A novel non-vascular system to treat resistant hypertension.

AIMS: To evaluate in a preclinical model the utility of a monopolar electrode catheter delivering radiofrequency (RF) energy placed into the renal pelvis in order to treat resistant hypertension (RH). METHODS AND RESULTS: Sixteen female domestic swine weighing 60-65 kg underwent renal pelvic denervation via ureteral access. Three animals were euthanised immediately after delivery of RF energy; five animals were allowed to survive for seven days, six animals were allowed to survive for 14 days and two animals were allowed to survive for 30 days. Renal cortical norepinephrine levels were measured in all groups of animals. Histopathology of the treated zone was performed to confirm nerve damage. Renal cortical tissue was harvested for determination of tissue norepinephrine by HPLC. The kidneys were then profusion-fixed and harvested for histopathologic analysis. Mean reduction of norepinephrine levels was 60.4% compared to control. Histopathology confirmed nerve ablation in the treated zone. CONCLUSIONS: In this small, preclinical study, we introduce a new non-vascular system to treat resistant hypertension. If the current clinical experience confirms efficacy and safety, this approach may be one way to treat patients who cannot be treated with the standard percutaneous arterial devices.

Migration patterns of the emerging plant pathogen Phytophthora ramorum on the West Coast of the United States of America.

Phytophthora ramorum (oomycetes) is the causal agent of sudden oak death and ramorum blight on trees, shrubs, and woody ornamentals in the forests of coastal California and southwestern Oregon and in nurseries of California, Oregon, and Washington. In this study, we investigated the genetic structure of P. ramorum on the West Coast of the United States, focusing particularly on population differentiation potentially indicative of gene flow. In total, 576 isolates recovered from 2001 to 2005 were genotyped at 10 microsatellite loci. Our analyses of genetic diversity and inferences of reproductive mode confirm previous results for the Oregon and California populations, with the strong majority of the genotypes belonging to the NA1 clonal lineage and showing no evidence for sexual reproduction. The high incidence of genotypes shared among populations and the lack of genetic structure among populations show that important large-scale, interpopulation genetic exchanges have occurred. This emphasizes the importance of human activity in shaping the current structure of the P. ramorum population on the West Coast of the United States.

First Report of Tobacco rattle virus in Peony in Alaska.

Peonies (Paeonia sp.) are highly valued for their large showy flowers in home gardens and commercially in the cut flower industry. In 2007, scattered peony (Paeonia lactiflora 'Sarah Bernhardt') plants cultivated on small plots at the University of Alaska Experimental Station in Fairbanks displayed distinct leaf ringspot patterns. Symptoms were more severe during the cooler months of the growing season (June and September), with symptom remission in the intervening warmer months. Leaf samples from six symptomatic plants were collected in July and from 20 symptomatic plants in September and assayed for viruses. Leaf samples (1 g) were assayed with a general protocol for plant virus extraction and partial purification with differential centrifugation followed by protein detection on stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1). No distinct proteins indicative of viral coat protein(s) were detected. Tomato spotted wilt virus (TSWV) and Tobacco rattle virus (TRV), known pathogens of peony, were then specifically targeted. Total RNA was extracted from each sample with an RNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and used as the template for reverse transcription (RT)-PCR with random primers. TSWV was not detected by RT-PCR with tospovirus group-specific primers (Agdia, Inc., Elkhart, IN). A nested set of primers designed from the TRV 16-kDa protein gene on RNA1 (4) amplified an ~600-bp fragment from one of the symptomatic plants. This DNA was directly sequenced (GenBank Accession No. FJ357572) and BLAST searches in GenBank revealed as much as 95% nucleotide (nt) identity with TRV accessions J04347 and X03685. Additional primer pairs specific for TRV (2) amplified overlapping fragments with expected sizes of ~818, ~515, and ~290 bp from the 29- and 16-kDa protein genes on the 3'-end of RNA1 that were directly sequenced. Assembly of these sequences in Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI) resulted in a 1,422-nt sequence (Accession No. FJ357571) and Clustal X analysis (3) showed 93 to 94% nt identity to TRV isolates, -ORY (AF034622), -PpK20 (AF314165), -Pp085 (AJ586803), and -SYM (D00155). Mechanical inoculation of partially purified virions from the confirmed TRV-infected peony plant to Nicotiana benthamiana gave no symptoms to occasional ringspots, faintly curled leaves, and chlorotic blotches on N. tabacum 'Samsun', and local lesions on Chenopodium amaranticolor. TRV infection of these hosts was confirmed by RT-PCR. With electron microscopy, rod-shaped particles similar to TRV with a distinct central canal characteristic of TRV were seen occasionally only from inoculated N. benthamiana. On the basis of the biological and molecular data, we have determined the virus in the peony to be an isolate of TRV, tentatively named TRV-Peony. TRV was confirmed in only one other peony based on a sequenced 290-nt PCR fragment with 95% identity with the sequence from the other TRV-infected peony. Lack of TRV detection in the other symptomatic peonies was possibly due to low viral concentrations and interfering plant substances. Documentation of TRV in peonies is especially important to help avoid distribution of virus-infected vegetative propagation material. To our knowledge, this is the first report of TRV in this host in Alaska, but also of this virus in Alaska. References: (1) L. C. Lane. Methods Enzymol. 118:687, 1986. (2) D. J. Robinson. J. Phytopathol. 152:286, 2004. (3) J. D. Thompson et al. Nucleic Acids Res. 24:4882, 1997. (4) F. Van Der Wilk et al. Eur. J. Plant Pathol.100:109, 1994.

Population dynamics of the sudden oak death pathogen Phytophthora ramorum in Oregon from 2001 to 2004.

Phytophthora ramorum (Oomycetes) is an emerging plant pathogen in forests in southwestern Oregon (Curry County). Moreover, since 2003 it has been repeatedly isolated from plants in Oregon nurseries. In this study, we analysed the genetic diversity of the P. ramorum population in Oregon from 2001 to 2004 by using microsatellites. A total of 323 isolates (272 from the infested forest; 51 from nurseries) were screened at 10 loci. The overall P. ramorum population in Oregon is characterized by low genetic diversity and has all the hallmarks of an introduced organism. All isolates within the A2 mating type belonged to the same clonal lineage and no recombinant genotypes were found. The forest population (24 genotypes) was dominated by a single multilocus genotype which persisted over years, indicating that eradication efforts in the forest have not completely eliminated inoculum sources. In contrast, genotypic evidence suggests that eradication was effective in nurseries. In 2003 and 2004, a total of 11 genotypes were found in the nurseries (one belonged to the European lineage of P. ramorum) but no genotype was recovered in both sampling years. Significant differentiation and low gene flow were detected between nursery and forest populations. Only two nursery genotypes were also found in the forest, and then at low frequency. Thus, the nursery infestation is not caused by the genotypes observed in Curry County, but likely resulted through introduction of novel genotypes from nurseries out-of-state. This highlights the continued importance of sanitation and quarantine in nurseries to prevent further introduction and spread of P. ramorum.

Occurrence of Late Blight Caused by Phytophthora infestans on Potato and Tomato in Alaska.

Phytophthora infestans, causal agent of late blight, was included in a list of plant pathogens found in Alaska in 1934 (1). No notes of symptoms, extent of disease, or dates were recorded. The only reference to the location was given as Wrangell, a town in southeast Alaska with subsistence gardening. Neither P. infestans nor late blight was noted again in the state for another 59 years. Late blight first appeared in Alaska's major potato-growing region in south-central Alaska's Matanuska Valley in 1995. Subsequent outbreaks have been sporadic, occurring only in 1998, 2005, and 2006. Each of these outbreaks was identified from rapidly enlarging brown foliar lesions with branched sporangiophores and lemon-shaped sporangia (~25 × 30 µm). The 1995 and 1998 potato late blight outbreaks in Alaska were not sampled extensively nor have they previously been formally reported. We recovered single isolates of P. infestans from symptomatic potato foliage in the 1995 and 1998 outbreaks. In 2005, symptomatic foliage was collected from individual potato plants in 10 commercial fields and from tomato plants in greenhouses at two locations. Sporulating stem and leaf tissue were used to inoculate semiselective rye medium and 147 isolates from potato and six from tomato were recovered. The isolates from the 1995, 1998, and 2005 outbreaks were analyzed to determine genotype at the allozyme loci GPI and PEP (3), mitochondrial haplotype (4), mating type, and metalaxyl sensitivity (2). The 1998 and 2005 outbreaks were similar because both were caused by the relatively aggressive US-11 allozyme genotype and had significant economic impact for commercial potato growers. All 153 isolates from potato and tomato in 2005 displayed the same allozyme pattern as the US-11 genotype, possessed the IIB mitochondrial haplotype, and were mating type A1. Of the 16 isolates tested, all were determined to be metalaxyl resistant because isolates grown on 5 and 100 µg/ml metalaxyl exhibited growth greater than 40% of the unamended control. The 1995 outbreak was caused by the relatively rare US-7 genotype and started so late during the season that economic impact was minimal. Similarly, the 2006 outbreak was noted from only one commercial potato field at the time of harvest in September 2006. However, the genotype of the 2006 isolate has not been determined because the patch was destroyed before adequate samples could be collected. Because the disease occurs so sporadically in Alaska, fungicides are not routinely in use, but it is unlikely that the pathogen has persisted locally between outbreaks. The source of P. infestans is unknown for each of the occurrences in Alaska. However, possible routes include seed potatoes for home gardens or commercial farms, tomato transplants, and retail vegetables shipped to Alaska from out of state. While potato is Alaska's main vegetable crop, there are less than 405 ha (1,000 acres) of potatoes planted in the state, with the majority planted in the Matanuska Valley. To our knowledge, this is the first formal report of P. infestans on both tomato and commercial potato in Alaska. References: (1) E. K. Cash. Plant Dis. Rep. 20:121, 1936. (2) D. E. L. Cooke et al. Plant Pathol. 52:181, 2003. (3) S. B. Goodwin et al. Plant Dis. 79:1181, 1995. (4) G. W. Griffith and D. S. Shaw. Appl. Environ. Microbiol. 64:4007, 1998.

Comparison of Biochemical, Molecular, and Visual Methods to Quantify Phaeocryptopus gaeumannii in Douglas-Fir Foliage.

ABSTRACT A recent epidemic of Swiss needle cast along the Oregon coast has prompted efforts to quantify foliar infection and colonization of the causal agent Phaeocryptopus gaeumannii. In this paper, we compare four methods to quantify colonization of Douglas-fir foliage by P. gaeumannii: fruiting body abundance, ergosterol content, dot blot analysis, and TaqMan based real-time quantitative polymerase chain reaction (PCR). Results from the four techniques were all significantly correlated. Fruiting body density and quantitative PCR are two methods least affected by the presence of other needle fungi and had the highest correlation. The methods also were used to compare foliage colonization in nine field sites exhibiting a range of disease severity. All four methods provided evidence that sites differed in the degree of fungal colonization, but only quantitative PCR consistently separated sites with moderate to severe levels of disease from sites with low disease estimated by foliage color, canopy density, and growth measurements.

First Report of A1 Mating Type of Phytophthora ramorum in North America.

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.

Simultaneous one-tube quantification of host and pathogen DNA with real-time polymerase chain reaction.

ABSTRACT Phaeocryptopus gaeumannii is a widespread foliar parasite of Douglas-fir. Although normally innocuous, the fungus also causes the defoliating disease Swiss needle cast in heavily infected needles. The extent of P. gaeumannii colonization in Douglas-fir foliage was estimated with real-time quantitative polymerase chain reaction (PCR) using TaqMan chemistry. In order to derive a normalized expression of colonization, both pathogen and host DNA were simultaneously amplified but individually detected by species-specific primers and TaqMan probes labeled with different fluorescent dyes. Detection of host DNA additionally provided an endogenous reference that served as both an internal positive control and adjusted for variation introduced by sample-to-sample differences in DNA extraction and PCR efficiencies. The genes employed for designing the TaqMan probes and primers were beta-tubulin for the pathogen and a LEAFY/FLORICAULA-like gene involved in floral development for the tree host. Both probe/primer sets exhibited high precision and reproducibility over a linear range of 4 orders of magnitude. This eliminated the need to analyze samples in multiple dilutions when comparing lightly with heavily infected needles. Quantification of the fungus within needles was successful as early as 1 month after initial infection. Real-time PCR is the only method currently available to quantify P. gaeumannii colonization early in the first year of the colonization process.

Incidence and Impact of Swiss Needle Cast in Forest Plantations of Douglas-fir in Coastal Oregon.

An epidemic of Swiss needle cast, caused by the ascomycete Phaeocryptopus gaeumannii, is causing defoliation and growth reductions in Douglas-fir forest plantations along the Oregon Coast. The area of symptomatic plantations has been monitored annually since 1996 by aerial survey; in spring 1999, 119,500 ha were affected. Pathogen and symptom development have also been monitored on nine permanent plots in stands of differing disease severity. Infection levels and symptom severity are greatest in low elevation plantations close to the coast. In areas of severe disease, trees retain only current year needles. Defoliation is proportional to the number of stomata occluded by pseudothecia of the fungus, with needles being shed when about 50% of stomata are occupied, regardless of needle age. Fungus sporulation and premature needle abscission are greatest on the upper branches of trees. Annual application of fungicides increases needle retention significantly. Tree height and diameter growth and total tree volume are reduced by disease, and tree volume is significantly correlated with needle retention on our plot trees. The epidemic continues to be most severe in Douglas-fir plantations established on sites where Sitka spruce and western hemlock or red alder predominated in earlier times.

Treatment of myeloid blastic crisis of chronic myelogenous leukemia.

Patients with myeloid blastic crisis of chronic myelogenous leukemia were treated by chemotherapy or by autologous hematopoietic reconstitution after aggressive chemotherapy. Chemotherapy alone failed to produce a second chronic phase. Autologous transplantation resulted in the establishment of a second chronic phase in two of ten patients treated with a four-drug regimen, while treatment with high-dose cytarabine with or without busulfan resulted in the establishment of a second chronic phase in three of six patients and the return of normal hematopoiesis in a fourth. Consolidation chemotherapy appeared to be beneficial.

Relationship between plasma adriamycin levels and the outcome of remission induction therapy for acute nonlymphocytic leukemia.

Plasma adriamycin and adriamycinol levels were measured in 45 patients with acute nonlymphocytic leukemia 3 h after the drug was administered. A wide range of levels as found. Plasma levels increased after the administration of each of the three daily doses of the drug. High plasma levels were associated with both death during remission induction therapy and, for patients who entered remission, long remissions.

Bladder calculi enveloped by extensive cystitis glandularis: case report.

We report a case of 2 large calculi encased within a 15 gm. tumor of cystitis cystica and cystitis glandularis. The patient presented with acute right epididymitis. An excretory urogram showed 2 bladder calculi. Cystoscopy revealed a mass around the stones resembling adenocarcinoma of the bladder base. Transurethral resection of the tumor and cystolitholapaxy were performed. The final diagnosis was bladder calculi within a mass of cystitis cystica and cystitis glandularis. A review of the literature failed to reveal a similar case.

Plantlets from aspen tissue cultures.

After 2 weeks in the dark, leafy shoots were initiated on subcultured callus tissue of triploid quaking aspen on Wolter's medium without auxin and with 6-benzylaminopurine at 0.05, 0.10., 0.15, or 0.20 milligram per liter substituted for kinetin. Shoots transferred to Wolter's medium under light (323 milliphots) grew large roots and were isolated as plantlets.