Regional Comparisons of Sensitivities of Phytophthora citrophthora and P. syringae Causing Citrus Brown Rot in California to Four New and Two Older Fungicides.

Isolates of the citrus brown rot pathogens Phytophthora citrophthora and P. syringae from the Inland Empire (IE) and Ventura Co. (VE) regions of southern California were evaluated for their sensitivity to ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin, and the previously published baselines that were generated for Central Valley (CV) isolates of California were expanded. Fungicides were generally more toxic to CV isolates of both species for all four fungicides. Specific differences were found in the toxicity of ethaboxam to P. syringae where CV isolates on average were 6.8 or 8.2 times more sensitive than those from the VE or IE regions, respectively. Based on the grouping of isolates in an unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, as well as fastStructure analyses and plotting of principal component analyses (PCAs), differences in ethaboxam sensitivity could be related to differences in genetic background of the isolates. Isolates of P. citrophthora from the IE and VE had slightly reduced (i.e., 1.5×) sensitivity to mandipropamid as compared with isolates from the CV and were found on distinct branches in the UPGMA dendrogram. Differences in genetic background of less sensitive isolates within each species indicate that these two phenotypes emerged multiple times independently. IE and VE isolates of both species were sensitive to mefenoxam. Moderate resistance to potassium phosphite (EC50 values of 25 to 75 µg/ml) was present in IE and VE isolates of P. syringae, whereas some IE isolates of P. citrophthora were considered resistant with EC50 values of up to 113.69 µg/ml. Resistance to potassium phosphite did not relate to distinct genotypes.

Diversity and Clonality in Populations of Phytophthora citrophthora and P. syringae Causing Brown Rot of Citrus in California.

Phytophthora citrophthora and P. syringae are currently the primary causal organisms of brown rot of citrus fruits in California. To possibly find an explanation for the prevalence of the previously minor species P. syringae, we determined the population structures of both pathogens in California using next-generation sequencing and population genomics analyses. Whole-genome sequencing and aligning with newly assembled reference genomes identified 972,266 variants in 132 isolates of P. citrophthora and 422,208 variants in 154 isolates (including 24 from noncitrus tree crops) of P. syringae originating from three major growing regions. The resulting data sets were visualized using principal component analysis, discriminant analysis of principal components, unweighted pair-group method with arithmetic mean dendrograms, fastStructure, and minimum spanning networks, and we obtained the index of association, diversity summary statistics, and genetic distance statistics values GST, G''ST, and Jost's D. Subpopulations of both species were mostly defined by their geographic origin indicating restricted dispersal of inoculum. Except for five isolates, the population structure of P. citrophthora (that is heterothallic and unlikely to reproduce sexually) was clonal to semi-clonal, with very little genetic diversity within and among subgroups. In contrast, the population structure of P. syringae was also clonal to semi-clonal, but isolates were placed into four main clusters of much higher diversity. Clonality in both species can be explained by a high level of asexual reproduction. The higher diversity in the homothallic P. syringae is likely due to commonly occurring sexual reproduction. One distinct cluster of P. syringae consisted solely of isolates from noncitrus hosts; therefore, the origin of P. syringae in citrus could not be resolved.

Root Absorption and Limited Mobility of Mandipropamid as Compared to Oxathiapiprolin and Mefenoxam After Soil Treatment of Citrus Plants for Managing Phytophthora Root Rot.

Phytophthora root rot can greatly impact citrus production worldwide, especially in newly established orchards by reducing crop yield and increasing the cost of disease management. Mandipropamid is an Oomycota fungicide that is currently registered as a soil treatment for citrus nursery container plants to manage Phytophthora root rot. In this study, we investigated the uptake of mandipropamid into citrus roots and its translocation to stems and leaves after soil application and evaluated its mobility in roots as compared to oxathiapiprolin and mefenoxam using split-root potted plants and trees in the field. A bioassay and liquid chromatography-tandem mass spectrometry were used to detect and quantify fungicides in citrus tissues, and overall, similar results were obtained using the two methods. When applied to the soil of potted, 6- to 7-month-old citrus plants using labeled rates, the majority of mandipropamid was found in root tissues (4.9 to 18.1 µg/g), but small amounts were also present in stems (0.18 to 0.32 µg/g) and leaves (0.03 to 0.22 µg/g). There was no significant increase in concentrations in all three tissues between 1 and 4 weeks after application. Concentrations in all tissues exceeded established EC50 values for mycelial growth inhibition of P. citrophthora and P. nicotianae, the main citrus root rot pathogens in California. In a split-root study where the root systems of single plants were separated, no basipetal phloem-based mobility of mandipropamid or oxathiapiprolin was observed, but relative uptake into roots was higher for mandipropamid. In contrast, low amounts of mefenoxam were also present in roots in the untreated soil. Similar results were obtained in a field study where part of the root system was treated, and fungicides were extracted from nontreated roots. All three fungicides persisted inside roots over the 8-week period of this study. Uptake and persistence inside roots, as well as the previously reported high efficacy against citrus root rot in greenhouse and field studies support the use of mandipropamid in citrus nurseries and potentially in the orchard.

An experimental approach for controlling confinement effects at catalyst interfaces.

Catalysts are conventionally designed with a focus on enthalpic effects, manipulating the Arrhenius activation energy. This approach ignores the possibility of designing materials to control the entropic factors that determine the pre-exponential factor. Here we investigate a new method of designing supported Pt catalysts with varying degrees of molecular confinement at the active site. Combining these with fast and precise online measurements, we analyse the kinetics of a model reaction, the platinum-catalysed hydrolysis of ammonia borane. We control the environment around the Pt particles by erecting organophosphonic acid barriers of different heights and at different distances. This is done by first coating the particles with organothiols, then coating the surface with organophosphonic acids, and finally removing the thiols. The result is a set of catalysts with well-defined "empty areas" surrounding the active sites. Generating Arrhenius plots with >300 points each, we then compare the effects of each confinement scenario. We show experimentally that confining the reaction influences mainly the entropy part of the enthalpy/entropy trade-off, leaving the enthalpy unchanged. Furthermore, we find this entropy contribution is only relevant at very small distances (<3 Å for ammonia borane), where the "empty space" is of a similar size to the reactant molecule. This suggests that confinement effects observed over larger distances must be enthalpic in nature.

Comparison of Primary Care Physician Reimbursement Rates in the United States.

With a growing shortage of physicians, particularly primary care physicians, the issue of adequate pay in Hawai'i is increasingly important. Anecdotal reports of low pay in Hawai'i have rarely been substantiated. Data from FAIR Health, a company that tracks private insurance reimbursement rates, is compared across the United States (US) for the CPT code 99213. In addition, FAIR Health and Medicare rates are compared for cities with both similar and disparate cost of living to Hawai'i. Hawai'i is in the second lowest quintile for payment in the US for private insurances, and providers are reimbursed significantly lower than in cities with similar cost of living by both Medicare and private insurances. Methods for increasing payment to physicians in Hawai'i are essential to recruiting the necessary workforce. Revising payment methodologies that increase pay for services in areas of unmet need, revising Medicare Geographic Price Cost Indices to better balance pay in areas of need, and making use of the 10% Medicare Bonus Program for physicians working in Health Professions Shortage Areas are first steps to creating a sustainable plan for physician payment in the future.

Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity.

Learning-related plasticity at excitatory synapses in the mammalian brain requires the trafficking of AMPA receptors and the growth of dendritic spines. However, the mechanisms that couple plasticity stimuli to the trafficking of postsynaptic cargo are poorly understood. Here we demonstrate that myosin Vb (MyoVb), a Ca2+-sensitive motor, conducts spine trafficking during long-term potentiation (LTP) of synaptic strength. Upon activation of NMDA receptors and corresponding Ca2+ influx, MyoVb associates with recycling endosomes (REs), triggering rapid spine recruitment of endosomes and local exocytosis in spines. Disruption of MyoVb or its interaction with the RE adaptor Rab11-FIP2 abolishes LTP-induced exocytosis from REs and prevents both AMPA receptor insertion and spine growth. Furthermore, induction of tight binding of MyoVb to actin using an acute chemical genetic strategy eradicates LTP in hippocampal slices. Thus, Ca2+-activated MyoVb captures and mobilizes REs for AMPA receptor insertion and spine growth, providing a mechanistic link between the induction and expression of postsynaptic plasticity.

Cell surface expression of GluR5 kainate receptors is regulated by an endoplasmic reticulum retention signal.

Kainate receptors (KARs) are mediators of excitatory neurotransmission in the mammalian central nervous system, and their efficient targeting and trafficking is critical for normal synaptic function. A key step in the delivery of KARs to the neuronal plasma membrane is the exit of newly assembled receptors from the endoplasmic reticulum (ER). Here we report the identification of a novel ER retention signal in the alternatively spliced C-terminal domain of the GluR5-2b subunit, which controls receptor trafficking in both heterologous cells and neurons. The ER retention motif consists of a critical arginine (Arg-896) and surrounding amino acids, disruption of which promotes ER exit and surface expression of the receptors, as well as altering their physiological properties. The Arg-896-mediated ER retention of GluR5 is regulated by a mutation that mimics phosphorylation of Thr-898, but not by PDZ interactions. Furthermore, two positively charged residues (Arg-900 and Lys-901) in the C terminus were also found to regulate ER export of the receptors. Taken together, our results identify novel trafficking signals in the C-terminal domain of GluR5-2b and demonstrate that alternative splicing is an important mechanism regulating KAR function.

Multiple trafficking signals regulate kainate receptor KA2 subunit surface expression.

The kainate receptor subunit KA2 does not form functional homomeric channels despite its structural similarity to the functional glutamate receptor 5-7subunits and high agonist binding affinity in in vitro assays. In this study, we first demonstrate that homomeric KA2 receptors fail to reach the plasma membrane and then identify the molecular mechanisms preventing surface expression. Specifically, we show that KA2 subunits form homooligomeric receptors that are confined to the endoplasmic reticulum (ER). We then demonstrate that, in both heterologous expression systems and primary neurons, the intracellular retention of KA2 is not caused by subunit misfolding but, rather, is mediated through discrete protein trafficking signals, including an arginine-rich ER retention/retrieval motif and a di-leucine endocytic sequence in the C terminus of the KA2 subunit. Disruption of these motifs results in ER exit and surface expression of KA2 homomeric receptors that remain nonfunctional. Furthermore, our data suggest that the ER retention/retrieval signal in KA2 is sterically shielded during heteromeric assembly, allowing delivery of functional heteromeric receptors to the plasma membrane. Taken together, our results illustrate novel regulatory mechanisms that control the intracellular trafficking and surface expression of kainate receptors.