Epac2a-null mice exhibit obesity-prone nature more susceptible to leptin resistance.

BACKGROUND: The exchange protein directly activated by cAMP (Epac), which is primarily involved in cAMP signaling, has been known to be essential for controlling body energy metabolism. Epac has two isoforms: Epac1 and Epac2. The function of Epac1 on obesity was unveiled using Epac1 knockout (KO) mice. However, the role of Epac2 in obesity remains unclear. METHODS: To evaluate the role of Epac2 in obesity, we used Epac2a KO mice, which is dominantly expressed in neurons and endocrine tissues. Physiological factors related to obesity were analyzed: body weight, fat mass, food intake, plasma leptin and adiponectin levels, energy expenditure, glucose tolerance, and insulin and leptin resistance. To determine the mechanism of Epac2a, mice received exogenous leptin and then hypothalamic leptin signaling was analyzed. RESULTS: Epac2a KO mice appeared to have normal glucose tolerance and insulin sensitivity until 12 weeks of age, but an early onset increase of plasma leptin levels and decrease of plasma adiponectin levels compared with wild-type mice. Acute leptin injection revealed impaired hypothalamic leptin signaling in KO mice. Consistently, KO mice fed a high-fat diet (HFD) were significantly obese, presenting greater food intake and lower energy expenditure. HFD-fed KO mice were also characterized by greater impairment of hypothalamic leptin signaling and by weaker leptin-induced decrease in food consumption compared with HFD-fed wild-type mice. In wild-type mice, acute exogenous leptin injection or chronic HFD feeding tended to induce hypothalamic Epac2a expression. CONCLUSIONS: Considering that HFD is an inducer of hypothalamic leptin resistance and that Epac2a functions in pancreatic beta cells during demands of greater work load, hypothalamic Epac2a may have a role in facilitating leptin signaling, at least in response to higher metabolic demands. Thus, our data indicate that Epac2a is critical for preventing obesity and thus Epac2a activators may be used to manage obesity and obesity-mediated metabolic disorders.

A multicenter study on anaphylaxis caused by peanut, tree nuts, and seeds in children and adolescents.

Peanut (PN) and tree nuts (TNs) are common causes of anaphylaxis in Western countries, but no information is available in Korea. To feature clinical characteristics of anaphylaxis caused by PN, TNs, and seeds, a retrospective medical record review was performed in 14 university hospitals in Korea (2009-2013). One hundred and twenty-six cases were identified, with the mean age of 4.9 years. PN, walnut (WN), and pine nut accounted for 32.5%, 41.3%, and 7.1%, respectively. The median values of specific IgE (sIgE) to PN, WN, and pine nut were 10.50, 8.74, and 4.61 kUA /l, respectively. Among 50 cases managed in the emergency department, 52.0% were treated with epinephrine, 66.0% with steroid, 94.0% with antihistamines, 36.0% with oxygen, and 48.0% with bronchodilator. In conclusion, WN, PN, and pine nut were the three most common triggers of anaphylaxis in Korean children, and anaphylaxis could occur at remarkably low levels of sIgE.

Biological characteristics of Bacillus amyloliquefaciens AK-0 and suppression of ginseng root rot caused by Cylindrocarpon destructans.

AIMS: The effect of Bacillus amyloliquefaciens AK-0 (AK-0) on ginseng root rot disease caused by Cylindrocarpon destructans was investigated. METHODS AND RESULTS: From 190 ginseng rhizosphere bacteria, AK-0 was selected for further analysis; its morphological characteristics were investigated by microscopy. AK-0 was identified as B. amyloliquefaciens using the Biolog system, 16S rRNA gene sequence analysis and examination of morphological and biochemical characteristics. Bacterial population and media optimization were estimated by the bacterial growth curve. The number of AK-0 cells was relatively higher in brain-heart infusion (BHI) medium than in other media. The potential antifungal effect of AK-0 culture filtrate on the in vitro conidial germination of C. destructans and root rot development on root discs and 4-year-old ginseng roots were assessed. Polymerase chain reaction (PCR) analysis of antibiotic biosynthesis gene expression suggested that the release of antibiotic compounds is involved in the antifungal effect of AK-0 and the suppression of ginseng root rot. CONCLUSION: These results indicate that the CF of AK-0 has antifungal effects on fungal pathogens of ginseng, resulting in the suppression of root rot disease caused by C. destructans. SIGNIFICANCE AND IMPACT OF THE STUDY: AK-0 is a potential source of novel bioactive metabolites. AK-0 CF exhibited antifungal effects against C. destructans on ginseng roots. PCR analysis indicated that the AK-0 harbours genes involved in the biosynthesis of antimicrobial compounds.

Fibroblast growth factor 21 analogue LY2405319 lowers blood glucose in streptozotocin-induced insulin-deficient diabetic mice by restoring brown adipose tissue function.

AIM: To investigate the effects of LY2405319, an analogue of fibroblast growth factor 21 (FGF21), on glucose homeostasis in streptozotocin (STZ)-induced insulin-deficient mice (STZ mice). METHODS: Nine-week-old male C57BL/6J mice were administered a single intraperitoneal injection of STZ (150 mg/kg). One week later, after confirmation of hyperglycaemia, saline or LY2405319 (5 mg/kg) was injected subcutaneously daily for 4 weeks. Changes in glucose homeostasis, energy metabolism and brown adipose tissue (BAT) function were assessed. RESULTS: The STZ mice had elevated blood glucose and reduced plasma FGF21 levels, impaired glucose uptake in the BAT, and BAT mitochondria with absent or swollen cristae and fewer lipid vacuoles. LY2405319 significantly reduced blood glucose levels and this was associated with increased BAT glucose uptake and changes in gene expression and morphology, indicating improved mitochondrial lipid metabolism in the BAT. Importantly, the ability of LY2405319 to lower blood glucose in STZ mice was compromised after removing interscapular BAT. CONCLUSIONS: Our results show that LY2405319 reduces blood glucose levels in insulin-deficient diabetes by improving BAT metabolism. Additional studies investigating the therapeutic potential of FGF21 for the treatment of type 1 diabetes are warranted.

Use of the Tailored Activities Program to reduce neuropsychiatric behaviors in dementia: an Australian protocol for a randomized trial to evaluate its effectiveness.

BACKGROUND: Behavioral and psychological symptoms of dementia (BPSD) are often considered to be the greatest challenge in dementia care, leading to increased healthcare costs, caregiver burden, and placement into care facilities. With potential for pharmacological intervention to exacerbate behaviors or even lead to mortality, the development and rigorous testing of non-pharmacological interventions is vital. A pilot of the Tailored Activities Program (TAP) for reducing problem behaviors in people with dementia was conducted in the United States with promising results. This randomized trial will investigate the effectiveness of TAP for reducing the burden of BPSD on persons with dementia and family caregivers within an Australian population. This trial will also examine the cost-effectiveness and willingness to pay for TAP compared with a control group. METHODS: This randomized trial aims to recruit 180 participant dyads of a person with dementia and their caregivers. Participants will have a diagnosis of dementia, exhibit behaviors as scored by the Neuropsychiatric Inventory, and the caregiver must have at least 7 h per week contact. Participants will be randomly allocated to intervention (TAP) or control (phone-based education sessions) groups, both provided by a trained occupational therapist. Primary outcome measure will be the revised Neuropsychiatric Inventory - Clinician rating scale (NPI-C) to measure BPSD exhibited by the person with dementia. CONCLUSIONS: This trial investigates the effectiveness and cost-effectiveness of TAP within an Australian population. Results will address a significant gap in the current Australian community-support base for people living with dementia and their caregivers.

First Report of Pectobacterium carotovorum subsp. carotovorum Causing Soft Rot of Orostachys japonica in Korea.

Orostachys japonica (Maxim) A. Berger is an important traditional medicine in Korea. The extract of this plant has antioxidant activity and suppresses cancer cell proliferation (1). From summer through fall of 2012 and 2013, a high incidence (~10% to 30%) of disease outbreaks of all plants characterized by water-soaked lesions and soft rot with a stinky odor was observed in cultivated O. japonica around Uljin (36°59'35.04″N, 126°24'1.51″E), Korea. Water-soaked lesions were first observed on the stem base of plants. Subsequently, the plants collapsed, although the upper portion remained asymptomatic. Thereafter, the lesions expanded rapidly over the entire plant. To isolate potential pathogens from infected leaves, small sections (5 to 10 mm2) were excised from the margins of lesions. Ten bacteria were isolated from ten symptomatic plants. Three representative isolates from different symptomatic plants were used for identification and pathogenicity tests. Isolated bacteria were gram negative, pectolytic on crystal violet pectate agar, nonfluorescent on King's medium B, and elicited a hypersensitive response in tobacco plants. All isolates caused soft rot of potato tubers. These isolates also differed from isolates of Erwinia chrysanthemi (Ech) that they were insensitive to erythromycin and did not produce phosphatase. These isolates differed from known strains of E. carotovora subsp. atroseptica in that they did not produce reducing substances from sucrose (2). Use of the Biolog GN microplate and the Release 4.0 system identified the isolate as Pectobacterium carotovorum subsp. carotovorum with 81.2% similarity. The 16S rRNA of the isolated bacteria was amplified by PCR and sequenced as described by Weisburg et al. (3). A BLAST analysis for sequence similarity of the 16S rRNA region revealed 99% similarity with nucleotide sequences for P. carotovorum subsp. carotovorum isolates (KC790305, KC790280, JF926758, JX196705, and AB680074). The pathogenicity of three bacterial isolates was examined on three 2-year-old O. japonica plants by adding 50 µl of a bacterial suspension containing 108 CFU/ml when wounding the leaves with sterile needles. Ten control plants were inoculated with sterilized water. After inoculation, plants were maintained in a growth chamber at 25°C with relative humidity ranging from 80 to 90%. After 2 to 3 days, tissue discoloration, water-soaked lesions, and soft rot developed around the inoculation point. Severe symptoms of soft rot and darkening developed on leaves of inoculated plants within 3 to 5 days after inoculation. All controls remained healthy during these experiments. The bacterial strains re-isolated from the parts of the leaf showing the symptoms and identified as P. carotovorum subsp. carotovorum on the basis of the biochemical and physiological tests, as well as Biolog system. The results obtained for pathogenicity, Biolog analysis, and molecular data corresponded with those for P. carotovorum subsp. carotovorum. To our knowledge, this is the first report of the presence of P. carotovorum on O. japonica in Korea. References: (1) C.-H. Kim et al. Kor. J. Med. Crop Sci. 11:31, 2003. (2) N. W. Schaad et al. Erwinia Soft Rot Group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al. eds. American Phytopathological Society, St. Paul. MN, 2001. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

First Report of Leaf Blight of Japanese Yew Caused by Pestalotiopsis microspora in Korea.

Worldwide, Japanese yew (Taxus cuspidata Sieb. & Zucc.) is a popular garden tree, with large trees also being used for timber. In July 2012, leaf blight was observed on 10% of Japanese yew seedling leaves planted in a 500-m2 field in Andong, Gyeongsangbuk-do Province, South Korea. Typical symptoms included small, brown lesions that were first visible on the leaf margin, which enlarged and coalesced into the leaf becoming brown and blighted. To isolate potential pathogens from infected leaves, small sections of leaf tissue (5 to 10 mm2) were excised from lesion margins. Eight fungi were isolated from eight symptomatic trees, respectively. These fungi were hyphal tipped twice and transferred to potato dextrose agar (PDA) plates for incubation at 25°C. After 7 days, the fungi produced circular mats of white aerial mycelia. After 12 days, black acervuli containing slimy spore masses formed over the mycelial mats. Two representative isolates were further characterized. Their conidia were straight or slightly curved, fusiform to clavate, five-celled with constrictions at the septa, and 17.4 to 28.5 × 5.8 to 7.1 µm. Two to four 19.8- to 30.7-µm-long hyaline filamentous appendages (mostly three appendages) were attached to each apical cell, whereas one 3.7- to 7.1-µm-long hyaline appendage was attached to each basal cell, matching the description for Pestalotiopsis microspora (2). The pathogenicity of the two isolates was tested using 2-year-old plants (T. cuspidata var. nana Rehder; three plants per isolate) in 30-cm-diameter pots filled with soil under greenhouse conditions. The plants were inoculated by spraying the leaves with an atomizer with a conidial suspension (105 conidia/ml; ~50 ml on each plant) cultured for 10 days on PDA. As a control, three plants were inoculated with sterilized water. The plants were covered with plastic bags for 72 h to maintain high relative humidity (24 to 28°C). At 20 days after inoculation, small dark lesions enlarged into brown blight similar to that observed on naturally infected leaves. P. microspora was isolated from all inoculated plants, but not the controls. The fungus was confirmed by molecular analysis of the 5.8S subunit and flanking internal transcribed spaces (ITS1 and ITS2) of rDNA amplified from DNA extracted from single-spore cultures, and amplified with the ITS1/ITS4 primers and sequenced as previously described (4). Sequences were compared with other DNA sequences in GenBank using a BLASTN search. The P. microspora isolates were 99% homologous to other P. microspora (DQ456865, EU279435, FJ459951, and FJ459950). The morphological characteristics, pathogenicity, and molecular data assimilated in this study corresponded with the fungus P. microspora (2). This fungus has been previously reported as the causal agent of scab disease of Psidium guajava in Hawaii, the decline of Torreya taxifolia in Florida, and the leaf blight of Reineckea carnea in China (1,3). Therefore, this study presents the first report of P. microspora as a pathogen on T. cuspidata in Korea. The degree of pathogenicity of P. microspora to the Korean garden evergreen T. cuspidata requires quantification to determine its potential economic damage and to establish effective management practices. References: (1) D. F. Farr and A. Y. Rossman, Fungal Databases, Syst. Mycol. Microbiol. Lab. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ (2) L. M. Keith et al. Plant Dis. 90:16, 2006. (3) S. S. N. Maharachchikumbura. Fungal Diversity 50:167, 2011. (4) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990.

First Report of Powdery Mildew Caused by Erysiphe alphitoides on Japanese Snailseed (Cocculus trilobus) in Korea.

Japanese snailseed (Cocculus trilobus DC.) has been known as a medicinal herb to treat dieresis, rheumatoid arthritis, and dropsy. In September 2011, severe powdery signs were found on several Japanese snailseed plants near Andong, Korea. Diseased leaves showed chlorotic or necrotic lesions, along with leaf distortion and senescence. Diseased leaves were associated with a fungus that resulted in what appeared to be white colonies, predominately associated with the upper leaf surfaces, and rarely on the lower surfaces. The colonies increased in size and coalesced, subsequently covering the entire surface. The fungus-produced chasmothecia were 92 to 123 µm in diameter, blackish brown, and had a depressed, globose shape. Each chasmothecium had approximately 8 to 12 appendages that were straight to mildly bent, and were four to six times dichotomously branched and often entwined. There were three to six asci per chasmothecium, 38 to 57 × 32 to 43 µm in size, each of which held six to eight ascospores. Conidiophores were single or sometimes two on a hyphal cell, arising from the upper part of mother cells, mostly positioned central, 6.5 to 8 µm with width. Conidiophores were erect and up to 150.5 µm long. Conidia were ellipsoidal or sometimes lemon-shaped. The conidial size was 31.5 to 40 × 19 to 24.5 µm with length/width. These morphological characteristics were identified as being similar to Erysiphe alphitoides (1). DNA was extracted from collected hyphae of infected leaves using the NucleoSpin Tissue Kit (Macherey-Nagel, Duren, Germany). The ITS region of rDNA was amplified using primers ITS4/ITS5 and sequenced (GenBank Accession No. KF734882). The isolate (APEC-F1203) was 99% homologous to other E. alphitoides isolates from oak trees in Japan (AB292704, AB292699, AB292697, and AB292701) and Europe (EF672350, AJ417497). In Korea, this fungus is an oak tree pathogen (2). As proof of pathogenicity, infected leaves having abundant sporulation were pressed onto leaves of five healthy plants. Inoculated and non-inoculated plants were incubated in a moist chamber for 48 h and then maintained in a greenhouse at 15 to 22°C. After 10 to 12 days, powdery mildew colonies developed on inoculated plants. Uninoculated control plants did not show powdery mildew. Microscopic observation of the pathogen growing on the inoculated plants revealed that it was the same as the original fungus. We also observed powdery mildews on oak tree leaves around Japanese snailseed and analyzed their ITS sequences with the above-mentioned methods. As a result, the ITS sequences of powdery mildew pathogens obtained from Japanese snailseed and oak tree were identical. To our knowledge, this is the first report of the presence of E. alphitoides on Japanese snailseed in Korea. This fungus has been reported in association with numerous oak (Quercus spp.) species in Korea, showing that it may be a potential source of inoculum in Japanese snailseed. References: (1) S. Takamatsu et al. British Mycol. Res. 111:809. 2007. (2) S. H. Yu. List of Plant Diseases in Korea, 5th ed. The Korean Society of Plant Pathology, 2009.

First Report of Gray Mold Caused by Botrytis cinerea on Greenhouse-Grown Zucchini in Korea.

In the winter of 2011, greenhouse-grown zucchini (Cucurbita pepo) in Andong City, Korea, showed severe disease symptoms on fruits and dying leaves of zucchini plants that resembled gray mold disease with about 20% yield loss. Symptoms included extensive growth of mycelia and gray conidia on stem and fruit lesions. Lesions expanded rapidly under cool, humid conditions. As the disease progressed, leaves, stems, and fruits became necrotic and were covered by an abundant, soft, gray, sporulating mycelium. Diseased fruit tissue was excised and surface sterilized by immersion in 2% NaOCl for 1 min, placed on PDA (potato dextrose agar), and incubated at 22°C. Fungal colonies were initially white and became gray to brown after 72 h. Analysis of light micrographs showed the presence of elliptical conidia on PDA that was 7.5 to 16.0 µm long and 5 to 10.5 µm wide. In culture, a few, black, small and large irregular sclerotia were produced. Microsclerotia were round, spherical or irregular in shape, and ranged from 1.0 to 3.3 and 1.2 to 3.4 mm (width and length). Conidiophores were slender and branched with enlarged apical cells bearing smooth, ash-colored conidia. These morphological characteristics identified the fungus as Botrytis cinerea (1). The internal transcribed spacer (ITS) region of rDNA was amplified using the ITS1 (forward) and ITS4 (reverse) primer set (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4: 5'-TCCTCCGCTTATTGATATGC-3') and sequenced (2). BLAST analysis of the PCR product showed that the sequence had 100% identity with the nucleotide sequences for B. cinerea. Pathogenicity tests were performed by placing mycelium fragments (1 cm2) of PDA cultures on zucchini fruits. Controls were treated with PDA alone. Five replicates for the inoculated and control plants were used. All fruits were covered with plastic bags and incubated in a growth chamber to maintain 90 to 100% relative humidity at 22°C. Typical symptoms appeared 2 to 6 days after inoculation. The inoculated plants developed typical gray mold symptoms with gray sporulating lesions, while controls remained healthy with no lesions. B. cinerea reisolated from the inoculated tissues was morphologically identical to the original isolates. In a cold outside (below 0°C), wet greenhouse, plants are likely to be exposed to resident Botrytis populations and if the gray mold disease occurs, it can spread on zucchini plants very fast, in 2 days to a week inside a 100 m2 greenhouse. Therefore, gray mold disease could have a significant impact on greenhouse production of zucchini. To our knowledge, this is the first report of B. cinerea causing gray mold of greenhouse-grown zucchini in Korea. References: (1) H. L. Barnett and B. B. Hunter. Illustrated Genera of Imperfect Fungi. Burgess Publishing Company, Minneapolis, MN, 1972. (2) T. J. White et al. PCR Protocols. Academic Press, Inc., New York, 1990.

First Report of Cane and Leaf Rust on Rubus fruticosus Caused by Kuehneola uredinis in Korea.

In September 2011, we observed a rust disease affecting about 90% of the leaves of several Rubus fruticosus L. plants at Andong (36°32'12.4″ N, 128°47'20.1″ E), Korea. Pustule formation occurred on cane surfaces and on the undersurfaces of leaves, with yellowing, reddening, and necrosis on the corresponding upper leaf surface. Leaf distortion and senescence also occurred. For light and scanning electron microscopy, uredinia were detached from leaf lesions using a razor blade and then mounted in water without staining. Leaves with lesions were prepared by gold sputtering and then observed by SEM (Hitachi S-2500C microscope, Japan). We observed the development of uredinial colonies with abundant sporulation. The echinulate urediniospores, which were 16.3 to 19.8 × 17.5 to 25.3 µm, were yellowish or occasionally pale brown, with a globose or subglobose shape (2). Telia were a floccose and pale yellowish-buff or almost white. Teliospores were four- to seven-celled. Cells were trapezoid-cylindric, 15 to 40 × 14 to 24 µm, with hyaline walls. Cell wall surface was smooth but often bearing coronate projections or bumps at the top (in apical cells) or upper rim (in intercalary cells). Cell wall thickness was about 0.5 µm at the sides and 2 to 4 µm at the apex; apical pores occurred on short cell-wall projections. Teliospore pedicels were short (0.17 to 1.34 × 0.78 to 1.45 µm), hyaline, and fragile (3). The pathogen was identified as Kuehneola uredinis L. on the basis of morphology (3), and this identification was confirmed by internal transcribed spacer (ITS) analysis. ITS rDNA of the fungus was amplified using the ITS1 and ITS4 primer set (ITS1: 5'-TCCGTAGGTGAACCTGCGG-3', ITS4: 5'-TCCTCCGCTTATTGATATGC-3'). BLAST analysis of the PCR product showed that the sequence shared 99% identity with the published nucleotide sequences for K. uredinis (Accession No. EU14069). The D1/D2 domain of 28S rDNA was amplified by PCR using the primer pair 5'-ACCCGCTGAAYTTAAGCATAT-3' and 5'-CTTCCTTGGTCCGTGTTTCAAGACGG-3' (1). Phylogenetic analysis using 28S rDNA sequences identified the causal fungus as K. uredinis. The identity of the fungus was confirmed as K. uredinis by DNA sequencing; the sequence was 99% similar to that of other K. uredinis (AF426218, AY745696, DQ354551, and GU058013). To the best of our knowledge, this is the first report of an outbreak of cane and leaf rust on Rubus fruticosus Linné caused by K. uredinis in Korea. References: (1) V. der Auwera et al. FEBS Microbiol. Lett. 338:133, 1994. (2) D. E. Gardner. Plant Dis. 67:963, 1983. (3) G. F. Laundon and A. F. Rainbow. CMI Descriptions of Pathogenic Fungi and Bacteria 202:1, 1969.

First Report of Branch Dieback of Walnut Caused by Neofusicoccum parvum in Korea.

Walnut (Juglans sinensis Dode) is an economically important tree in the world, both for its wood and its fruit. Walnut fruits, as rich sources of omega-3 essential fatty acid, are valuable nutritionally. Consumer interest in Korea for walnuts has increased in recent years, and production has increased to 1,042 ha with the Kyoungbuk region consisting of 402 ha (2). In May 2012, lethal dieback disease of walnut tree was detected in two orchards in Andong, Kyoungbuk region, Korea, each with an incidence of 25 to 30%. Disease symptoms included blight and dieback of the stems, flowing resin, dark decay inside the bark of dead twigs, and defoliation. The bark of dead twigs was removed and sliced thinly using a razor blade, and water-mounted, without staining, for observation of fungal structures, if present. Pycnidia were found embedded within the bark of dead twigs and conidia were mostly characterized by fusoid, hyaline, smooth, thin-walled, unicellular and 16.25 to 21.25 µm long and 4.37 to 6.87 µm wide. These characteristics are consistent with those reported previously for Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (1). Diseased branch tissues collected from the two locations were surface sterilized with 1% NaOCl, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA). The fungal isolates, recovered from the two different orchards, produced white, aerial mycelium and became light gray within a week after incubating plates at 25°C. To confirm the identities of the isolates, the complete internal transcribed spacer (ITS) rDNA of the fungi was amplified and sequenced using PCR. The sequences were compared with other DNA sequences in the GenBank database, using a BLAST search. BLAST analysis of the PCR product showed that the sequence had 99% identity with the nucleotide sequences for N. parvum (JQ411396.1 and GU997688.1). Additionally, the chitin synthase 1 gene was sequenced and analyzed using the BLAST server. The sequence of PCR product had 100% identity with the nucleotide sequences of N. parvum strain CMW9080 chitin synthase 1 gene (EU339501). Thus, both morphological and molecular characters confirmed this species as N. parvum. Pathogenecity tests were performed by inoculating 2-year-old J. sinensis trees. Inoculations consisted of inserting 5-mm-diamter agar plug bearing fresh mycelium of the fungal isolates into the wounds. Within 2 weeks, black lesions appeared on all inoculated plants accompanied by defoliation, whereas no symptoms were observed in the control plants. N. parvum has been reported a member of Botryosphaeriaceae, commonly associated with dieback and cankers of woody plants (1). To our knowledge, this study is the first report of N. parvum as a pathogen of Juglans sinensis in Korea. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) Statistics Korea. Forest Households by Growing Area of Walnut/Total Area. 2010.

First Report of Anthracnose Caused by Colletotrichum gloeosporioides on Malus prunifolia in Korea.

In 2010 and 2011, crab apples in Andong Province, Korea were found with dark brown spots on the fruit and mummified fruit on a tree. The fruit surface had red, circular spots that contained smaller, white spots; the color of the inner spots later changed to brown or black. Eventually, the rotten fruit dried and became mummified. Microscopic examination revealed the presence of acervuli and dark brown-to-black, needle-shaped setae. To isolate potential pathogens from infected fruit, small sections (5 to 10 mm2) were excised from the margins of lesions. These sections were surface sterilized with 70% ethanol and 1% NaOCl for 1 min and then rinsed three times with sterile distilled water. The fungus that was isolated produced whitish mycelia when grown on potato dextrose agar (PDA); the mycelia later became gray to dark gray with aerial mycelia in tufts and numerous conidia were produced. The conidia were straight, cylindrical with an obtuse apex and a truncated base, and measured 11.4 to 17.5 × 4.2 to 7.1 µm. The measurements and taxonomic characteristics coincide with those of Colletotrichum gloeosporioides (Penz.) (1). The isolated fungus was tested for pathogenicity on crab apples and cv. Fuji apples by inoculation with a conidial suspension (105 conidia/ml) prepared from 20-day-old PDA cultures. A 20-µl drop of the conidial suspension was placed onto crab apple and apple fruits that had been wounded by piercing them 1 to 2 mm deep with a pin. Small, dark lesions were observed on the artificially inoculated fruit 3 days after inoculation. Nine days after inoculation, dark lesions with salmon-colored masses of conidia were observed on fruit, which were also soft and sunken. Abundant masses of conidia were produced in the decayed tissues. The fungus was reisolated from the parts of the fruits showing the symptoms. The internal transcribed spacer (ITS) rDNA of the isolated fungus was amplified and sequenced by PCR as described by White et al. (2). The resulting 582-bp of ITS rDNA sequence was deposited in GenBank (Accession No. JQ405742). A BLAST analysis for sequence similarity of the ITS region revealed 100% identity with nucleotide sequences for C. gloeosporioides isolates (Accession Nos. HQ645080 and AB458667). The results obtained on morphological characteristics, pathogenicity, and molecular data corresponded with those of C. gloeosporioides described by Sutton (1). To our knowledge, this is the first report of the presence of C. gloeosporioides on crab apple in Korea (3). Crab apple is used as a pollinator for single-cultivar apple orchards and may become a possible source of inoculum for cultivated apple. References: (1) T. B. Sutton. Compendium of Apple and Pear Diseases. The American Phytopathological Society, St. Paul, MN, 1990. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc., New York, 1990. (3) S. H. Yu. List of Plant Diseases in Korea. 5th ed. (in Korean). The Korean Society of Plant Pathology, 2009.

Regional and annual patterns in respiratory virus co-infection etiologies and antibiotic prescriptions for pediatric mycoplasma pneumoniae pneumonia.

OBJECTIVE: Mycoplasma pneumoniae (M. pneumoniae) pneumonia is the second-most common cause of community-acquired pneumonia (CAP). This study aimed at investigating into the prevalence of macrolide-resistant M. pneumoniae (MRMP) with respiratory virus co-infection and the antibiotic prescriptions in children with CAP in four provinces in Korea, and to assess the variations in the findings across regions and throughout the year. PATIENTS AND METHODS: This prospective study was conducted in 29 hospitals in Korea between July 2018 and June 2020. Among the enrolled 1,063 children with CAP, all 451 patients with M. pneumoniae underwent PCR assays of M. pneumoniae and respiratory viruses, and the presence of point mutations of residues 2063 and 2064 was evaluated. RESULTS: Gwangju-Honam (88.6%) showed the highest prevalence of MRMP pneumonia, while Daejeon-Chungcheong (71.3%) showed the lowest, although the differences in prevalence were not significant (p=0.074). Co-infection of M. pneumoniae pneumonia and respiratory virus was observed in 206 patients (45.4%), and rhinovirus co-infection (101 children; 22.2%) was the most frequent. The prevalence of MRMP pneumonia with respiratory virus co-infection and the antibiotic prescriptions differed significantly among the four provinces (p < 0.05). The monthly rate of MRMP pneumonia cases among all cases of M. pneumoniae pneumonia and tetracycline or quinolone prescriptions did not differ significantly among the four regions (trend p > 0.05) during the study period. CONCLUSIONS: The prevalence of M. pneumoniae pneumonia with virus co-infection and antibiotic prescriptions could differ according to region, although the MRMP pneumonia rate showed no difference within Korea.

Effects of initial inoculation density of Paenibacillus polymyxa on colony formation and starch-hydrolytic activity in relation to root rot in ginseng.

AIMS: To examine the relationships between population growth and biological characters of the plant-growth-promoting rhizobacterium Paenibacillus polymyxa GBR-1. METHODS AND RESULTS: Population growth, colony formation, starch-hydrolytic activity, and ginseng root rot caused by P. polymyxa GBR-1 isolated from a rotten ginseng root were examined in vitro and in vivo at high [1 x 10(8) colony-forming units (CFU) ml(-1)] and low (1 x 10(6) CFU ml(-1)) initial inoculum densities. Paenibacillus polymyxa GBR-1 showed strong starch-hydrolytic activity on modified starch agar with relatively low starch content, but only at certain incubation temperatures (18 and 23 degrees C); the high-density inoculum produced bacterial colonies about nine times thicker than those formed from the lower inoculum density. Light, scanning electron, and transmission electron microscopy showed that the thick colonies from the high-density inoculum were filled with extracellular polymeric substances (EPS), in which a relatively small number of ovoid-rod-shaped bacterial cells (mostly endospore-bearing cells) were distributed. In contrast, the thin colonies from the low-density inoculum were composed of massive vegetative cells with a rectangular rod shape and minimum EPS. Fluorescent in situ hybridization (FISH) revealed that the beta-amylase gene was expressed only in bacterial cells from the thick colonies formed from the high-density inoculum, but not in those from the low-density inoculum. The culture filtrate from the thick colonies produced a hydrolytic clear zone on modified starch agar, degraded starch granules in various manners, and produced rot symptoms on ginseng root tissues. CONCLUSIONS: The biological properties of colony formation, starch hydrolysis, and ginseng tissue rotting by P. polymyxa GBR-1 are interrelated; they are influenced by the initial bacterial population density but not by the in situ and the final population densities. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of disease-inducing characters of P. polymyxa GBR-1 can be used in the development of biocontrol strategies.

Effects of sevoflurane on collagen production and growth factor expression in rats with an excision wound.

BACKGROUND: Sevoflurane is a widely used inhalation anesthetic, but there are no studies on its effect on the wound-healing process. This study was undertaken to evaluate the effect of exposure time to sevoflurane on wound healing. METHOD: Male Sprague-Dawley rats were used. Two circular full-thickness skin defects 8 mm in diameter were made on the dorsum of the rats. The animals were divided into six groups according to exposed gas type and time: S1 (sevoflurane, 1 h), S4 (sevoflurane, 4 h), S8 (sevoflurane, 8 h), O1 (oxygen, 1 h), O4 (oxygen, 4 h), and O8 (oxygen, 8 h). The surface area of the wounds was measured 0, 1, 3, and 7 days after surgery. Separately, the mean blood pressures (MBP) and arterial oxygen pressures (PaO(2)) were monitored during the sevoflurane exposure. Collagen type I production and transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF) expression on the wound surface were analyzed. Routine histological analysis was also performed. RESULT: Exposure duration to sevoflurane had no influence on MBP and PaO(2). The reduction in wound size and collagen type I production was delayed in S8. The expression of TGF-beta1 and bFGF on the wound surface in S8 was significantly attenuated in S8. The histology of the S8 demonstrated a delayed healing status. CONCLUSIONS: Prolonged exposure to sevoflurane might alter the inflammatory phase of the wound-healing process by attenuation of growth factor expression such as TGF-beta1 and bFGF and subsequently by reduced collagen production.

Solution structure of the activator contact domain of the RNA polymerase alpha subunit.

The structure of the carboxyl-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alpha CTD), which is regarded as the contact site for transcription activator proteins and for the promoter UP element, was determined by nuclear magnetic resonance spectroscopy. Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins. The UP element binding site was found on the surface comprising helix 1, the amino-terminal end of helix 4, and the preceding loop. Mutation experiments indicated that the contact sites for transcription activator proteins are also on the same surface.

A plant growth promoting rhizobacterium, Paenibacillus polymyxa strain GBR-1, suppresses root-knot nematode.

Exposure of root-knot nematode, Meloidogyne incognita to various concentrations (5-100%) of culture filtrate of Paenibacillus polymyxa GBR-1 under in vitro conditions significantly reduced egg hatch and caused substantial mortality of its juveniles. The increase in the exposure durations of juveniles to culture filtrate and its concentrations increased the mortality rate. Similarly, higher concentrations increased its inhibitory effect on egg hatch. In higher concentrations (25-100%) egg hatch was inhibited by 84-91% after 2 days of exposures as compared to control in sterile distilled water. Application of various concentrations of culture filtrate extract or bacterial suspension of P. polymyxa GBR-1 into potting soil infested with 2000 J2 of M. incognita, reduced the root galling and nematode populations and increased tomato plant growth and root-mass production compared with untreated control (P< or = 0.05). The beneficial effect of P. polymyxa GBR-1 into potted soil increased exponentially with the increase in dose concentrations. Root gall index was reduced from 4.8 to 1.4 and 1.8 when potting soil was treated with 10% concentrations of culture filtrate extract and bacterial suspension, respectively, compared with untreated control. Application of bacterial suspension of P. polymyxa GBR-1 into potted soil at 3 day pre-inoculation of nematode was the most effective followed by simultaneously and at 2 days post-inoculation; as root galling was reduced by 62.5%, 58.3% and 50.0%, respectively, compared with untreated control.

Flexible linker in the RNA polymerase alpha subunit facilitates the independent motion of the C-terminal activator contact domain.

The dynamic properties of the C-terminal one-third of the alpha subunit of RNA polymerase were investigated. The intact alpha subunit exhibited almost the same NMR spectral pattern as the isolated C-terminal fragment, indicating that the C-terminal domain retains the same conformation as the isolated fragment, and that its motion is independent of that of the associated N-terminal domain. Analysis of the NMR dynamics data for the intact alpha subunit indicated that at least 13 residues between the N and C-terminal domains show distinctly higher motional flexibility than the structured parts. This flexible linker may endow the C-terminal domain with locational freedom in different kinds of initiation complex. The dynamics data also revealed that the residues in the contact site for DNA and transcription factors exhibited higher mobility than other secondary structural elements.

Suppressed cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with Toxoplasma gondii tachyzoites.

Mechanisms of host immunosuppression after infection with Toxoplasma gondii are unclear. This study was performed to observe cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with live, nonreplicating (irradiated) RH tachyzoites on stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS). For lymphocyte cultivation, 3 groups were prepared: coculture with live nonirradiated tachyzoites separated by a transwell (group T), live irradiated tachyzoites without a transwell (group R), and no tachyzoites (group C). Compared with group T, groups R and C, on stimulation with Con A, revealed significantly (P < 0.05) lower levels of interleukin-2 (IL-2) and IFN-gamma, but not IL-10. The levels of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were also significantly (P < 0.05) lower in groups R and C than in group T after stimulation with LPS. The results suggest that intracellular infection of murine splenic lymphocytes with T. gondii tachyzoites could impair their capacity to produce cytokine and immunoglobulin secretions.