The major light-harvesting pigment-protein complex of Mamiella gilva (Parke et Rayns) Moestrup (Micromonadophyceae, Chlorophyta) was isolated by lithium dodecyl sulphate polyacrylamide gel electrophoresis at 4°C. This complex is similar to that of Mantoniella squamata (Manton et Parke) Desikachary and Micromonas pusilla (Butcher) Manton et Parke with regard to molecular weight, absorption spectrum, and immunochemical cross-reactivity. On the other hand, the light-harvesting complex of Chlamydomonas reinhardtii P. Dangeard is quite different. The distribution of this unusual light-harvesting complex, which has to date been reported only in Mamiella, Mantoniella, and Micromonas, suggests a line of evolution in the Micromonadophyceae separate from all other green algae. In addition, the inclusion of the naked Micromonas in this group suggests that Micromonas should be included with Mamiella and Mantoniella in the Mamiellales, an order which is presently defined by scale type.
Sudden oak death, caused by Phytophthora ramorum (1,2), has been found for the first time in Oregon, killing tanoak, Lithocarpus densiflorus, trees. To our knowledge, this is the first report of the disease outside of the San Francisco to Monterey area in California, (300 km to the south). Nine areas of infestation, all within a 24-km2 area, were discovered on forest lands near Brookings, in southwest Oregon. Mortality centers ranged in size from 0.2 to 4.5 ha and included 5 to approximately 40 diseased trees. P. ramorum was isolated from stem cankers using Phytophthora-selective medium. Isolates had distinctive morphological features characteristic of P. ramorum, including abundant production of chlamydospores and caducous, semipapillate sporangia on solid media. Internal transcribed spacer (ITS) sequences of isolates of P. ramorum from Oregon were identical to ITS sequences of isolates from California (1). The pathogen also was isolated from necrotic lesions on leaves and stems of native Rhododendron macrophyllum and Vaccinium ovatum growing beneath diseased tanoaks. In July 2001, the disease was located by an aerial survey conducted cooperatively by the USDA Forest Service and Oregon Department of Forestry. All lands within 1.6 km (1 mile) of the mortality centers are subject to Oregon quarantine, which bars the transport of any host plant materials. An eradication effort is currently underway. Symptomatic plants and all known host plants within 15 to 30 m of symptomatic plants are being cut and burned in the first phase of this operation. The total treated area is approximately 16 ha. References: (1) D. M. Rizzo et al. Plant Dis. In press. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.
Phytophthora ramorum attacks many plant species important to the nursery industry and to the natural environment. USDA-APHIS requires all West Coast nursery stock to be inspected and, if necessary, tested for P. ramorum prior to export. The plants must be tested using a commercial DAS-ELISA kit and, if confirmed positive, the species-specific methods of PARP and PCR. This paper compares these diagnostic methods for the detection of P. ramorum in Rhododendron (L.) nursery stock. Accepted for publication 4 March 2005. Published 14 March 2005.
ABSTRACT The biosynthesis of the light‐harvesting complex (LHC) polypeptides of the green flagellate Mantoniella squamata (Manton et Parke) Desikachary (Micromonadophyceae, Chlorophyta) was examined by in vivo polypeptide labeling and immunoprecipitation of in vitro translation products. Using protein synthesis inhibitors, the LHC polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes and not in the chloroplasts of cells. Poly (A)+ RNA was isolated and proteins were synthesized by a rabbit reticulocyte lysate system, with antisera raised against M. squamata LHC used for immunoprecipitation from the translation products. One polypeptide 3‐5 kDa larger than mature LHC polypeptides was immunoprecipitated. These studies indicate that although the LHC of M. squamata is quite different from the LHC of most green plants, the LHC polypeptides are synthesized as precursors in the cytoplasm of the cell and suggest that the genes encoding these polypeptides are located in the nucleus.
Phytophthora ramorum is known in Europe and the western United States (1). In Europe, it is found in nurseries and landscape plantings. In the United States, it has been confined to coastal forests, and in California, it is found in a few horticultural nurseries. All European isolates tested have been A1 mating type, while all North American isolates were A2 mating type (2). Amplified fragment length polymorphism markers also indicated that the populations on the two continents are distinct, and nearly all North American isolates are from one clone (Kelly Ivors, unpublished). In June 2003, P. ramorum was isolated from diseased Viburnum and Pieris spp. cultivars from a Clackamas County nursery in northern Oregon and diseased Camellia sp. cultivar from a Jackson County nursery in southern Oregon. Representative isolates were submitted to the American Type Culture Collection, Manassas, VA. As part of the effort to determine the origin of these new infestations, we tested the nursery isolates for mating type. Seven Oregon nursery isolates, three Oregon forest isolates (from the predominant North American clone), and two European isolates were paired. Agar plugs from 3-day-old colonies were placed in close proximity on carrot agar plates, and then the plates were examined for oogonia after 3 and 10 days as advised by C. M. Brasier (personal communication). Oogonia and antheridia typical of P. ramorum (2) formed when isolates from the Clackamas County nursery were paired with the Oregon forest isolates and also when isolates from the Jackson County nursery were paired with the European isolates. Gametangia also formed in pairings between Oregon forest isolates and European isolates, but not in any other combinations. We developed polymerase chain reaction (PCR) primers for four microsatellite loci and determined allele sizes for the same set of isolates (unpublished). Microsatellite alleles of the Clackamas County isolates were identical to the European tester isolates, and alleles of the Jackson County isolates were identical to the Oregon forest isolates. These results indicate that the recent Oregon nursery infestations are of separate origins. The Clackamas County isolates are A1 mating type and have microsatellite alleles like the European testers, but according to shipping records, the nursery has received no host nursery stock directly from Europe. However, host nursery stock has been received from a Canadian nursery. The Jackson County isolates are of A2 mating type and have microsatellite alleles like the forest isolates of Oregon, which is consistent with the reported origin of these plants from a California nursery. These preliminary microsatellite results need to be validated against a larger isolate set but are congruent with the mating type results. The Oregon nursery infestations highlight the dangers of unregulated or underregulated transport of host nursery stock from infested areas to noninfested areas. All host plants from infested nursery blocks at the affected Oregon nurseries have been destroyed by incineration, and a monitoring program has been implemented. Other host nursery stock on site has been taken "off-sale" pending verification that it is disease free, per the United States Department of Agriculture, APHIS requirements. References: (1) J. M. Davidson et al. On-line publication. doi:10.1094/PHP-2003-0707-01-DG. Plant Health Progress, 2003. (2) S. Werres et al. Mycol. Res. 105:1155, 2001.
The Oregon Department of Agriculture's official virus testing program for Vaccinium has grown 915% since its inception. To address this increased workload, we evaluated a high-throughput ELISA protocol that uses a 96-well format for sap extraction. To verify the efficacy of the protocol, it was used to test leaves from known infected and healthy plants (positive and negative controls) and from 940 Vaccinium plants grown in a nursery with Blueberry shock virus (BlShV) problems. Each leaf collected was cut in half; one-half was macerated in an extraction buffer using mesh sample bags and a roller press (standard protocol) and the other half was placed into 10 × 96 collection microtubes and macerated in the extraction buffer using a Mixer Mill MM300 (high-throughput protocol). All sample sap extractions were then tested with a commercial ELISA kit. In experiments with the BlShV positive and negative control plants, the high-throughput protocol had high sensitivity (100%) and specificity (100%), matching the standard protocol. In experiments with the blueberry plants of unknown BlShV infection status, the high-throughput protocol had a sensitivity of 94.85% and a specificity of 96.70%, compared to the standard protocol. It is concluded the high-throughput protocol can be used successfully with ELISA to detect BlShV in Accepted for publication 17 May 2009. Published 10 July 2009. Accepted for publication 17 May 2009. Published 10 July 2009.
Phytophthora ramorum is a pathogen of regulatory concern in North America and Europe. In 2004, potentially infected plants were shipped from large, wholesale nurseries on the West Coast (California, Oregon, and Washington) throughout the U.S. This prompted a nationwide survey effort and the adoption of a federal order requiring mandatory inspection and testing of all West Coast nurseries shipping P. ramorum host and associated host plants (HAP) interstate. In Oregon, this required the testing of 51,645 samples from 1,034 growing areas and 79,930 samples from 1,394 growing areas in 2005 and in 2006, respectively. Because all testing must be completed before nurseries can ship HAP interstate, the Oregon Department of Agriculture developed a high throughput system using a 96-well format to enable testing of large numbers of samples in accordance with federally validated protocols (ELISA, nested PCR, and qPCR). To verify the efficacy of the system, healthy leaves from four HAP species were wounded and then artificially inoculated with P. ramorum; healthy control leaves were wounded and then inoculated with a sterile agar plug. Samples were collected from the inoculated and control leaves and placed into 10 X 96 collection microtubes. Subsamples from each HAP were bulked five per microtube in varying ratios of inoculated to noninoculated tissue (5:0, 1:4, and 0:5). Sample tissues were macerated and tested with ELISA according to the manufacturer?s protocol. The OD readings of all inoculated samples were consistently 5X the negative control. All non-inoculated controls were below the 2X threshold with one exception. In the second replicate, the OD reading of the Pieris japonica noninoculated control was >2X the negative control. DNA was then extracted from the remaining sample tissue in the GEB2 buffer. All inoculated samples were positive using nested PCR while non-inoculated controls were negative. Sample DNA was then tested with qPCR. All inoculated samples were positive while all non-inoculated controls were negative with one exception. In the first replicate, the Camellia non-inoculated control had a Ct value of 40.08 for the P. ramorum-specific probe. According to USDA protocol, this sample would be tested with nested PCR to confirm this negative test result. The high throughput, 96-well format system was also used successfully with environmental samples. Samples from four HAP species and six HAP genera were identified as positive with ELISA and subsequently as P. ramorum-positive by nested PCR and/or qPCR. USDA officially confirmed these test results. These preliminary results indicate that the high throughput system successfully detected P. ramorum in infected plant tissue using the USDA-validated ELISA, nested PCR, and qPCR protocols.
Phytophthora ramorum, the causal agent of sudden oak death, was first discovered in Oregon in July 2001 by aerial survey (Goheen and others 2002). Alerted to the situation in California and experienced in aerial tree mortality surveys, cooperators from the USDA Forest Service and the Oregon Department of Forestry planned a pilot survey for P. ramorum to become familiar with what was perceived to be the high risk type: forest stands with tanoak components within approximately 16 km of the Oregon coastline and along highly traveled road corridors leading to and from California. One hundred thirty seven thousand hectares were surveyed in total, but almost immediately upon starting the fixed-wing based portion of the survey, patches of recently killed tanoaks were spotted and mapped.
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