen was applied as lyophilized cells but in none of the trials in which the pathogen was applied as freshly harvested cells. In contrast, Pseudomonas fluorescens A506 reduced disease significantly in only one trial. A mixture of the two strains also suppressed fire blight, but the magnitude of disease suppression over all field trials (averaging 32%) was less than that attained by C9-1 alone (42%). The two biological control agents did not antagonize one another on blossom surfaces, and application of the mixture of A506 and C9-1 to blossoms resulted in a greater proportion of flowers having detectable populations of at least one bacterial antagonist than the application of individual strains. Therefore, the mixture of A506 and C9-1 provided less disease control than expected based upon the epiphytic population sizes of the antagonists on blossom surfaces. We speculate that the biocontrol mixture was less effective than anticipated due to incompatibility between the mechanisms by which A506 and C9-1 suppress disease. © 2010 The American Phytopathological Society.


Stockwell V.O.,Oregon State University | Johnson K.B.,Oregon State University | Sugar D.,Oregon State University | Loper J.E.,Oregon State University | Loper J.E.,Horticultural Crops Research Laboratory
Phytopathology | Year: 2011

Mixtures of biological control agents can be superior to individual agents in suppressing plant disease, providing enhanced efficacy and reliability from field to field relative to single biocontrol strains. Nonetheless, the efficacy of combinations of Pseudomonas fluorescens A506, a commercial biological control agent for fire blight of pear, and Pantoea vagans strain C9-1 or Pantoea agglomerans strain Eh252 rarely exceeds that of individual strains. A506 suppresses growth of the pathogen on floral colonization and infection sites through preemptive exclusion. C9-1 and Eh252 produce peptide antibiotics that contribute to disease control. In culture, A506 produces an extracellular protease that degrades the peptide antibiotics of C9-1 and Eh252. We hypothesized that strain A506 diminishes the biological control activity of C9-1 and Eh252, thereby reducing the efficacy of biocontrol mixtures. This hypothesis was tested in five replicated field trials comparing biological control of fire blight using strain A506 and A506 aprX::Tn5, an extracellular proteasedeficient mutant, as individuals and combined with C9-1 or Eh252. On average, mixtures containing A506 aprX::Tn5 were superior to those containing the wild-type strain, confirming that the extracellular protease of A506 diminished the biological control activity of C9-1 and Eh252 in situ. Mixtures of A506 aprX::Tn5 and C9-1 or Eh252 were superior to oxytetracycline or single biocontrol strains in suppressing fire blight of pear. These experiments demonstrate that certain biological control agents are mechanistically incompatible, in that one strain interferes with the mechanism by which a second strain suppresses plant disease. Mixtures composed of mechanistically compatible strains of biological control agents can suppress disease more effectively than individual biological control agents. © 2011 The American Phytopathological Society.


Lee J.C.,Horticultural Crops Research Laboratory
Environmental Entomology | Year: 2010

Methyl salicylate (MeSA) is a common herbivore-induced plant volatile that, when applied to crops, has the potential to enhance natural enemy abundance and pest control. The impacts of MeSA in strawberry were unknown and examined in the spring and midsummer period. Strawberry plots contained no lures (control) or two 30-d MeSA lures (Predalure) in the center: one lure 0.61 m aboveground over a sticky trap, and one lure on a plant near the ground. Arthropod abundance was monitored at the point source, 5 m and 10 m away from lures over 31 d with white sticky traps, pitfall traps, and leaf inspection. Twenty-seven and nine comparisons were made among beneficial and pest arthropods, respectively. Overall positive responses were found among Chrysopidae in JulyAugust 2008 and Orius tristicolor (White) in MayJune 2009 to MeSA based on sticky traps. Chrysopidae showed attraction to the point source, but not at 5 m and 10 m. Ground-dwelling predators collected in pitfall traps such as Araneae, the carabid beetles, Pterostichus melanarius (Illiger), and Nebria brevicollis (Fabricius) did not respond. Increased abundance of six natural enemy groups appeared on various dates between 3 and 24 d after placement of lures in the field based on leaf inspection and sticky traps. Conversely, fewer Coccinellidae were captured on sticky traps on days 03, and fewer natural enemies were observed on leaves on day 28 in MeSA plots. MeSA did not increase nor decrease pest abundance.


PubMed | Horticultural Crops Research Laboratory
Type: Journal Article | Journal: PloS one | Year: 2013

Phytophthora plant pathogens contain many hundreds of effectors potentially involved in infection of host plants. Comparative genomic analyses have shown that these effectors evolve rapidly and have been subject to recent expansions. We examined the recent sequence evolution of RXLR-class effector gene families in the sudden oak death pathogen, P. ramorum. We found that P. ramorum RXLR effectors have taken multiple evolutionary paths, including loss or gain of repeated domains, recombination or gene conversion among paralogs, and selection on point mutations. Sequencing of homologs from two subfamilies in P. ramorums closest known relatives revealed repeated gene duplication and divergence since speciation with P. lateralis. One family showed strong signatures of recombination while the other family has evolved primarily by point mutation. Comparison of a small number of the hundreds of RXLR-class effectors across three clonal lineages of P. ramorum shows striking divergence in alleles among lineages, suggesting the potential for functional differences between lineages. Our results suggest future avenues for examination of rapidly evolving effectors in P. ramorum, including investigation of the functional and coevolutionary significance of the patterns of sequence evolution that we observed.

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