Diurnal effects on sporangium and zoospore production by Phytophthora ramorum on Rhododendron 'Cunningham's White'.

We evaluated sporangium and zoospore production by three isolates of Phytophthora ramorum on Rhododendron 'Cunningham's White' leaves under light and dark conditions at both variable and constant (14 C) temperature. P. ramorum-infected leaves were detached and placed in funnels inside of a 62-L plastic storage container located in a growth chamber. Cool mist was introduced to the container to create a high-humidity environment. Sporangia and zoospores were collected over a 4-day period by misting leaves with 5 mL of distilled water, which was collected in conical test tubes that also contained runoff from the misting. Spores were collected daily just before a 13-h light period and again just before an 11-h dark period. Sporangia and zoospores in the collection tubes were counted using a dissecting microscope following staining with lactoglycerin/aniline blue. Large differences in sporangium and zoospore numbers observed for the dark versus light periods were observed on days 2, 3, and 4. A diurnal effect has been observed for production of propagules of other oomycetes, but such effects have not been previously reported for P. ramorum. This information will help provide a better understanding of patterns of inoculum production by P. ramorum and resulting fluctuations in inoculum density that will influence sudden oak death epidemics in forest ecosystems in the United States and other countries where it occurs.

The Effect of Leaf Wetness on Phytophthora ramorum Zoospore Infection of Rhododendron 'Cunningham's White' and Viburnum tinus.

We performed studies using zoospore inoculum combined from nine isolates of Phytophthora ramorum and determined the effect of leaf wetness on infection of whole plants of Rhododendron 'Cunningham's White' and Viburnum tinus. The mean percentage of infected leaves for both host species increased gradually across a dew chamber moisture period of 1 to 6 h, reaching approximately 80% infection by 6 h. We also evaluated the effect of a postinoculation drying period on infectivity of the two host species with zoospore inoculum. With a 30-min postinoculation drying period, Rhododendron 'Cunningham's White' sustained less than 40% infected leaves, whereas V. tinus had an infection rate of almost 75% infected leaves. Disease percentages for both host species declined sharply with drying periods longer than 30 min. Knowledge of infectivity parameters for P. ramorum will provide a better understanding of epidemic development and lead to improved recommendations for control.

Sporangia Production Over Time by Phytophthora ramorum on Rhododendron 'Cunningham's White' After Placement at Different Relative Humidities.

We examined the impact of relative humidity (RH) on Phytophthora ramorum sporangia production on Rhododendron 'Cunningham's White'. When diseased plants were maintained under continuous moisture in a mist tent, sporangia were collected from some plants for 22 weeks. More than 3,000 sporangia/leaf/week were collected over the first 3 weeks but levels declined to <100 sporangia/leaf/week after 7 weeks. We also examined the impact of drying on P. ramorum sporangia production. Diseased, detached leaves were maintained in humidity chambers (100, 96.2, 84.5, 74.9, and 56.2% RH) for up to 9 weeks and removed weekly to assess sporulation. For comparison, diseased leaves were harvested from plants maintained with dry foliage or subjected to 10 h of simulated dew nightly. All leaves supported sporulation following 5 weeks at 100% RH, 3 weeks at 96.2% RH, and 1 week at 84.5% RH. All leaves collected from plants subjected to nightly dew supported sporulation for 3 weeks; however, only 66.7% of leaves collected from plants with dry foliage supported sporulation after 1 week. Knowledge of the effects of RH levels on P. ramorum sporulation capacity will prove useful in terms of disease management recommendations and for development of predictive models and pest risk assessments.

Pyracantha 'Mohave' Fruit Infection by Phytophthora ramorum and Transmission of the Pathogen from Infected Fruit to Roots of Viburnum tinus.

Colonization of the fleshy fruit of Cornus florida, C. kousa, Laurus nobilis, Malus hupehensis, and Pyracantha 'Mohave' was observed following inoculation with sporangia of Phytophthora ramorum. However, abundant production of chlamydospores was only observed in the fruit of Pyracantha 'Mohave'. Pyracantha 'Mohave' fruit that had been inoculated with a P. ramorum sporangia suspension were placed in pots containing rooted cuttings of Viburnum tinus in a misting tent or in water-filled trays in a climate-controlled greenhouse. Runoff was collected for 24 to 30 days, and roots were plated after the final collection. Mean percent recovery from infected roots was not significantly different (P = 0.05, Tukey's test) between bottom-watered treatments in trays and misted treatments, averaging 58% for bottom-watered and 54% for mist treatments. The number of CFU collected in runoff from bottom-watered plants was consistently lower than that obtained from plants held under mist, likely due to desiccation of the fruit. The results show that root infection of V. tinus can occur by P. ramorum via infected fruit of Pyracantha 'Mohave'. This phenomenon represents a pathway of infection for P. ramorum not previously reported, which may play a role in disease epidemiology.

The effect of temperature on germination of chlamydospores of Phytophthora ramorum.

Mycelium-free chlamydospores of 12 isolates of P. ramorum representing three clonal lineages were produced with a method involving incubation in nonsterile sand at 20 C in darkness for 30 d. Chlamydospores were incubated on selective agar medium at 5, 10, 15, 20, 25 and 30 C and germination assessed after 1, 2, 4, 6 and 8 d incubation. The optimal temperature for germination based on 8 d incubation was 20 C for all three clonal lineages tested (NA1, NA2, EU1). Mean germination rates were 2, 21, 44, 67, 32 and 0 percent at 5, 10, 15, 20, 25 and 30 C respectively for all isolates combined. The highest mean germination rate was scored by isolates of the EU1 clonal lineage at 20 C (85%) after 8 d incubation However, substantial variation was observed among isolates within each clonal lineage. Overall temperatures and days of incubation on which germination was assessed isolates of the NA1 clonal lineage had the lowest mean germination, even though one isolate had the highest germination of any isolate in any lineage. The results indicate that 20 C is the optimal germination temperature for P. ramorum chlamydospores and that a great disparity in germination percentage can exist within isolates, even within a single clonal lineage.

Expanded molecular features of carnitine acyl-carnitine translocase (CACT) deficiency by comprehensive molecular analysis.

Carnitine-acylcarnitine translocase (CACT) deficiency is a rare autosomal recessive disease of fatty acid oxidation, mainly affecting long chain fatty acid utilization. The disease usually presents at neonatal period with severe hypoketotic hypoglycemia, hyperammonemia, cardiomyopathy and/or arrhythmia, hepatic dysfunction, skeletal muscle weakness, and encephalopathy. Definitive diagnosis of CACT deficiency by molecular analysis of the SLC25A20 gene has recently become clinically available. In contrast to biochemical analysis, sequence analysis is a more rapid and reliable method for diagnosis of CACT deficiency. In this study, we used Sanger sequencing and target array CGH to identify molecular defects in the SLC25A20 gene of patients with clinical features and an acylcarnitine profile consistent with CACT deficiency. Eight novel mutations, including a large 25.9 kb deletion encompassing exons 5 to 9 of SLC25A20 were found. Review of the published cases revealed that CACT deficiency is a pan-ethnic disorder with a broad mutation spectrum. Mutations are distributed along the entire gene without a hot spot. Two thirds of them are nonsense, frame-shift, or splice site mutations resulting in premature stop codons. This study underscores the importance of comprehensive molecular analysis, including sequencing and targeted array CGH of the SLC25A20 gene when CACT deficiency is suspected.

Parents' decision-making in newborn screening: opinions, choices, and information needs.

OBJECTIVE: Our objective was to describe how parents consider disease and test characteristics when making decisions about newborn screening. METHODS: We conducted focus groups with parents from primary care clinics and interviews of parents from a genetics clinic (total of 45 participants). Participants discussed 7 vignettes about newborn screening that we developed and refined with the assistance of an expert panel. Two coders coded the data independently, compared coding, and resolved disagreements through discussion. Using framework analysis, we analyzed the data and identified how parents' preferences varied according to disease characteristics, test characteristics, and perceptions of the associated risks and benefits. RESULTS: Study participants strongly supported population-wide screening for disorders with well-defined, effective treatments, even if the treatment (eg, a bone marrow transplant) had significant morbidity. However, particularly among primary care clinic participants, there were more-varied preferences and greater difficulty making decisions about disorders associated with older age at onset, less-accurate screening tests, or less-effective treatment. In those cases, many participants suggested optional screening. For all disorders, participants expressed a desire for more information to facilitate decision-making. CONCLUSIONS: Participants supported newborn screening for treatable disorders but suggested optional screening for other disorders. The variable influences on parents' decision-making suggest that parents with diverse experiences, if they were included in decision-making regarding screening policies, could provide critical perspectives and help screening programs address parents' preferences and meet parents' information needs.

Ocular manifestations of Fabry disease within in a single kindred.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder that causes progressive complications within the kidneys, brain, and heart. Ocular manifestations of this disease are often present at a very young age, thereby facilitating early diagnosis, before the signs and symptoms of renal disease, stroke, or hypertrophic cardiomyopathy. Early diagnosis by the eye care provider may eventually reduce the morbidity and mortality of this disease through the institution of therapy before the development of sclerotic end organ damage. This study evaluated 23 Fabry-affected members of a single cohort for the presence of ocular signs of Fabry disease. METHODS: Twenty-three patients of a single family were seen on a single day. Patients were given comprehensive ophthalmic examinations and completed a health and lifestyle questionnaire. RESULTS: Eight hemizygous men (mean age, 32.3 years) and 15 heterozygous women (mean age, 26.9 years) from a single family of 43 known Fabry patients were evaluated. Corneal verticillata was present in all patients. Additional findings in the male patients included anterior capsule opacity (25% total) and Fabry cataract (12.5%). Thinning of the retinal nerve fiber layer was observed in one man whose medical history was significant for stroke. Conjunctival and/or retinal vessel tortuosity was present in the majority of patients (62.5% and 75% of hemizygotes, respectively; 40% and 13.3% heterozygotes, respectively). Additional findings in the women included anterior capsule opacity. The majority of patients (87.5% hemizygotes, 60% heterozygotes) felt Fabry disease had an impact on their quality of life. CONCLUSIONS: All evaluated patients who had Fabry disease had corneal verticillata, which generally does not affect vision and is readily recognizable by slit lamp examination. Greater than 60% showed conjunctival and/or retinal vessel tortuosity. The eye care provider can play a crucial role in the early recognition of ocular manifestations of Fabry disease and decrease both the time to accurate diagnosis and the delay in the institution of disease-modifying therapy.

Macroautophagy is defective in mucolipin-1-deficient mouse neurons.

Mucolipidosis type IV is a neurodegenerative lysosomal disease clinically characterized by psychomotor retardation, visual impairment, and achlorhydria. In this study we report the development of a neuronal cell model generated from cerebrum of Mcoln1(-/-) embryos. Prior functional characterization of MLIV cells has been limited to fibroblast cultures gleaned from patients. The current availability of the mucolipin-1 knockout mouse model Mcoln1(-/-) allows the study of mucolipin-1-defective neurons, which is important since the disease is characterized by severe neurological impairment. Electron microscopy studies reveal significant membranous intracytoplasmic storage bodies, which correlate with the storage morphology observed in cerebral cortex of Mcoln1(-/-) P7 pups and E17 embryos. The Mcoln1(-/-) neuronal cultures show an increase in size of LysoTracker and Lamp1 positive vesicles. Using this neuronal model system, we show that macroautophagy is defective in mucolipin-1-deficient neurons and that LC3-II levels are significantly elevated. Treatment with rapamycin plus protease inhibitors did not increase levels of LC3-II in Mcoln1(-/-) neuronal cultures, indicating that the lack of mucolipin-1 affects LC3-II clearance. P62/SQSTM1 and ubiquitin levels were also increased in Mcoln1(-/-) neuronal cultures, suggesting an accumulation of protein aggregates and a defect in macroautophagy which could help explain the neurodegeneration observed in MLIV. This study describes, for the first time, a defect in macroautophagy in mucolipin-1-deficient neurons, which corroborates recent findings in MLIV fibroblasts and provides new insight into the neuronal pathogenesis of this disease.

Systematic evidence review of newborn screening and treatment of severe combined immunodeficiency.

CONTEXT: Severe combined immunodeficiency (SCID) is a group of disorders that leads to early childhood death as a result of severe infections. Recent research has addressed potential newborn screening for SCID. OBJECTIVE: To conduct a systematic review of the evidence for newborn screening for SCID, including test characteristics, treatment efficacy, and cost-effectiveness. METHODS: We searched Medline and the OVID In-Process & Other Non-Indexed Citations databases. We excluded articles if they were reviews, editorials or other opinion pieces, or case series of fewer than 4 patients or if they contained only adult subjects or nonhuman data. The remaining articles were systematically evaluated, and data were abstracted by 2 independent reviewers using standardized tools. For topics that lacked published evidence, we interviewed experts in the field. RESULTS: The initial search resulted in 719 articles. Twenty-six met inclusion criteria. The results of several small studies suggested that screening for SCID is possible. Interviews revealed that 2 states have begun pilot screening programs. Evidence from large case series indicates that children receiving early stem-cell transplant for SCID have improved outcomes compared with children who were treated later. There is some inconclusive evidence regarding the need for donor-recipient matching and use of pretransplant chemotherapy. Few data on the cost-effectiveness of a SCID-screening program. CONCLUSIONS: Evidence indicates the benefits of early treatment of SCID and the possibility of population-based newborn screening. Better information on optimal treatment and the costs of treatment and screening would benefit policy makers deciding among competing health care priorities.

Intragenic rearrangements in NRXN1 in three families with autism spectrum disorder, developmental delay, and speech delay.

NRXN1 is highly expressed in brain and has been shown recently to be associated with ASD, schizophrenia, cognitive and behavioral abnormalities, and alcohol and nicotine dependence. We present three families, in whom we identified intragenic rearrangements within NRXN1 using a clinical targeted oligonucleotide array CGH. An approximately 380 kb deletion was identified in a woman with Asperger syndrome, anxiety, and depression and in all four of her children affected with autism, anxiety, developmental delay, and speech delay but not in an unaffected child. An approximately 180 kb tandem duplication was found in a patient with autistic disorder and cognitive delays, and in his mother and younger brother who have speech delay. An approximately 330 kb tandem duplication was identified in a patient with autistic features. As predicted by conceptual translation, all three genomic rearrangements led to the premature truncation of NRXN1. Our data support previous observations that NRXN1 may be pathogenic in a wide variety of psychiatric diseases, including autism spectrum disorder, global developmental delay, anxiety, and depression.

Functional multimerization of mucolipin channel proteins.

MCOLN1 encodes mucolipin-1 (TRPML1), a member of the transient receptor potential TRPML subfamily of channel proteins. Mutations in MCOLN1 cause mucolipidosis-type IV (MLIV), a lysosomal storage disorder characterized by severe neurologic, ophthalmologic, and gastrointestinal abnormalities. Along with TRPML1, there are two other TRPML family members, mucolipin-2 (TRPML2) and mucolipin-3 (TRPML3). In this study, we used immunocytochemical analysis to determine that TRPML1, TRPML2, and TRPML3 co-localize in cells. The multimerization of TRPML proteins was confirmed by co-immunoprecipitation and Western blot analysis, which demonstrated that TRPML1 homo-multimerizes as well as hetero-multimerizes with TRPML2 and TRPML3. MLIV-causing mutants of TRPML1 also interacted with wild-type TRPML1. Lipid bilayer re-constitution of in vitro translated TRPML2 and TRPML3 confirmed their cation channel properties with lower single channel conductance and higher partial permeability to anions as compared to TRPML1. We further analyzed the electrophysiological properties of single channel TRPML hetero-multimers, which displayed functional differences when compared to individual TRPMLs. Our data shows for the first time that TRPMLs form distinct functional channel complexes. Homo- and hetero-multimerization of TRPMLs may modulate channel function and biophysical properties, thereby increasing TRPML functional diversity.

Effect of temperature and moisture period on infection of Rhododendron 'Cunningham's White' by Phytophthora ramorum.

We investigated the temperature and moisture conditions that allow Phytophthora ramorum to infect Rhododendron 'Cunningham's White'. Most experiments were performed with a single P. ramorum isolate from the NA1 clonal lineage. For whole plants incubated in dew chambers at 10 to 31 degrees C, the greatest proportion of diseased leaves, 77.5%, occurred at the optimum temperature of 20.5 degrees C. Disease occurred over the entire range of temperatures tested, although amounts of disease were minor at the temperature extremes. For whole plants exposed to varying dew periods at 20 degrees C and then incubated at 20 degrees C for 7 days, a dew period as short as 1 h resulted in a small amount of disease; however, at least 4 h of dew were required for >10% of the leaves to become diseased. Moisture periods of 24 and 48 h resulted in the greatest number of diseased leaves. In detached-leaf, temperature-gradient-plate experiments, incubation at 22 degrees C resulted in the greatest disease severity, followed by 18 degrees C and then 14 degrees C. In detached-leaf, moisture-tent experiments, a 1-h moisture period was sufficient to cause disease on 67 to 73% of leaves incubated for 7 days at 20 degrees C. A statistical model for disease development that combined the effects of temperature and moisture period was generated using nonlinear regression. Our results define temperature and moisture conditions which allow infection by P. ramorum on Cunningham's White rhododendron, and show that P. ramorum is able to infect this host over a wide range of temperatures and moisture levels. The results indicate that P. ramorum has the potential to become established in parts of the United States that are outside its current range.

Effect of sample collection on alpha-galactosidase A enzyme activity measurements in dried blood spots on filter paper.

BACKGROUND: Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha galactosidase A (AGAL, EC 3.2.1.22). Despite increasing utilization of dried blood spot (DBS) as samples for AGAL enzyme assays, the effects of blood sample collection techniques on enzyme activity have not been studied. METHODS: DBS samples were prepared by spotting blood collected into an ethylenediaminetetraacetic acid (EDTA) tube and by direct application of blood from a finger prick or a venipuncture syringe. AGAL activity was measured quantitatively by detecting the fluorescence of 4-methylumbelliferone (4-MU) generated using the substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (4-MUGal) in an acidic pH for 20 h. N-acetyl-D-galactosamine (GalNAc) was used to inhibit alpha-galactosidase B (EC 3.2.1.49). RESULTS: We studied 88 previously diagnosed Fabry disease patients and 690 healthy controls. Average AGAL activity in DBS samples prepared using EDTA tubes was higher compared to those spotted directly irrespective of disease status. CONCLUSIONS: The study confirms the need for collection method-specific reference ranges using DBS samples.

Susceptibility to Phytophthora ramorum and Inoculum Production Potential of Some Common Eastern Forest Understory Plant Species.

Twenty-five plant species (21 genera, 14 families), which comprise a portion of the understory in forests of the Eastern United States, were evaluated for susceptibility to infection by Phytophthora ramorum. The degree to which P. ramorum is able to form sporangia and chlamydospores was also assessed on these hosts. Seedlings were spray-inoculated with a mixture (4,000 sporangia/ml) of four P. ramorum isolates followed by incubation in a dew chamber at 20°C in darkness for 5 days. Percent infection on individual leaves/leaflets was assessed visually. Mean percent leaf area infected ranged from 0.7% for Smilax rotundifolia to 93.8% for Kalmia latifolia. Eight plant species tested developed significantly larger lesion areas than those found on susceptible control Rhododendron 'Cunningham's White'. Fourteen species in addition to the susceptible control exhibited infection of over 90% of their leaves. Sporangia production by P. ramorum varied considerably among plant species, ranging from 36 per cm2 lesion area on Myrica pennsylvannica to 2,001 per cm2 lesion area on Robinia pseudoacacia. Numbers of chlamydospores produced per 6-mm-diameter leaf disk incubated in a P. ramorum sporangia suspension ranged from 25 on Ilex verticillata to 493 on Rhus typhina. The results indicate that many common understory species in Eastern U.S. forests are susceptible to P. ramorum and capable of providing ample sources of inoculum (sporangia and chlamydospores) for forest epidemics should the pathogen be introduced and should temperature and moisture conditions exist that are conducive to disease development.

Survival of Phytophthora ramorum hyphae after exposure to temperature extremes and various humidities.

We examined the effect of short-term exposure to high and low temperatures and a range of relative humidity (RH) on survival of Phytophthora ramorum hyphae. Spore-free hyphal colonies were grown on dialysis squares atop V8 medium. Colonies were transferred to water agar plates positioned at 27.5-50 C on a thermal gradient plate and incubated 2.5-480 min. For low temperature trials colonies were transferred to vials of distilled water and incubated in a water bath at -5 to -25 C for 1-24 h. In the relative humidity trials hyphal colonies were transferred to sealed humidity chambers containing various concentrations of glycerin for 1-8 h. Relative humidity was 41-93% at 20 C and 43-86% at 28 C. Survival in all trials was characterized by growth from dialysis squares into V8 medium. Temperatures of 37.5-40 C were lethal to P. ramorum hyphae within several hours, and temperatures of 42.5-50 C were lethal within minutes. Exposure to 32.5 and 35 C resulted in reduced survival over 8 h, while 30 C had no effect on three of four isolates. Hyphal colonies demonstrated considerable tolerance to cold, with all isolates surviving a 24 h exposure to -5 C. Survival diminished over time at lower temperatures, however a few colonies survived 24 h exposure to -25 C. Temperature also affected the ability of hyphal colonies to withstand reduced humidity. A RH of 41-43% was lethal in 2 h at 28 C compared to 8 h at 20 C. Three of four isolates were unaffected by an 8 h exposure to 81 and 95% RH at 20 C, and 73 and 86% RH at 28 C. Isolate differences were apparent in tolerance to freezing temperatures and reduced humidity. From these results it is apparent that the cold temperatures found in the northeastern USA are not likely to prevent the establishment of P. ramorum. There is also the potential for hyphae, and presumably spores, to survive periods of high humidity on the leaf surface in the absence of free water.

A novel mutation in JARID1C/SMCX in a patient with autism spectrum disorder (ASD).

We describe a nondysmorphic patient with developmental delay and autism spectrum disorder who has a missense mutation in the Jumonji AT-rich interactive domain 1C (JARID1C) gene. This child first presented at 30 months of age with stereotyped and repetitive behaviors, impairment in social reciprocity and in the use of multiple nonverbal behaviors, and developmental delay primarily in the language domain. A diagnosis of autism was made and subsequently confirmed at the current age of 47 months. Cytogenetic and fragile X studies were normal. Mutational analysis revealed a novel missense mutation in exon 16 of the JARID1C gene that results in an arginine to tryptophan substitution at amino acid 766 (R766W). Sequence alignment analysis with multiple available eukaryotic sequences including the homologous proteins of mouse and zebrafish demonstrated that the affected amino acid is conserved. JARID1C has not previously been implicated in autism susceptibility. Recent novel molecular evidence suggests that it is a histone demethylase specific for di- and trimethylated histone 3 lysine 4 (H3K4) and functions as a transcriptional repressor by fostering REST-mediated neuronal gene regulation. The JARID1C-regulated genes SCN2A, CACNA1H, BDNF, and SLC18A1 have previously been associated with autism and cognitive dysfunction. This patient brings the total number of reported JARID1C mutations to 14. This presentation both extends the range of neurocognitive phenotypes attributable to mutations in this gene and illustrates the importance of molecular studies and DNA sequence analysis for accurate diagnosis of monogenic causes of autism.

Recovery of Phytophthora ramorum Following Exposure to Temperature Extremes.

We examined the impact of exposure to high and low temperature extremes on recovery of Phytophthora ramorum both as free chlamydospores and within infected rhododendron tissue over a 7-day period. Chlamydospores held in moistened sand were incubated at 30, 35, 40, 0, -10, and -20°C for up to 7 days. Infected Rhododendron 'Cunningham's White' leaf disks held in sandy loam, loam, or sand at two different soil moisture levels also were subjected to these temperatures for up to 7 days, and to a variable temperature regimen for 12 weeks. Recovery was characterized by growth of P. ramorum on selective agar medium following exposures to temperature treatments. Chlamydospores held in moistened sand showed a high rate of recovery at 30°C, steadily declining recovery at 35°C, and no recovery at 40°C over the 7-day period. Chlamydospores were recovered from 0°C after 7 days, with little or no recovery observed at -10 or -20°C. In infected rhododendron tissue, P. ramorum was recovered at 20 and 30°C after 7 days but, at 35°C, the pathogen showed a decline within 2 days and no recovery by 4 days. A 40°C treatment allowed no recovery of P. ramorum from infected tissue after 2 days. For cold treatments, P. ramorum was recovered from infected leaf disks at 0 and -10°C after 7 days. At -20°C, recovery declined rapidly after 1 to 3 days and no recovery was obtained after 4 days. P. ramorum showed nearly 100% recovery from leaf disks subjected to a 12-week variable temperature treatment based on ambient summer temperatures in Lewisburg, TN. The results suggest that P. ramorum is capable of surviving some highly adverse temperature conditions for at least 7 days both as free chlamydospores in sand and within infected host tissue. Thus, P. ramorum present as free chlamydospores or within tissue of infected plants shipped to the eastern United States has the potential to survive some of the adverse conditions encountered in summer and winter in many eastern states.