An expanded concept of Ceratocystis manginecans and five new species in the Latin American clade of Ceratocystis.

The genus Ceratocystis contains a number of emerging plant pathogens, mostly members of the Latin American Clade (LAC), in which there are several unresolved taxonomic controversies. Among the most important are Brazilian pathogens in the C. fimbriata complex, C. manginecans and C. eucalypticola. Representatives of C. manginecans and C. eucalypticola from India and China, respectively, were shown to be fully interfertile in laboratory matings, and hybrids between the putative species were identified on Punica in India. An Indian tester strain was sexually compatible with representatives of what has been considered C. fimbriata on numerous hosts across Brazil. In this revision of the LAC, the name C. fimbriata is restricted to the widely dispersed Ipomoea strain, and C. manginecans is recognized as a Brazilian species that is important on Mangifera, Eucalyptus, and many other crops. C. mangivora and C. mangicola are also considered synonyms of C. manginecans. Based on phylogenetics and mating studies, two other Brazilian species are recognized: C. atlantica, sp. nov., and C. alfenasii, sp. nov., each with wide host ranges. Three new Caribbean species are recognized based on phylogenetics and earlier inoculation studies: C. costaricensis, sp. nov., on Coffea, C. cubensis, sp. nov., on Spathodea, and C. xanthosomatis, sp. nov., on the vegetatively propagated aroids Xanthosoma and Syngonium. Some of the other Ceratocystis species were based primarily on unique internal transcribed spacer (ITS) rDNA sequences, but the unreliability of rDNA sequences was demonstrated when intraspecific crossing of isolates with differing ITS sequences generated single-ascospore progeny with intragenomic variation in ITS sequences and others with new ITS sequences. Species recognition in Ceratocystis should use phenotype, including intersterility tests, to help identify which lineages are species. Although some species remain under-studied, we recognize 16 species in the LAC, all believed to be native to Latin America, the Caribbean region, or eastern USA.

Asymmetric Evolution of Protein Domains in the Leucine-Rich Repeat Receptor-Like Kinase Family of Plant Signaling Proteins.

The coding sequences of developmental genes are expected to be deeply conserved, with cis-regulatory change driving the modulation of gene function. In contrast, proteins with roles in defense are expected to evolve rapidly, in molecular arms races with pathogens. However, some gene families include both developmental and defense genes. In these families, does the tempo and mode of evolution differ between genes with divergent functions, despite shared ancestry and structure? The leucine-rich repeat receptor-like kinase (LRR-RLKs) protein family includes members with roles in plant development and defense, thus providing an ideal system for answering this question. LRR-RLKs are receptors that traverse plasma membranes. LRR domains bind extracellular ligands; RLK domains initiate intracellular signaling cascades in response to ligand binding. In LRR-RLKs with roles in defense, LRR domains evolve faster than RLK domains. To determine whether this asymmetry extends to LRR-RLKs that function primarily in development, we assessed evolutionary rates and tested for selection acting on 11 subfamilies of LRR-RLKs, using deeply sampled protein trees. To assess functional evolution, we performed heterologous complementation assays in Arabidopsis thaliana (Arabidopsis). We found that the LRR domains of all tested LRR-RLK proteins evolved faster than their cognate RLK domains. All tested subfamilies of LRR-RLKs had strikingly similar patterns of molecular evolution, despite divergent functions. Heterologous transformation experiments revealed that multiple mechanisms likely contribute to the evolution of LRR-RLK function, including escape from adaptive conflict. Our results indicate specific and distinct evolutionary pressures acting on LRR versus RLK domains, despite diverse organismal roles for LRR-RLK proteins.

Corticioid basidiomycetes associated with bark beetles, including seven new Entomocorticium species from North America and Cylindrobasidium ipidophilum, comb. nov.

Seven new Entomocorticium species (Peniophoraceae) are described based on morphology and phylogenetic analyses. Along with the type species (E. dendroctoni), Entomocorticium comprises eight species of nutritional symbionts of pine bark beetles in North America. Entomocorticium cobbii is the mycangial associate of the southern pine beetle, Dendroctonus frontalis, and E. parmeteri is the mycangial associate of the western pine beetle, D. brevicomis. Entomocorticium whitneyi, E. portiae, E. kirisitsii, E. oberwinkleri and the previously described E. dendroctoni have been isolated from galleries of D. ponderosae and D. jeffreyi in western North America. Entomocorticium sullivanii forms an ambrosia-like layer of basidia and basidiospores in the pupal chambers of Ips avulsus in the southeastern USA. Entomocorticium is phylogenetically placed within Peniophora, a corticioid genus of wood decay fungi with wind-dispersed basidiospores. At least four species of Entomocorticium produce basidiospores on basidia with reduced sterigmata that apparently do not forcibly discharge basidiospores. Another basidiomycete, Gloeocystidium ipidophilum, was described from Ips typographus galleries in Europe, but it is phylogenetically and taxonomically placed in another genus of wood decay fungi as Cylindrobasidium ipidophilum (Physalacriaceae). The free-living wood-decay fungus Phlebiopsis gigantea (Phanerochaetaceae) has been occasionally associated with bark beetles but is unrelated to C. ipidophilum or Entomocorticium.

Patterns of coevolution between ambrosia beetle mycangia and the Ceratocystidaceae, with five new fungal genera and seven new species.

Ambrosia beetles farm specialised fungi in sapwood tunnels and use pocket-like organs called mycangia to carry propagules of the fungal cultivars. Ambrosia fungi selectively grow in mycangia, which is central to the symbiosis, but the history of coevolution between fungal cultivars and mycangia is poorly understood. The fungal family Ceratocystidaceae previously included three ambrosial genera (Ambrosiella, Meredithiella, and Phialophoropsis), each farmed by one of three distantly related tribes of ambrosia beetles with unique and relatively large mycangium types. Studies on the phylogenetic relationships and evolutionary histories of these three genera were expanded with the previously unstudied ambrosia fungi associated with a fourth mycangium type, that of the tribe Scolytoplatypodini. Using ITS rDNA barcoding and a concatenated dataset of six loci (28S rDNA, 18S rDNA, tef1-α, tub, mcm7, and rpl1), a comprehensive phylogeny of the family Ceratocystidaceae was developed, including Inodoromyces interjectus gen. & sp. nov., a non-ambrosial species that is closely related to the family. Three minor morphological variants of the pronotal disk mycangium of the Scolytoplatypodini were associated with ambrosia fungi in three respective clades of Ceratocystidaceae: Wolfgangiella gen. nov., Toshionella gen. nov., and Ambrosiella remansi sp. nov. Closely-related species that are not symbionts of ambrosia beetles are accommodated by Catunica adiposa gen. & comb. nov. and Solaloca norvegica gen. & comb. nov. The divergent morphology of the ambrosial genera and their phylogenetic placement among non-ambrosial genera suggest three domestication events in the Ceratocystidaceae. Estimated divergence dates for the ambrosia fungi and mycangia suggest that Scolytoplatypodini mycangia may have been the first to acquire Ceratocystidaceae symbionts and other ambrosial fungal genera emerged shortly after the evolution of new mycangium types. There is no evidence of reversion to a non-ambrosial lifestyle in the mycangial symbionts.

Taxonomic revision and multi-locus phylogeny of the North American clade of Ceratocystis.

The North American clade (NAC) of Ceratocystis includes pathogenic species that infect a wide range of woody hosts. Previous phylogenetic analyses have suggested that this clade includes cryptic species and a paraphyletic C. variospora. In this study, we used morphological data and phylogenetic analyses to characterize NAC taxa, including Ceratocystis isolates causing a serious disease of almond trees in California. Phylogenetic analyses based on six gene regions supported two new species of Ceratocystis. Ceratocystis destructans is introduced as the species causing severe damage to almond trees in California, and it has also been isolated from wounds on Populus and Quercus in Iowa. It is morphologically similar to C. tiliae, a pathogen on Tilia and the most recently characterized species in the NAC. Ceratocystis betulina collected from Betula platyphylla in Japan is also newly described and is the sister taxon to C. variospora. Our six-locus phylogenetic analyses and morphological characterization resolved several cryptic species in the NAC.

New Ceratocystis species associated with rapid death of Metrosideros polymorpha in Hawai'i.

The native 'ohi'a lehua (Metrosideros polymorpha) has cultural, biological and ecological significance to Hawai'i, but it is seriously threatened by a disease commonly referred to as rapid 'ohi'a death (ROD). Preliminary investigations showed that a Ceratocystis species similar to C. fimbriata s.lat. was the cause of the disease. In this study, we used a combination of the phylogenetic, morphological and biological species concepts, as well as pathogenicity tests and microsatellite analyses, to characterise isolates collected from diseased 'ohi'a trees across Hawai'i Island. Two distinct lineages, representing new species of Ceratocystis, were evident based on multigene phylogenetic analyses. These are described here as C. lukuohia and C. huliohia. Ceratocystis lukuohia forms part of the Latin American clade (LAC) and was most closely associated with isolates from Syngonium and Xanthosoma from the Caribbean and elsewhere, including Hawai'i, and C. platani, which is native to eastern USA. Ceratocystis huliohia resides in the Asian-Australian clade (AAC) and is most closely related to C. uchidae, C. changhui and C. cercfabiensis, which are thought to be native to Asia. Morphology and interfertility tests support the delineation of these two new species and pathogenicity tests show that both species are aggressive pathogens on seedlings of M. polymorpha. Characterisation of isolates using microsatellite markers suggest that both species are clonal and likely represent recently-introduced strains. Intensive research is underway to develop rapid screening protocols for early detection of the pathogens and management strategies in an attempt to prevent the spread of the pathogens to the other islands of Hawai'i, which are currently disease free.

Oral immunotherapy with omalizumab reverses the Th2 cell-like programme of regulatory T cells and restores their function.

BACKGROUND: Oral immunotherapy (OIT) successfully desensitizes patients with food allergies, but the immune mechanisms mediating its efficacy remain obscure. OBJECTIVES: We tested the hypothesis that allergen-specific regulatory T (Treg) cell function is impaired in food allergy and is restored by anti-IgE antibody (omalizumab)-supplemented OIT. METHODS: Peanut-specific T effector (Teff) and Treg cell proliferative responses, activation markers and cytokine expression were analysed by flow cytometry in 13 peanut-allergic subjects before the start of omalizumab-supplemented OIT and periodically in some subjects thereafter for up to 2 years. Peripheral blood regulatory T cells (Treg cells) were analysed for their peanut-specific suppressor function before and at 1 year following OIT. This study was registered on ClinicalTrials.gov (NCT01290913). RESULTS: Proliferation of allergen-specific Teff and Treg cells precipitously declined following the initiation of omalizumab therapy prior to OIT, followed by partial recovery after the initiation of OIT. At baseline, peanut-specific Treg cells exhibited a Th2 cell-like phenotype, characterized by increased IL-4 expression, which progressively reversed upon OIT. Peanut-specific Treg cell suppressor activity was absent at the start of omalizumab/OIT therapy but became robust following OIT. Absent peanut-specific Treg cell function could also be recovered by the acute blockade of IL-4/IL-4R receptor signalling in Treg cells, which inhibited their IL-4 production. CONCLUSIONS AND CLINICAL RELEVANCE: OIT supplemented by omalizumab promotes allergen desensitization through an initial omalizumab-dependent step that acutely depletes allergen-reactive T cells, followed by an increase in allergen-specific Treg cell activity due to the reversal of their Th2 cell-like programme. Improved Treg cell function may be a key mechanism by which OIT ameliorates food allergy.

A re-evaluation of Tubakia, including three new species on Quercus and six new combinations.

Morphological comparisons and phylogenetic analyses of Tubakia species from leaves of Quercus spp. in Iowa and other areas of eastern USA revealed three novel species: Tubakia hallii, Tubakia macnabbii, and Tubakia tiffanyae. These species, as well as Tubakia dryina and Tubakia iowensis, are common leaf endophytes and pathogens on Quercus and Castanea in eastern USA, as is Tubakia americana comb. nov, originally described from Quercus in New Jersey as Actinopelte americana. New combinations of species on leaves of other hosts in the eastern USA include Tubakia gloeosporioides, Tubakia liquidambaris, and Tubakia nyssae. Asian species of Tubakia are phylogenetically compared, and the new combination Tubakia supraseptata is made to accommodate a Japanese endophyte known only by its sexual state. The earlier description of Dicarpella dryina as the sexual state of T. dryina is questioned. The new combination Tubakia stellata is made to accommodate an unusual species from Brazil.

Patient Navigators Connecting Patients to Community Resources to Improve Diabetes Outcomes.

BACKGROUND: Despite the recognized importance of lifestyle modification in reducing risk of developing type 2 diabetes and in diabetes management, the use of available community resources by both patients and their primary care providers (PCPs) remains low. The patient navigator model, widely used in cancer care, may have the potential to link PCPs and community resources for reduction of risk and control of type 2 diabetes. In this study we tested the feasibility and acceptability of telephone-based nonprofessional patient navigation to promote linkages between the PCP office and community programs for patients with or at risk for diabetes. METHODS: This was a mixed-methods interventional prospective cohort study conducted between November 2012 and August 2013. We included adult patients with and at risk for type 2 diabetes from six primary care practices. Patient-level measures of glycemic control, diabetes care, and self-efficacy from medical records, and qualitative interview data on acceptability and feasibility, were used. RESULTS: A total of 179 patients participated in the study. Two patient navigators provided services over the phone, using motivational interviewing techniques. Patient navigators provided regular feedback to PCPs and followed up with the patients through phone calls. The patient navigators made 1028 calls, with an average of 6 calls per patient. At follow-up, reduction in HbA1c (7.8 ± 1.9% vs 7.2 ± 1.3%; P = .001) and improvement in patient self-efficacy (3.1 ± 0.8 vs 3.6 ± 0.7; P < .001) were observed. Qualitative analysis revealed uniformly positive feedback from providers and patients. CONCLUSIONS: The patient navigator model is a promising and acceptable strategy to link patient, PCP, and community resources for promoting lifestyle modification in people living with or at risk for type 2 diabetes.

Detection of Colletotrichum acutatum Sensu Lato on Strawberry by Loop-Mediated Isothermal Amplification.

Colletotrichum acutatum, one of the most economically damaging pathogens of strawberry, is the primary causal agent of anthracnose fruit rot (AFR). A key challenge in managing AFR is detecting the pathogen on asymptomatic plants. To meet this need, a loop-mediated isothermal amplification (LAMP) assay was developed that incorporated two sets of primers: LITSG1, targeted on the intergenic transcribed spacer (ITS) region of ribosomal DNA, and Ltub2, on the ß-tubulin 2 gene. In pure culture assays, Ltub2 was specific for detection of C. acutatum, whereas LITSG1 detected C. acutatum and two additional anthracnose pathogens, C. gloeosporioides and C. fragariae. LITSG1 had 10-fold lower detection threshold (20 pg of mycelial DNA) than Ltub2 (200 pg mycelial DNA) in detection of C. acutatum from pure culture. For detection on asymptomatic leaves, two protocols for dislodging C. acutatum for DNA extraction were compared: i) the sonicate-agitate (SA) method and ii) the freeze-incubate-sonicate-agitate (FISA) method, which initially freezes tissues, followed by 2 days of incubation at 26°C in darkness, and then, sonication and agitation. Both methods were used for greenhouse-grown plant leaves that had been spray inoculated with serial dilutions ranging from 1.5 × 106 to 1.5 conidia ml-1. The FISA method produced more repeatable results than the SA method. For the FISA method, detection limits (expressed as initial inoculum concentrations) using LITSG1 and Ltub2 were 1.5 × 101 and 1.5 × 102 conidia ml-1, respectively. For composite samples comprised of inoculated (1.5 × 106 conidia ml-1) and noninoculated leaves of greenhouse-grown strawberry, the two sets of LAMP primers were compared using the SA method. Primer set LITSG1 consistently detected the pathogen from a single inoculated leaf in bulk samples of 50 or fewer pathogen-free leaves, whereas Ltub2 consistently detected one inoculated leaf in 20 or fewer pathogen-free leaves. Using primer set LITSG1, FISA was more sensitive than SA for detecting C. acutatum on leaves of field-grown plants from Florida. In an Iowa field trial using the FISA method, both primer sets detected C. acutatum in samples of asymptomatic leaves 6 days before fruit symptoms appeared. The results indicate that the LAMP assay has potential to provide a simplified method for detection of C. acutatum on asymptomatic strawberry plants.

Phenology of Infection on Apple Fruit by Sooty Blotch and Flyspeck Species in Iowa Apple Orchards.

Sooty blotch and flyspeck (SBFS) is a fungal disease complex that can cause significant economic losses to apple growers by blemishing the fruit surface with dark-colored colonies. Little is known about the phenology of host infection for this diverse group of epiphytes. In 2009 and 2010, we investigated the timing of infection of apple fruit by SBFS species in six commercial apple orchards in Iowa. Five trees in each orchard received no fungicide sprays after fruit set. Within 3 weeks after fruit set, 60 apples per tree were covered with Japanese fruit bags to minimize inoculum deposition. Subsequently, a subsample of bagged apples was exposed for a single 2-week-long period and then rebagged for the remainder of the growing season. Experimental treatments included seven consecutive 2-week-long exposure periods; control treatments were apples that were either bagged or exposed for the entire season. After apples had been stored at 2°C for 6 weeks following harvest, all SBFS colonies on the apples were identified to species using a PCR-RFLP protocol. A total of 15 species were identified. For the seven most prevalent species, the number of infections per cm2 of fruit surface was greatest on apples that had been exposed early in the season. Two SBFS species, Peltaster fructicola and Colletogloeopsis-like FG2, differed significantly from each other in time required to attain 50% of the total number of colonies per apple, and analysis of variance indicated a significant interaction of SBFS taxon with exposure period. Our findings are the first evidence of species-specific patterns in timing of SBFS inoculum deposition and infection on apple fruit, and strengthen previous observations that most SBFS infections resulting in visible colonies at harvest develop from infections that occur early in the fruit development period. By defining taxon-specific phenological patterns of fruit infection, our findings, when combined with knowledge of region-specific patterns of taxon prevalence, provide a foundation for development of more efficient and cost-effective SBFS management tactics.

Early Magnesium Treatment After Aneurysmal Subarachnoid Hemorrhage: Individual Patient Data Meta-Analysis.

BACKGROUND AND PURPOSE: Delayed cerebral ischemia (DCI) is an important cause of poor outcome after aneurysmal subarachnoid hemorrhage (SAH). Trials of magnesium treatment starting <4 days after symptom onset found no effect on poor outcome or DCI in SAH. Earlier installment of treatment might be more effective, but individual trials had not enough power for such a subanalysis. We performed an individual patient data meta-analysis to study whether magnesium is effective when given within different time frames within 24 hours after the SAH. METHODS: Patients were divided into categories according to the delay between symptom onset and start of the study medication: <6, 6 to 12, 12 to 24, and >24 hours. We calculated adjusted risk ratios with corresponding 95% confidence intervals for magnesium versus placebo treatment for poor outcome and DCI. RESULTS: We included 5 trials totaling 1981 patients; 83 patients started treatment<6 hours. For poor outcome, the adjusted risk ratios of magnesium treatment for start <6 hours were 1.44 (95% confidence interval, 0.83-2.51); for 6 to 12 hours 1.03 (0.65-1.63), for 12 to 24 hours 0.84 (0.65-1.09), and for >24 hours 1.06 (0.87-1.31), and for DCI, <6 hours 1.76 (0.68-4.58), for 6 to 12 hours 2.09 (0.99-4.39), for 12 to 24 hours 0.80 (0.56-1.16), and for >24 hours 1.08 (0.88-1.32). CONCLUSIONS: This meta-analysis suggests no beneficial effect of magnesium treatment on poor outcome or DCI when started early after SAH onset. Although the number of patients was small and a beneficial effect cannot be definitively excluded, we found no justification for a new trial with early magnesium treatment after SAH.

Ceratocystis tiliae sp. nov., a wound pathogen on Tilia americana.

Species in the North American clade (NAC) of the Ceratocystis fimbriata complex are mostly weak pathogens that infect native tree hosts through fresh wounds. Isolations from discolored tissue of wounded Tilia americana (basswood) in Iowa and Nebraska yielded a Ceratocystis species that was similar to but distinct from isolates of C. variospora from other hosts. Sequences of 28S rDNA showed that isolates from basswood did not differ from C. variospora, but there were minor differences in ITS rDNA sequences. The DNA sequences of a portion of the Cerato-platanin gene and TEF1α showed the basswood fungus to be a unique lineage. Cross inoculations in two experiments showed that the basswood isolates and C. variospora isolates from Quercus spp. were most aggressive to their respective hosts. Isolates from basswood grew slower and were less pigmented than C. variospora isolates from Quercus spp. The basswood fungus thus is distinguished from C. variospora based on phylogenetic analyses and phenotype and is herein described as C. tiliae sp. nov.

Intraspecific and intragenomic variability of ITS rDNA sequences reveals taxonomic problems in Ceratocystis fimbriata sensu stricto.

Fourteen new species in the Latin American Clade (LAC) of the Ceratocystis fimbriata complex recently were distinguished from C. fimbriata sensu stricto largely based on variation in ITS rDNA sequences. Among the 116 isolates representing the LAC, there were 41 ITS haplotypes. Maximum parsimony (MP) analysis of ITS sequences produced poorly resolved trees. In contrast, analyses of mating-type genes (MAT1-1-2 and MAT1-2-1) resolved a single MP tree with branches of high bootstrap and posterior probability support. Four isolates showed intragenomic variation in ITS sequences. Cloning and sequencing of PCR products from a single haploid strain identified two or more ITS sequences differing at up to 16 base positions and representing two described species. Isolates from introduced populations that appeared to be clonal based on microsatellite markers varied at up to 14 bp in ITS sequence. Strains of seven Brazilian ITS haplotypes and an isolate from Ipomoea batatas (on which the species name C. fimbriata was based) were fully interfertile in sexual crosses. These analyses support three phylogenetic species that differ in pathogenicity: C. platani, C. cacaofunesta and C. colombiana. Five ITS species (C. manginecans, C. mangicola, C. mangivora, C. acaciivora, C. eucalypticola) appear to be ITS haplotypes that have been moved from or within Brazil on nursery stock. The taxonomic status of other species delineated primarily by ITS sequences (C. diversiconidia, C. papillata, C. neglecta, C. ecuadoriana, C. fimbriatomima, C. curvata) needs further study, but they are considered doubtful species.

Catastrophic antiphospholipid syndrome with concurrent thrombotic and hemorrhagic manifestations.

Antiphospholipid syndrome (APS) is a distinct autoimmune prothrombotic disorder due to pathogenic autoantibodies directed against proteins that bind to phospholipids. APS is characterized by arterial and venous thrombosis and their clinical sequelae. Catastrophic antiphospholipid syndrome (CAPS) is a rare and often fatal form of APS characterized by disseminated intravascular thrombosis and ischemic injury resulting in multiorgan failure. Rarely, intravascular thrombosis in CAPS is accompanied by hemorrhagic manifestations such as diffuse alveolar hemorrhage. Here, we report a 43-year-old woman who presented with anemia, acute gastroenteritis, abnormal liver function tests, bilateral pulmonary infiltrates, and a systemic inflammatory response syndrome. The patient developed respiratory failure as a result of diffuse alveolar hemorrhage followed by acute renal failure. Laboratory tests disclosed hematuria, proteinuria, and reduced platelet count. Microbiologic tests were negative. A renal biopsy demonstrated acute thrombotic microangiopathy and extensive interstitial hemorrhage. Serologic tests disclosed antinuclear antibodies and reduced serum complement C4 concentration. Coagulation studies revealed the lupus anticoagulant and autoantibodies against cardiolipin, beta 2-glycoprotein I, and prothrombin. High-dose glucocorticoids and plasma exchange resulted in rapid resolution of pulmonary, renal, and hematological manifestations. This rare case emphasizes that CAPS can present with concurrent thrombotic and hemorrhagic manifestations. Rapid diagnosis and treatment may result in complete recovery.

Using tablet computers compared to interactive voice response to improve subject recruitment in osteoporosis pragmatic clinical trials: feasibility, satisfaction, and sample size.

INTRODUCTION: Pragmatic clinical trials (PCTs) provide large sample sizes and enhanced generalizability to assess therapeutic effectiveness, but efficient patient enrollment procedures are a challenge, especially for community physicians. Advances in technology may improve methods of patient recruitment and screening in PCTs. Our study looked at a tablet computer versus an integrated voice response system (IVRS) for patient recruitment and screening for an osteoporosis PCT in community physician offices. MATERIALS AND METHODS: We recruited women ≥ 65 years of age from community physician offices to answer screening questions for a hypothetical osteoporosis active comparator PCT using a tablet computer or IVRS. We assessed the feasibility of these technologies for patient recruitment as well as for patient, physician, and office staff satisfaction with the process. We also evaluated the implications of these novel recruitment processes in determining the number of primary care practices and screened patients needed to conduct the proposed trial. RESULTS: A total of 160 women (80% of those approached) agreed to complete the osteoporosis screening questions in ten family physicians' offices. Women using the tablet computer were able to complete all screening questions consistently and showed a nonsignificant trend towards greater ease of use and willingness to spend more time in their physician's office compared to those using IVRS. Using the proportion of women found to be eligible in this study (almost 20%) and other eligibility scenarios, we determined that between 240 and 670 community physician offices would be needed to recruit ample patients for our hypothetical study. CONCLUSION: We found good satisfaction and feasibility with a tablet computer interface for the recruitment and screening of patients for a hypothetical osteoporosis PCT in community office settings. In addition, we used this experience to estimate the number of research sites needed for such a study.

First Report of Laurel Wilt, Caused by Raffaelea lauricola, on Sassafras (Sassafras albidum) in Alabama.

Laurel wilt, caused by Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus, is responsible for extensive mortality of native redbays (Persea borbonia and P. palustris) in the coastal plains of the southeastern United States (1). The wilt also affects the more widespread sassafras, Sassafras albidum, particularly in areas where diseased redbays are common and populations of X. glabratus are high. Because sassafras stems were thought to lack chemicals that are attractive to the beetle, and sassafras tends to be widely scattered in forests, it was believed that the advance of the laurel wilt epidemic front might slow once it reached the edge of the natural range of redbay, which is restricted to the coastal plains of the Gulf and Atlantic Coasts (2). In July and August of 2011, wilt-like symptoms (i.e., wilted and dead leaves, and streaks of black discoloration in the xylem) were observed on 1 to 10 sassafras trees (15 to 23 cm diameter; 6 to 9 m height) at each of three locations, which were approximately 6 km from one another in Marengo Co., Alabama. Samples of the discolored wood from five trees were plated on malt agar amended with cycloheximide and streptomycin (CSMA), and a fungus morphologically identical to R. lauricola was isolated from each tree (1). For confirmation, a portion of the large subunit (28S) of the rDNA region of three of the isolates was sequenced (3); in each case, the sequence matched exactly that of other isolates of R. lauricola (EU123077) from the United States. Symptomatic trees were found at all three sites when revisited in April 2012, and approximately 20 sassafras trees in various stages of wilt were observed at one location, where only one diseased tree had been noted in 2011. Bolts were cut from the main stem of a symptomatic tree, and eggs, larvae, and adults of X. glabratus were commonly found in tunnels, and R. lauricola was isolated from the discolored xylem. Three container-grown sassafras saplings (mean height 193 cm, mean diameter 2.1 cm at groundline) were inoculated as previously described (1) with conidia (~600,000) from an isolate of R. lauricola. Three additional sassafras saplings were inoculated with sterile, deionized water, and all plants were placed in a growth chamber at 25°C with a 15-h photoperiod. Inoculated plants began to exhibit wilt symptoms within 14 days, and at 30 days all inoculated plants were dead and xylem discoloration was observed. Control plants appeared healthy and did not exhibit xylem discoloration. Pieces of sapwood from 15 cm above the inoculation points were plated on CSMA, and R. lauricola was recovered from all wilted plants but not from control plants. This is the first record of laurel wilt in Alabama and is significant because the disease appears to be spreading on sassafras in an area where redbays have not been recorded (see http://www.floraofalabama.org ). The nearest previously documented case of laurel wilt is on redbay and sassafras in Jackson Co., Mississippi (4), approximately 160 km to the south. The exact source of the introduction of X. glabratus and R. lauricola into Marengo Co. is not known. The vector may have been transported into the area with storms, moved with infested firewood, or shipped with infested timber by companies that supply mills in the area. References: (1) S. Fraedrich et al. Plant Dis. 92:215, 2008. (2) J. Hanula et al. Econ. Ent. 101:1276, 2008. (3) T. Harrington et al. Mycotaxon 111:337, 2010. (4) J. Riggins et al. Plant Dis. 95:1479, 2011.

Temporal patterns in appearance of sooty blotch and flyspeck fungi on apples.

Sooty blotch and flyspeck (SBFS) is a complex of about 80 fungal species that blemish the surface of apple fruit in humid regions worldwide. The dark colonies become visible in mid- to late summer, reducing the value of fresh fruit. Although many SBFS species can co-occur in the same orchard and even on the same apple, little is known about temporal patterns of these species, including the timing of colony appearance. To test the hypothesis that colonies of SBFS species appear on apples at characteristic times during the growing season, 50 apples were monitored weekly at three Iowa orchards in 2006 and six orchards in 2007 and 2008. However, a mean of 24.3 apples per orchard was assessed at harvest because of apple drop throughout the season. Colonies were marked with colored pens as they appeared. After harvest and after storage of apples at 2 °C for 3 months, SBFS colonies on each fruit were counted and classified by morphology, and a representative subset of colonies was excised from the fruit and preserved on dried peels for species identification using rDNA. Seventeen species were identified. Stomiopeltis spp. RS1 and RS2 appeared on apples 10 to 14 days before other SBFS taxa. Dissoconium aciculare was generally the last species to appear on apple fruit, and it continued to appear during postharvest storage. The most prevalent taxa in Iowa orchards were also the most abundant. Diversity of SBFS fungi in an orchard was positively correlated with cumulative hours of surface wetness hours due to rainfall or dew, which is believed to favor growth of SBFS fungi. Species-specific information about temporal patterns of appearance on apple fruit may lead to improved SBFS management strategies.