Hypoxaemia during tracheal intubation in patients with hypertensive disorders of pregnancy: analysis of data from an obstetric airway management registry.

BACKGROUND: In South Africa, hypertensive disorders of pregnancy are the leading cause of maternal mortality. More than 50% of anaesthesia-related maternal deaths are attributed to complications of airway management. We compared the prevalence and risk factors for hypoxaemia during induction of general anaesthesia in parturients with and without hypertensive disorders of pregnancy. We hypothesised that hypertensive disorders of pregnancy are associated with desaturation during tracheal intubation. METHODS: Data from 402 cases in a multicentre obstetric airway management registry were analysed. The prevalence of peri-induction hypoxaemia (SpO2 <90%) was compared in patients with and without hypertensive disorders of pregnancy. Quantile regression of SpO2 nadir was performed to identify confounding variables associated with, and mediators of, hypoxaemia. RESULTS: In the cohort of 402 cases, hypoxaemia occurred in 19% with and 9% without hypertension (estimated risk difference, 10%; 95% CI 2% to 17%; P=0.005). Quantile regression demonstrated a lower SpO2 nadir associated with hypertensive disorders of pregnancy as body mass index increased. Room-air oxygen saturation, Mallampati grade, and number of intubation attempts were associated with the relationship. CONCLUSIONS: Clinically significant oxygen desaturation during airway management occurred twice as often in patients with hypertensive disorders of pregnancy, compounded by increasing body mass index. Intermediary factors in the pathway from hypertension to hypoxaemia were also identified.

Inflation of 430-parsec bipolar radio bubbles in the Galactic Centre by an energetic event.

The Galactic Centre contains a supermassive black hole with a mass of four million Suns1 within an environment that differs markedly from that of the Galactic disk. Although the black hole is essentially quiescent in the broader context of active galactic nuclei, X-ray observations have provided evidence for energetic outbursts from its surroundings2. Also, although the levels of star formation in the Galactic Centre have been approximately constant over the past few hundred million years, there is evidence of increased short-duration bursts3, strongly influenced by the interaction of the black hole with the enhanced gas density present within the ring-like central molecular zone4 at Galactic longitude |l| < 0.7 degrees and latitude |b| < 0.2 degrees. The inner 200-parsec region is characterized by large amounts of warm molecular gas5, a high cosmic-ray ionization rate6, unusual gas chemistry, enhanced synchrotron emission7,8, and a multitude of radio-emitting magnetized filaments9, the origin of which has not been established. Here we report radio imaging that reveals a bipolar bubble structure, with an overall span of 1 degree by 3 degrees (140 parsecs × 430 parsecs), extending above and below the Galactic plane and apparently associated with the Galactic Centre. The structure is edge-brightened and bounded, with symmetry implying creation by an energetic event in the Galactic Centre. We estimate the age of the bubbles to be a few million years, with a total energy of 7 × 1052 ergs. We postulate that the progenitor event was a major contributor to the increased cosmic-ray density in the Galactic Centre, and is in turn the principal source of the relativistic particles required to power the synchrotron emission of the radio filaments within and in the vicinity of the bubble cavities.

Hypothesis: Can drug-loaded platelets be used as delivery vehicles for blood-brain barrier penetration?

Neurovascular conditions are disorders associated with the blood vessels of the brain that are extremely difficult to treat successfully due to the selectivity and fastidious nature of the blood- brain barrier. Consequently, the efficacy of the pharmacological treatments for these conditions are greatly reduced thereby resulting in large amounts of neurovascular-related morbidity and mortality. Platelets are an important component of blood that actively respond to neurovascular distress in the body. Recent research has proven the effectiveness of platelets as drug delivery vehicles, during circumstances where the body naturally elicits a platelet response. This hypothesis highlights the theoretical use of platelets as drug delivery vehicles, able to penetrate the blood-brain barrier, for the treatment of two neurovascular conditions; glioblastoma multiforme and ischemic stroke. The success of the hypothesised system may lead to the development of a novel and extremely necessary delivery mechanism.

The developing world of pre-operative optimisation: a systematic review of Cochrane reviews.

Pre-operative optimisation is a heterogenous group of interventions aimed at improving peri-operative outcomes. To understand the evidence for pre-operative optimisation in the developing world, we systematically reviewed Cochrane reviews on the topic according to the Human Developmental Index (HDI) of the country where patient recruitment occurred. We used summary statistics and cartograms to describe the HDI, year of publication, timing of pre-operative intervention and risk of bias associated with each included trial. We assessed the impact of multinational trials on the risk of bias introduced by countries of differing HDI. Four-hundred and nine trials representing 51 countries and 89,389 randomly allocated participants were summarised in this review. Four-hundred and nineteen out of 451 (93%) trial populations (i.e. a group of study participants from one country) were from high and very high HDI countries. The median (IQR [range]) HDI of countries were 0.862 (0.806-0.892 [0.445-0.949]). Three of the 409 included trials were multinational, representing 32 countries and 37,736 out of 89,389 (42.2%) included participants. Africa was the least represented continent, with only 4 included trials and 566 participants, of which 62.3% were from one multinational trial. The overall risk of bias was high or unclear in 381 out of 409 (93%) trials. Inclusion of multinational trials decreased the proportion of trial populations introducing high or unclear risk of bias by 9.4% (95%CI 5.1-13.7; p < 0.0001). Half of the world's population live in low- and middle-HDI countries. This population is poorly represented in systematically reviewed evidence on pre-operative optimisation. Multinational trials increase the knowledge contribution from low- and middle-HDI countries and decrease risk of bias in systematic reviews.

The African Surgical OutcomeS-2 (ASOS-2) Pilot Trial, a mixed-methods implementation study

Background: The African Surgical Outcomes Study (ASOS) showed that surgical patients in Africa have a mortality twice the global average. The working hypothesis is that patients die as a result of failure to rescue following complications in the postoperative period. The African Surgical OutcomeS-2 (ASOS-2) Trial plans to test the efficacy of increased postoperative surveillance in high risk patients for decreasing perioperative morbidity and mortality. This pilot trial aimed i) to evaluate the adequacy of data produced by the data collection strategies of the ASOS-2 Trial, ii) to evaluate the fidelity of implementation of the increased postoperative surveillance intervention, and iii) to understand the acceptability, appropriateness and feasibility of the intervention and the trial processes. Methods: The ASOS-2 Pilot Trial was a mixed-methods (quantitative-qualitative) implementation study focusing on the intervention arm of the proposed ASOS-2 Trial. The intervention is increased postoperative surveillance for high-risk surgical patients. The intervention protocol was implemented at all sites for a seven-day period. A post pilot trial survey was used to collect data on the implementation outcomes. Results: 803 patients were recruited from 16 hospitals in eight African countries. The sampling and data collection strategies provided 98% complete data collection. Seventy-three percent of respondents believed that they truly provided increased postoperative surveillance to high risk patients. In reality 83/125 (66%) of high-risk patients received some form of increased postoperative surveillance. However, the individual components of the increased postoperative surveillance intervention were implemented in less than 50% of high-risk patients (excepting increasing nursing observations). The components most frequently unavailable were the ability to provide care in a higher care ward (32.1%) and assigning the patient to a bed in view of the nurses' station (28.4%). Failure to comply with available components of the intervention ranged from 27.5% to 54.3%. The post pilot survey had a response rate of 30/40 (75%). In Likert scale questions about acceptability, appropriateness, and feasibility of the ASOS-2 intervention, 63% to 87% of respondents indicated agreement. Respondents reported barriers related to resources, trial processes, teamwork and communication as reasons for disagreement. Conclusions: The proposed ASOS-2 Trial appears to be appropriate, acceptable and feasible in Africa. This pilot trial provides support for the proposed ASOS-2 Trial. It emphasises the need for establishing trial site teams which address the needs of all stakeholders during the trial. A concerted effort must be made to help participating hospitals to increase compliance with all the components of the proposed intervention of 'increased postoperative surveillance' during the ASOS-2 Trial

Synthesis and biocompatibility of dual-responsive thermosonic injectable organogels based on crosslinked N-(isopropyl acrylamide) for tumour microenvironment targeting.

A series of three dual-responsive 'thermosonic' (thermo- and ultrasound-responsive) injectable organogels (TIOs) based on crosslinked N-(isopropyl acrylamide) (NIPAM) bearing biocompatible polymeric constituents were investigated for strong gelation in response to tumour temperature, and sol-like fluid gel formation upon the application of an ultrasonic stimulus. A time-efficient free radical polymerisation reaction of ˂15 min resulted in TIO formation. Moreover, the formulation of the TIOs integrated green chemistry principles to ensure enhanced biocompatibility. Fourier Transform Infrared (FTIR) spectral analysis revealed the presence of new molecular vibrations at 847 and 771 cm-1 (CH deformation), which were indicative of the functionalisation of the NIPAM backbone with hydrophobic and ultrasound-responsive aromatic moieties. Thermo- and ultrasound-response analysis and rheological analysis demonstrated that the TIOs displayed a temperature-induced transition to a strong highly-structured gel, and an ultrasound-triggered increase in gel flowability dependant on the composition of the formulation. Cell proliferation studies were undertaken for the TIOs, which verified that the designed TIOs were all non-cytotoxic and promoted cell proliferation over 1, 3, and 5 day intervals. The rational design and formulation of a biocompatible injectable in-situ depot drug delivery system for ultimate application in tumour targeting was successfully achieved and warrant further investigation.

Decreased neutrophil-associated miRNA and increased B-cell associated miRNA expression during tuberculosis.

MicroRNAs are short non-coding RNAs that regulate gene expression by binding to, and suppressing the expression of genes. Research show that microRNAs have potential to be used as biomarkers for diagnosis, treatment response and can be used for therapeutic interventions. Furthermore, microRNA expression has effects on immune cell functions, which may lead to disease. Considering the important protective role of neutrophils and B-cells during M.tb infection, we evaluated the expression of microRNAs, known to alter function of these cells, in the context of human TB. We utilised real-time PCR to evaluate the levels of microRNA transcripts in the peripheral blood of TB cases and healthy controls. We found that neutrophil-associated miR-197-3p, miR-99b-5p and miR-191-5p transcript levels were significantly lower in TB cases. Additionally, B-cell-associated miR-320a, miR-204-5p, miR331-3p and other transcript levels were higher in TB cases. The miRNAs differentially expressed in neutrophils are predominantly implicated in signalling pathways leading to cytokine productions. Here, the decreased expression in TB cases may imply a lack of suppression on signalling pathways, which may lead to increased production of pro-inflammatory cytokines such as interferon-gamma. Furthermore, the miRNAs differentially expressed in B-cells are mostly involved in the induction/suppression of apoptosis. Further functional studies are however required to elucidate the significance and functional effects of changes in the expression of these microRNAs.

A meta-analysis of the efficacy of preoperative surgical safety checklists to improve perioperative outcomes

BACKGROUND:Meta-analyses of the implementation of a surgical safety checklist (SSC) in observational studies have shown a significant decrease in mortality and surgical complications.OBJECTIVE:To determine the efficacy of the SSC using data from randomised controlled trials (RCTs). METHODS:This meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines and was registered with PROSPERO (CRD42015017546). A comprehensive search of six databases was conducted using the OvidSP search engine.RESULTS:Four hundred and sixty-four citations revealed three eligible trials conducted in tertiary hospitals and a community hospital; with a total of 6 060 patients. All trials had allocation concealment bias and a lack of blinding of participants and personnel. A single trial that contributed 5 295 of the 6 060 patients to the meta-analysis had no detection; attrition or reporting biases. The SSC was associated with significantly decreased mortality (risk ratio (RR) 0.59; 95% confidence interval (CI) 0.42 - 0.85; p=0.0004; I2=0%) and surgical complications (RR 0.64; 95% CI 0.57 - 0.71; petlt;0.00001; I2=0%). The efficacy of the SSC on specific surgical complications was as follows: respiratory complications RR 0.59; 95% CI 0.21 - 1.70; p=0.33; cardiac complications RR 0.74; 95% CI 0.28 - 1.95; p=0.54; infectious complications RR 0.61; 95% CI 0.29 - 1.27; p=0.18; and perioperative bleeding RR 0.36; 95% CI 0.23 - 0.56; petlt;0.00001.CONCLUSIONS:There is sufficient RCT evidence to suggest that SSCs decrease hospital mortality and surgical outcomes in tertiary and community hospitals. However; randomised evidence of the efficacy of the SSC at rural hospital level is absent

Screening for Retinopathy of Prematurity in a Provincial Hospital in Port Elizabeth; South Africa

Background. Retinopathy of prematurity (ROP) is an emerging public health problem in many middle-income countries where improved neonatal survival rates coupled with inadequate health resources have created a new epidemic. There are limited available data on the magnitude of the problem; and screening in South African (SA) hospitals has not been uniformly practised.OBJECTIVE:To describe the results of various interventions implemented over a 6-year period while developing a new ROP screening service in a provincial hospital in Port Elizabeth; SA.METHOD: A retrospective case folder review of ROP screening at Dora Nginza Hospital; Port Elizabeth; SA; over the 6-year period 2009 - 2014 was conducted.RESULTS:A total of 919 new cases were seen. Fifteen patients received treatment for type 1 ROP (T1ROP); 223 had type 2 (T2) or earlier ROP; 1 had stage 4 ROP and 6 had stage 5 ROP. The combination of healthcare worker education; improved equipment and human resources and the introduction of dual responsibility for case referrals resulted in an increase in the number of new infants screened from 33 in year 1 to 292 in year 6. The number of infants who were screened late decreased from 33/33 (100%) in year 1; prior to the interventions; to 23/292 in the final year (7.9%). Improved oxygen delivery and adequate oxygen saturation monitoring contributed to a decrease in the incidence of T1ROP from 1.5% to 1% over 1 year and in the incidence of T2 or earlier ROP from 30.3% to 24%.CONCLUSIONS:Better management of ROP can be achieved through adequate provision of healthcare professionals and material resources coupled with education and a well-supported referral system. A close working relationship between paediatricians and ophthalmologists results in a more efficient screening programme

Design of a novel crosslinked HEC-PAA porous hydrogel composite for dissolution rate and solubility enhancement of efavirenz.

The purpose of this research was to synthesize, characterize and evaluate a Crosslinked Hydrogel Composite (CHC) as a new carrier for improving the solubility of the anti-HIV drug, efavirenz. The CHC was prepared by physical blending of hydroxyethylcellulose (HEC) with poly(acrylic acid) (PAA) (1:1) in the presence of poly(vinyl alcohol) (PVA) (as a crosslinker) (1:5) under lyophilization. Efavirenz was loaded in situ into the CHC in varying proportions (200-600 mg). The CHC demonstrated impressive rheological properties (dynamic viscosity=6053 mPa; 500 s(-1)) and tensile strength (2.5 mPa) compared with the native polymers (HEC and PAA). The physicochemical and thermal behavior also confirmed that the CHC was compatible with efavirenz. The incorporation of efavirenz in the CHC increased the surface area (4.4489-8.4948 m(2)/g) and pore volume (469.547-776.916Å) of the hydrogel system which was confirmed by SEM imagery and BET surface area measurements. The solubility of efavirenz was significantly enhanced (150 times) in a sustained release manner over 24h as affirmed by the in vitro drug release studies. The hydration medium provided by the CHC network played a pivotal role in improving the efavirenz solubility via increasing hydrogen bonding as proved by the zeta potential measurements (-18.0 to +0.10). The CHC may be a promising alternative as an oral formulation for the delivery of efavirenz with enhanced solubility.

First Report of Bacterial Blight of Crucifers Caused by Pseudomonas cannabina pv. alisalensis in Minnesota on Arugula (Eruca vesicaria subsp. sativa).

In 2011, bacterial blight of arugula (Eruca vesicaria subsp. sativa; cv. Roquette) was observed in organically grown plants under overhead irrigation in a field near Delano, MN. Approximately 80 to 100% of each planting was affected, with greater rates of infection occurring after periods of high humidity. Small, water-soaked, angular spots apparent on both sides of the leaves comprised the initial symptoms, which sometimes expanded and coalesced. Lesions maintained a dark water-soaked appearance or dried and turned a brown/tan color. Additionally, some lesions were outlined by a purple margin. Blue-green fluorescent pseudomonads were isolated consistently on King's Medium B agar (KMB) from symptomatic leaf tissue surface-disinfested with sodium hypochlorite (0.525%). The isolates nucleated ice and produced levan. Isolates were oxidase and arginine dihydrolase negative. They did not rot potato slices but did induce a hypersensitive reaction in tobacco (Nicotiana tabacum cv. Samsun). These data indicated that the bacteria belonged to Lelliott's LOPAT group 1 (2). DNA fragment banding patterns generated by amplifying DNA of the arugula isolates using repetitive extragenic palindromic sequence-polymerase chain reaction (rep-PCR) and the BOX A1R primer were identical and nearly identical to the banding patterns of the Pseudomonas cannabina pv. alisalensis (formerly P. syringae pv. alisalensis) (1) strain (CFBP1637) and the pathotype strain (CFBP 6866PT), respectively. Pathogenicity was confirmed on the arugula cv. My Way in two independent experiments, each with three replicate plants per treatment. Four isolates were grown on KMB for 48 h at 27°C, suspended in 0.01M potassium phosphate buffer (pH 7.0), and adjusted to 0.6 optical density at 600 nm (approximately 1 × 108 CFU/ml). Five- to six-week old plants were spray-inoculated until run-off, incubated in a humidity chamber for 48 h, and then placed in a greenhouse at 20 to 25°C for symptom development. For negative and positive control treatments, a similar number of plants each were sprayed with sterile buffer or P. cannabina pv. alisalensis strains CFBP1637 and CFBP 6866PT, respectively. Water-soaked and brown/tan lesions similar to the original symptoms appeared on plants inoculated with the arugula isolates and P. cannabina pv. alisalensis strains 7 to 14 days postinoculation. No symptoms developed on plants treated with sterile buffer. The bacterial strains re-isolated from surface-disinfested symptomatic tissue were identical by rep-PCR to the isolates used to inoculate the plants, thus, confirming Koch's postulates. Identical replicated experiments conducted on broccoli raab indicated that the arugula isolates were also pathogens of broccoli raab (Brassica rapa subsp. rapa, the original host from which P. cannabina pv. alisalensis was isolated). To our knowledge, this is the first report of bacterial blight of crucifers caused by P. cannabina pv. alisalensis in Minnesota. Arugula germplasm is being evaluated for resistance to this pathogen as an acceptable management method for organic cropping systems. References: (1) C. T. Bull et al. Syst. Appl. Microbiol. 33:105, 2010. (2) R. A. Lelliott. J. Appl. Bacteriol. 29:470, 1966.

Multiplex real-time PCR assays for detection of four seedborne spinach pathogens.

AIMS: To develop multiplex TaqMan real-time PCR assays for detection of spinach seedborne pathogens that cause economically important diseases on spinach. METHODS AND RESULTS: Primers and probes were designed from conserved sequences of the internal transcribed spacer (for Peronospora farinosa f. sp. spinaciae and Stemphylium botryosum), the intergenic spacer (for Verticillium dahliae) and the elongation factor 1 alpha (for Cladosporium variabile) regions of DNA. The TaqMan assays were tested on DNA extracted from numerous isolates of the four target pathogens, as well as a wide range of nontarget, related fungi or oomycetes and numerous saprophytes commonly found on spinach seed. Multiplex real-time PCR assays were evaluated by detecting two or three target pathogens simultaneously. Singular and multiplex real-time PCR assays were also applied to DNA extracted from bulked seed and single spinach seed. CONCLUSIONS: The real-time PCR assays were species-specific and sensitive. Singular or multiplex real-time PCR assays could detect target pathogens from both bulked seed samples as well as single spinach seed. SIGNIFICANCE AND IMPACT OF THE STUDY: The freeze-blotter assay that is currently routinely used in the spinach seed industry to detect and quantify three fungal seedborne pathogens of spinach (C. variabile, S. botryosum and V. dahliae) is quite laborious and takes several weeks to process. The real-time PCR assays developed in this study are more sensitive and can be completed in a single day. As the assays can be applied easily for routine seed inspections, these tools could be very useful to the spinach seed industry.

First Report of Pythium sulcatum Causing Cavity Spot in Processing Carrot Crops in the Columbia Basin of Washington State.

In October 2012, symptoms of cavity spot (1) were observed on roots of two 50 ha, Red Core Chantenay processing carrot (Daucus carota L. subsp. sativus (Hoffm.)) crops in the Columbia Basin of central Washington. Symptoms consisted of sunken, elliptical lesions (3 to 15 mm long) on the root surface. Approximately 6% of the roots in each crop were affected, which was sufficient to present sorting problems for the processor. Symptomatic roots were washed thoroughly in tap water, and then small sections of tissue from the lesion margins were removed aseptically and plated onto water agar (WA) without surface-sterilization. Isolates with morphological characteristics typical of Pythium sulcatum Pratt & Mitchell (2) were obtained consistently from the symptomatic tissue. The genus and species identity of seven isolates was confirmed by sequence analysis of the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA (rDNA) using universal eukaryotic primers UN-UP18S42 and UN-LO28S576B with the PCR protocol described by Schroeder et al. (3). The ITS consensus sequences of the seven isolates (Accession Nos. KF509939 to KF509945) were 98 to 99% homologous to ITS sequences of P. sulcatum in GenBank. Pathogenicity of all seven isolates was confirmed by inoculating mature carrot roots of cv. Bolero. Each root was washed with tap water, sprayed to runoff with 70% isopropanol, and dried in a laminar flow hood on sterilized paper toweling. The roots were then placed in plastic bins lined with paper toweling moistened with sterilized, deionized water. Each root was inoculated by placing two 5 mm-diameter agar plugs, taken from the edge of an actively growing WA culture of the appropriate isolate, on the root surface approximately 3 cm apart. Non-colonized agar plugs were used for a non-inoculated control treatment. Four replicate roots were inoculated for each isolate and the control treatment. After inoculation, the roots were misted with sterilized, deionized water, a lid was placed on each bin, and the roots were incubated in the dark at 22°C. Roots were misted daily to maintain high relative humidity. Dark, sunken lesions were first observed 3 days post-inoculation on roots inoculated with the P. sulcatum isolates, and all inoculated roots displayed cavity spot lesions by 7 days. No symptoms were observed on the non-inoculated control roots. Colonies with morphology typical of P. sulcatum were re-isolated from the symptomatic tissue of roots inoculated with the P. sulcatum isolates, and the species identity of the re-isolates was confirmed by ITS rDNA sequence analysis, as described above. Although P. sulcatum is one of several Pythium species that can cause cavity spot of carrot (1), to our knowledge, this is the first report of P. sulcatum causing cavity spot in Washington State, which has the largest acreage of processing carrot crops in the United States (4). References: (1) R. M. Davis and R. N. Raid. Compendium of Umbelliferous Crop Diseases. The American Phytopathological Society, St. Paul, MN, 2002. (2) A. J. van der Plaats-Niterink. Monograph of the Genus Pythium. Stud. Mycol. No. 21. CBS, Baarn, The Netherlands, 1981. (3) K. L. Schroeder et al. Phytopathology 96:637, 2006. (4) E. J. Sorensen. Crop Profile for Carrots in Washington State. U.S. Dept. Agric. National Pest Manage. Centers, 2000.

First Report of Bacterial Blight of Carrot in Indiana Caused by Xanthomonas hortorum pv. carotae.

In summer 2012, bacterial blight symptoms (2) were observed on leaves of carrot plants in 7 out of 70 plots of carrot breeding lines at the Purdue University Meig Horticulture Research Farm, Lafayette, IN. Symptoms included small to large, variably shaped, water-soaked to dry, necrotic lesions, with or without chlorosis, at <5% incidence. Microscopic examination of symptomatic leaf sections revealed bacterial streaming from the cut ends of each leaf piece. For each of the seven plots, symptomatic leaf sections (each 5 to 10 mm2) were surface-sterilized in 1.2% NaOCl for 60 s, triple-rinsed in sterilized, deionized water, dried on sterilized blotter paper, macerated in sterilized water, and a loopful of the suspension was streaked onto yeast dextrose carbonate (YDC) agar medium (1). Colonies with morphology similar to that of strain Car001 of Xanthomonas hortorum pv. carotae from California (3) were obtained consistently from all seven plots, and serial dilutions streaked onto YDC agar medium to obtain pure cultures. One bacterial strain/plot was then subjected to a PCR assay for X. hortorum pv. carotae using the protocol of Meng et al. in (5), except for an annealing temperature of 60°C. All seven Indiana strains and Car001 produced a 355-bp DNA fragment indicative of X. hortorum pv. carotae. The Indiana strains and Car001 were each tested for pathogenicity on five 11-week-old carrot plants of a proprietary Nantes inbred line grown from a seed lot that tested negative for X. hortorum pv. carotae (1,3). Each strain was grown for 16 h in 523 broth (4) on a shaker (200 rpm) at 28°C, and diluted in 0.0125M phosphate buffer to 108 CFU/ml. Approximately 24 h prior to inoculation, the five plants for each strain were enclosed in a large plastic bag to create a moist chamber. The plants were inoculated by atomizing 30 ml of the appropriate bacterial suspension onto the foliage using an airbrush. Five plants inoculated with sterilized phosphate buffer served as a negative control treatment. The plants were re-sealed in plastic bags for 72 h, and placed in a randomized complete block design in a greenhouse set at 25 to 28°C. Symptoms of bacterial blight were first observed 14 days after inoculation, and developed on all inoculated plants by 21 to 28 days after inoculation, with slight variation in severity of symptoms among strains. Symptoms did not develop on negative control plants. Re-isolations were done 32 days after inoculation from symptomatic leaves of three replicate plants/strain and from three plants of the negative control treatment, using the protocol described for the original samples. Bacterial colonies typical of X. hortorum pv. carotae were obtained from symptomatic leaves for all seven Indiana strains and the control strain, but not from the negative control plants. Identity of the re-isolated strains as X. hortorum pv. carotae was confirmed by PCR assay. To our knowledge, this is the first report of bacterial blight of carrot in Indiana. References: (1) M. Asma. Detection of Xanthomonas hortorum pv. carotae on Daucus carota. 7-020. International Rules for Seed Testing, Annex to Chapter 7: Seed Health Testing Methods. Internat. Seed Testing Assoc., Bassersdorf, Switzerland, 2006. (2) R. M. Davis and R. N. Raid. Compendium of Umbelliferous Crop Diseases. The American Phytopathological Society, St. Paul, MN, 2002. (3) L. J. du Toit et al. Plant Dis. 89:896, 2005. (4) E. I. Kado and M. G. Heskett. Phytopathology 60:969, 1970. (5) X. Q. Meng et al. Plant Dis. 88:1226, 2004.

White Rust of Echinacea angustifolia and E. purpurea in North America Caused by a Pustula Species.

In 2012 and 2013, foliar symptoms were observed in certified organic, 2- to 4-ha crops of Echinacea angustifolia and E. purpurea in Grant and Klickitat counties, WA. White pustules were predominant on the abaxial leaf surface, increased in number, and coalesced on E. angustifolia, with 100% infection by the end of the season; in contrast, symptoms remained sparse on E. purpurea. Symptomatic leaves of each species were collected in May 2013 in Grant Co. Sori and sporangia were typical of those of white rust on Asteraceae caused by Pustula obtusata (1), originally named Albugo tragopogonis, then P. tragopogonis (4). Hyaline sporangia (n = 50) averaged 21 ± 2 × 20 ± 2 µm (16 to 25 × 16 to 24 µm) with a 2.6 ± 0.8 µm (1.0 to 4.0 µm) thick wall. Honey-colored to dark brown oospores were embedded in the abaxial leaf surface surrounding sori on older leaves. Oospores (n = 50) averaged 75 ± 7 × 63 ± 6 µm (60 to 96 × 52 to 76 µm) and 52 ± 4 × 51 ± 4 µm (44 to 65 × 44 to 60 µm) with (including protruberances) and without the hyaline outer wall, respectively. Sori were excised and shaken in 100 ml cold (4°C), deionized water at 400 rpm for 15 min on a gyrotory shaker. DNA extracted from the resulting spore suspension was subjected to a PCR assay using oomycete specific primers (2) to amplify the cytochrome oxidase subunit II (cox2) region of mtDNA (3). The 511-nt consensus sequence of the PCR product (GenBank Accession No. KF981439) was 98% identical to a cox2 sequence of A. tragopogonis from sunflower (Helianthus annuus) (AY286221.1), and 96% identical to cox2 sequences of P. tragopogonis (GU292167.1 and GU292168.1) (= P. obtusata) (1,2,4). Pathogenicity of the white rust isolate was confirmed by inoculating 49-day-old plants of E. angustifolia and E. purpurea with a spore suspension prepared as described above. One plant/species was placed in each of six clear plastic bags in a growth chamber at 18°C with a 12-h day/12-h night cycle for 48 h. Five replicate sets of one plant/species were each inoculated with 2.2 × 105 spores/ml on the adaxial and abaxial leaf surfaces using an airbrush (8 psi). One plant/species was sprayed with water as a control treatment. The plants were resealed in the bags for 48 h. After 7 days, white pustules were observed on at least one plant species. The plants were placed in plastic bags again overnight, and re-inoculated with 2.9 × 105 spores/ml. In addition, two sunflower plants at the 4-true-leaf stage were incubated in each of two plastic bags overnight, and inoculated with the spore suspension. Two additional sunflower plants were treated with water as control plants. All plants were removed from the bags after 48 h. White rust sori with sporangia developed on all inoculated Echinacea plants within 10 days, but not on control plants of either species, nor inoculated and non-inoculated sunflower plants, verifying that the pathogen was not P. helianthicola (1,2). Since the cox2 sequence was closest to that of a sunflower white rust isolate, the pathogen appears to be closer to P. helianthicola than P. obtusata, and may be a new Pustula species. To our knowledge, this is the first documentation of white rust on E. angustifolia and E. purpurea in North America. The severity of white rust on E. angustifolia highlights the need for effective management practices. References: (1) C. Rost and M. Thines. Mycol. Progress 11:351, 2012. (2) O. Spring et al. Eur. J. Plant Pathol 131:519, 2011. (3) S. Telle and M. Thines. PloS ONE 3(10):e3584, 2008. (4) M. Thines and O. Spring. Mycotaxon 92:443, 2005.

Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae.

Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3-14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada's agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

Stunting of Onion in the Columbia Basin of Oregon and Washington Caused by Rhizoctonia spp.

During 2009 and 2010, 45 isolates of Rhizoctonia spp. were recovered from onion bulb crops in the semiarid Columbia Basin of Oregon and Washington, in which patches of severely stunted onion plants developed following rotation with winter cereal cover crops. Characterization of isolates recovered from naturally infested soil and roots was performed by sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer region, with the majority of isolates (64%) identified as Rhizoctonia solani. In steam-pasteurized field soil, stunting of onion was caused by isolates of R. solani anastamosis groups (AGs) 2-1, 3, 4, and 8, as well as Waitea circinata var. circinata and binucleate Rhizoctonia AG E evaluated at 13 and 8 or 15 and 15°C day and night temperatures, respectively, typical of spring planting conditions in the Columbia Basin. Isolates of R. solani AG 5 as well as binucleate AG A and I were nonpathogenic. The most virulent isolates belonged to AG 8, although an AG 3 and an AG E isolate were also highly virulent. Isolates of AG 2-1 and 3 caused moderate levels of disease, while isolates of AG 4 and W. circinata var. circinata caused low levels of disease. Emergence was reduced by isolates of AG 2-1, 3, and E. When the various AGs were grown at temperatures of 5 to 30°C, the relative growth rate of the Rhizoctonia isolates was not positively correlated with virulence on onion within an AG.

First Report of Cladosporium Leaf Spot of Spinach Caused by Cladosporium variabile in the Winter Spinach Production Region of California and Arizona.

In December 2011, symptoms typical of Cladosporium leaf spot caused by Cladosporium variabile (4) were observed in organic "baby leaf" spinach (Spinacia oleracea) crops of the cultivars Amazon, Missouri, Tasman, and Tonga in the Imperial Valley (Imperial County, CA and Yuma County, AZ). Leaves had small, circular lesions (1 to 3 mm in diameter), some of which had progressed to necrotic, bleached lesions surrounded by a thin dark margin. The incidence of symptoms in affected crops was ≤20%. Fungal isolates resembling C. variabile were recovered by surfacesterilizing sections (5 mm2) of symptomatic leaf tissue in 0.6% NaOCl, triple-rinsing the sections in sterile water, and plating the sections onto water agar and potato dextrose agar amended with 100 ppm chloramphenicol (cPDA). Single-spore transfers made onto cPDA were maintained at 24 ± 2°C with a natural day/night cycle. Each isolate produced slow growing cultures consisting of dense masses of dark conidiophores (≤350 µm long) with chains of up to three dematiaceous (olive) conidia, and almost no mycelium. Torulose (coiled) aerial hyphae developed from the apices of conidiophores after 5 to 7 days, and distinguished the isolates as C. variabile, not C. macrocarpum (2,4). Pathogenicity was tested for each of six single-spore isolates using 36-dayold plants of the spinach cultivar Carmel. The plants were enclosed in clear plastic bags overnight and inoculated the next day with the isolates of C. variabile by atomizing approximately 30 ml of a spore suspension (1.0 × 106 conidia/ml in sterile water amended with 0.01% Tween 20) of the appropriate isolate onto the upper and lower leaf surfaces of each of five plants/isolate. Five control plants were inoculated similarly with sterile water + 0.01% Tween 20. The plants were resealed in plastic bags for 72 h and then placed on a greenhouse bench. Pinpoint, sunken lesions developed within 4 to 7 days on the leaves of plants inoculated with each of the six test isolates. Lesions developed into dry, circular spots typical of Cladosporium leaf spot. Symptoms were not observed on control plants. After 20 days, C. variabile was reisolated from lesions caused by all six isolates, but not from control plants. Although Cladosporium leaf spot has been reported in the Salinas Valley of California (4), to our knowledge, this is the first report of the disease on spinach crops in the Imperial Valley of California and Arizona, the primary winter, fresh market spinach production region of the United States. Inoculum of C. variabile may have been introduced to this region on spinach seed lots (3), because even seed infestation levels <0.1% could lead to seed transmission (1) under the dense planting populations (≤9 million seeds/ha) and overhead irrigation typical of "baby leaf" spinach crops in this region. Fungicides can be used to manage Cladosporium leaf spot in conventional spinach crops (1), but management in certified organic crops may be more challenging. References: (1) L. J. du Toit et al. Fung. Nemat. Tests 59:V115, 2004. (2) M. B. Ellis. Page 315 in: Dematiaceous Hyphomycetes. Commonwealth Mycological Institute, Surrey, England, 1971. (3) P. Hernandez-Perez. Page 79 in: Management of Seedborne Stemphylium botryosum and Cladosporium variabile Causing Leaf Spot of Spinach Seed Crops in Western Washington, MS thesis, Pullman, WA, 2005. (4) P. Hernandez-Perez and L. J. du Toit. Plant Dis. 90:137, 2006.