Analyzing endodontic infections by deep coverage pyrosequencing.

Bacterial diversity in endodontic infections has not been sufficiently studied. The use of modern pyrosequencing technology should allow for more comprehensive analysis than traditional Sanger sequencing. This study investigated bacterial diversity in endodontic infections through taxonomic classification based on 16S rRNA gene sequences generated by 454 GS-FLX pyrosequencing and conventional Sanger capillary sequencing technologies. Sequencings were performed on 7 specimens from endodontic infections. On average, 47 vs. 28,590 sequences were obtained per sample for Sanger sequencing vs. pyrosequencing, representing a 600-fold difference in "depth-of-coverage". Based on Ribosomal Database Project (RDP II) Classifier analysis, pyrosequencing identified 179 bacterial genera in 13 phyla, which was significantly more than Sanger sequencing. The phylum Bacteroidetes was the most prevalent bacterial phylum. These results indicate that bacterial communities in endodontic infections are more diverse than previously demonstrated. In addition, deep-coverage pyrosequencing of the 16S rRNA gene revealed low-abundance micro-organisms with potential clinical implications.

First Report of Fruit Basal Rot by Ceratocystis paradoxa on Coconut in Taiwan.

Since 2005, coconut fruits (Cocos nucifera L.) harvested from trees in southern Taiwan have shown symptoms of a fruit rot disease. The disease occurs only on harvested coconut fruits and is more serious during warmer seasons. Principal symptoms are blackening of the exocarps, mesocarps, and then the endocarps of coconut fruits from pedicel end or wounds on fruits. A fungus was consistently recovered from diseased fruits. Colonies on potato dextrose agar (PDA) were white, becoming black 1 to 2 days later, with a strong fruity aroma. The fungus produced two asexual spores, including phialospore-type conidia, which were hyaline to mid brown, cylindrical to somewhat oval and thick walled when mature, 8.5 to 16.0 × 4.7 to 6.7 µm, released from phialides measuring 80.0 to 155.0 × 5.0 to 7.8 µm, and chlamydospores, which were oval, black, 13.4 to 25.0 × 8.9 to 12.5 µm, and formed in short chains from specialized hyphal tips. Perithecia were produced after 2 months by mating isolates on coconut exocarp placed on PDA. Perithecia were brown, globose, 280 µm in diameter, with a 1,100-µm long neck. Ascospores generally were ellipsoid, but some were unequally bent, 12 × 3 µm. The temperature for hyphal growth of this fungus was 25 to 30°C, with a growth rate of 3.3 cm per day. It did not grow at temperatures lower than 10°C or higher than 35°C. On the basis of these characteristics, this fungus was identified as Ceratocystis paradoxa (Dade) C. Moreau (anamorph Thielaviopsis paradoxa (de Seynes) Höhn) (1). The internal transcribed spacer (ITS) region of mycelial DNA of two isolates was amplified with ITS5 and ITS4 primers and the PCR products were sequenced (GenBank Accession Nos. GU358207 and GU358206). BLAST analysis of both sequences showed 99% sequence similarity with C. paradoxa strain WIN(M) 925 (Accession No. DQ318203). Two of the isolates (BCRC 34425 and BCRC 34426) were deposited in the Bioresource Collection and Research Center, Hsinchu, Taiwan. Twenty-four detached healthy coconut fruits were inoculated by placing spore suspensions (1 × 105 spores/ml) of eight wild type isolates onto wounds created by removing the calyx. Symptoms similar to those observed in the field developed on inoculated fruits after incubating at room temperature for 10 days, but did not develop on six fruits that were wounded but not inoculated. The same pathogen was reisolated from the inoculated fruits but not the control fruits. C. paradoxa has been reported as causing disease of coconut fruits in Brazil (2), but to our knowledge, this is the first report of the disease in Taiwan. References: (1) G. Morgan-Jones. No. 143 in: CMI Descriptions of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Surrey, England, 1967. (2) V. Rossetti. "Crostas Pretas" das folhas e "Podridão Basal" dos frutos de coqueiro. O Biológico 21:54, 1955.

First Report of Rhizoctonia Blight of a Coastal Redwood Tissue-Culture-Derived Saplings Caused by Rhizoctonia solani AG-IV in Taiwan.

Coastal redwood (Sequoia sempervirens (D. Don) Endl.) is native to North America. This tall tree species is used for forestation and lumber; its wood is also used for furniture, its burls for art ware, and its bark for fuel, insulation, and mulch. In August 2005, an instance of wilt was observed among 2-year-old tissue-culture-cloned plants (2) in the Sitou Forest of central Taiwan. Essentially, all plants were infected. The leaves or stems near the ground were affected first, but the wilt soon spread over the entire plant with the leaves becoming grayish brown and water soaked, and then wilting, drying, and finally defoliation occurred. Aerial hyphae were present over the affected areas, aerial mycelium was cob-web-like, hyaline, later becoming slightly brown. Hyphae were 6.5 to 10.4 µm wide with right-angle branching and septal constriction at their bases. Sclerotia were hemispherical, subglobose, to irregular in shape, 1 to 2 mm, and brown. The perfect stage of the fungus was not found. The fungus was identified as Rhizoctonia solani Kühn (3). Vegetative cells were stained with alkaline safranin solution and identified as multinucleate (1). Portions of the stem that displayed symptoms, together with adjacent healthy tissue, were disinfested for 1 min in 0.5% NaOCl and plated on to potato-dextrose agar (PDA) (Merck, Darmstadt, Germany) supplemented with 100 mg/L of ampicillin (Sigma, St. Louis, MO). Single hyphal tips were transferred to PDA and two isolates were established as pure cultures. On the basis of hyphal anastomosis with AG-IV tester isolates (exfop234, exfop241, and exfop250) (1), the fungus was identifed as R. solani AG-IV. Pathogenicity of the fungal isolates was confirmed by inoculating 2-month-old tissue-culture-derived S. sempervirens plants that were grown in pots and incubated in a growth chamber maintained at 28°C with a relative humidity above 95%. Inoculum consisted of a single mycelial 5-day-old 0.5-cm disc grown on PDA of the pathogen placed on the soil surface touching the base of each plant. Four plants were inoculated with mycelium and the four control plants were noninoculated. Inoculated plants wilted gradually over 4 days and all plants developed severe stem rot and were dead in 6 days, whereas control plants remained symptomless. The Rhizoctonia solani AG-IV was reisolated from all inoculated plants. This fungus has been observed to cause disease in many species of plants (4), but to our knowledge, this is the first report of Rhizoctonia blight of coastal redwood tissue-culture-derived saplings caused by Rhizoctonia solani AG-IV in Taiwan. References: (1) T. T. Chang. Taiwan J. For. Sci. 12:47, 1997. (2) L. C. Huang et al. Plant Physiol. 98:166, 1992. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society, St. Paul, MN, 1991. (4) S. T. Su et al. List of Plant Diseases in Taiwan. The Phytopathological Society of the Republic of China, 2002.

First Report of Southern Blight of Silvery Messerschmidia Seedlings in Taiwan.

Silvery messerschmidia, Messerschmidia argentea (L.) Johnston, of the Boraginaceae, is indigenous to Taiwan and grown as an ornamental, for windbreaks, or as a shade tree. During the summer of 2005, a sudden wilt of 1-year-old plants was observed in a nursery in central Taiwan. Initial symptoms included stem necrosis at the collar, leaf yellowing, and tan discoloration of leaves. As stem necrosis progressed, infected plants wilted, defoliated, and died. Necrotic tissues were covered with whitish mycelium with clamp connections that formed reddish brown spherical (1 to 2.2 mm in diameter) sclerotia. A fungus was consistently recovered from the interface of diseased and healthy stem tissue, disinfested for 1 min in 0.5% NaOCl, and plated on Difco (Sparks, MD) potato dextrose agar (PDA) amended with 100 ppm of ampicillin. Pure cultures were prepared by transferring single hyphal tips to PDA, and Sclerotium rolfsii (Sacc.) was identified (1). Pathogenicity of two S. rolfsii isolates was confirmed by inoculating 3-month-old silvery messerschmidia seedlings grown in pots. Inoculum consisted of a single agar disk of a 7-day-old culture used per pot or a single sclerotium produced in 10 days on PDA and added per pot. Both the mycelium on the 0.5-cm-diameter agar plug and the sclerotium touched the base of the plant stem. Four plants were inoculated with mycelia, four with sclerotia, and four were noninoculated controls. All plants were kept in a growth chamber at 25 to 35°C with relative humidity of more than 95%. Initially, the basal stems were covered by whitish mycelia growth with a fanlike pattern from the inoculum, and brown, water-soaked necrotic lesions developed near the soil line. Inoculated plants developed symptoms within 4 days, wilted gradually in 7 days, and all were eventually killed in 11 days. Plants inoculated with sclerotia developed disease at a slower rate and control plants remained symptomless. Sclerotia developed on diseased tissues and S. rolfsii was reisolated. This disease has been observed on many species of plants (2), but to our knowledge, this is the first report of southern blight of silvery messerschmidia seedlings caused by S. rolfsii in Taiwan. References: (1) R. K. Jones and D. M. Benson, eds. Diseases of Woody Ornamentals and Trees in Nurseries. The American Phytopathological Society, St. Paul, MN, 2001. (2) Y. P. Tsai, ed. List of Plant Diseases in Taiwan. The Plant Protection Society of the Republic of China and The Phytopathological Society of the Republic of China, 1991.

First Report of White Root Rot of Japanese Serissa Caused by Rosellinia necatrix in Taiwan.

Japanese serissa (Serissa japonica (Thunb.) Thunb.) is a very popular ornamental in Taiwan. During the summer of 2005, serissa plants in a central Taiwan nursery had decaying roots, leaf yellowing, and were wilting. Wilted plants had white fan-like mycelium under the bark. The disease caused 70% loss of seedlings at the nursery. Associated synnemata were rigid, erect, dark, setaceous, 0.8 to 2.1 mm long, 90 to 200 µm wide, and tapering to enlarged whitish gray heads composed of geniculate conidiophores and conidia. Conidia were 3.1 to 5.8 × 2.6 to 3.4 µm, unicellular, hyaline, and subglobal with a truncated base. Root rots were washed, disinfested for 1 min in 0.5% NaOCl, cut into 3 mm3 pieces, plated on Merck (Darmstadt, Germany) potato dextrose agar (PDA) amended with 100 ppm of ampicillin (Sigma, St. Louis, MO), and incubated at 24°C in the dark until hyphae emerged. Single hyphal tips were transferred to PDA, and two isolates were established as pure cultures. Mycelia were cut, stained with 1% cotton blue in lactophenol, and pear-shaped hyphal swellings adjacent to the septa were observed. According to these hyphal, synnematal, and conidial characteristics, the fungus was identified as Dematophora necatrix Hartig, the anamorph of Rosellinia necatrix Prill. Inoculum for pathogenicity tests were produced on oat-wheat medium composed of 20 ml of oat grain and 20 ml of wheat grain, mixed, and soaked in water for 3 h. The grains were placed in 200-ml flasks, autoclaved at 121°C for 30 min, inoculated with two isolates of D. necatrix separately, and grown for 14 days. Six 3-month-old Japanese serissa seedlings were grown in pots. The grain inoculum was added to unsterilized field soil and the plants were transplanted into this mix. Control plants were transplanted into a similar mix without the inoculum. Two replicates were used for a total of 24 inoculated plants and 24 control plants. All plants were kept in a growth chamber at 25 to 35°C with 20 min of irrigation per day, 12 h of irradiation, and relative humidity at more than 95%. Inoculated plants developed root rots after 1 month, and after 4 months, all plants were dead, while control plants remained healthy. D. necatrix was reisolated, hyphal characteristics confirmed, and synnemata were observed on collars of dead plants. The teleomorph was not formed by our cultures, and the identification of Rosellinia necatrix was confirmed by molecular studies. The nuclear ribosomal internal transcribed spacer (ITS) amplified with two primers, ITS1 and ITS4, from our representative isolate demonstrated 99.63, 99.81, and 99.27% similarity to two R. necatrix isolates from Japan and one R. necatrix isolate from Italy, respectively. This disease has been reported on many species of plants (1), but to our knowledge, this is the first report of white root rot of Japanese serissa seedlings caused by R. necatrix in Taiwan. Reference: (1) S. T. Su et al. List of Plant Diseases in Taiwan. The Phytopathological Society of the Republic of China, 2002.

First Report of Southern blight of Formosan Michelia Seedlings in Taiwan.

Formosan michelia (Michelia compressa (Maxim.) Sargent) is a native, giant evergreen tree in Taiwan. This species is used for afforestation, provision of shade in domestic situations, used in parks, and also for furniture. During the summer of 2004, a sudden wilt of 1-year-old plants was observed in a nursery of northern Taiwan. Initial symptoms included stem necrosis at the soil line, yellowing, and tan discoloration of leaves. As stem necrosis progressed, infected plants wilted and died. Necrotic tissues were covered with whitish mycelium that differentiated into reddish brown spherical (1 to 2 mm in diameter) sclerotia. Sclerotium rolfsii was consistently recovered from the surface of symptomatic stem sections that were disinfected for 1 min in 0.5% NaOCl and then plated on potato-dextrose agar amended with 100 ppm ampicillin. Pathogenicity of two S. rolfsii isolates was confirmed by inoculating 1-year-old Formosan michelia seedlings grown in pots. Inoculum consisted of mycelium and sclerotia of the pathogen placed on the soil surface around the base of each plant. Noninoculated plants served as controls. All plants were kept in a growth chamber at 25 to 35°C with relative humidity >95%. Inoculated plants developed symptoms within 14 days while control plants remained symptomless. Sclerotia developed on infected tissues, and S. rolfsii was reisolated from symptomatic tissues. This disease has been observed on many species of plants (1), but to our knowledge, this is the first report of southern blight of Formosan michelia seedlings caused by S. rolfsii in Taiwan. Reference: (1) Y. P. Tsai, ed. List of Plant Diseases in Taiwan. The Plant Protection Society of the Republic of China and The Phytopathological Society of the Republic of China, 1991.

First Report of Anthracnose Caused by Colletotrichum gloeosporioides on Taxus mairei in Taiwan.

Taxus mairei (Lemee & Levl.) S.Y. Hu ex Liu is a giant evergreen tree native to Taiwan. T. mairei and the Pacific yew, T. brevifolia, produce taxol, a highly effective antitumor drug. Anthracnose was observed on cuttings and seedlings of T. mairei in nurseries and on larger plants grown in plantations in Taiwan. Circular or irregular, brown leaf spots were associated with defoliation. Stems lesions and tip dieback were also observed. Colletotrichum gloeosporioides (Penz.) Sacc. was isolated from diseased tissues, and this fungus grew well on potato dextrose agar and malt extract agar (MEA), with a growth rate of 6.3 mm per day on MEA at 32°C in the dark. Colonies were white to grayish white and became dark gray with age. Acervuli produced on leaves were 115 to 155 µm in diameter, with one or several brown-to-black setae and pale salmon conidial masses. Conidia were guttulate, straight, cylindrical, obtuse at the apex and truncate at the base, and approximately 12 to 17 × 3.5 to 6.0 µm. The disease was reproduced by spraying T. mairei seedlings with a suspension of 104 to 105 conidia per ml, and the control plants were inoculated with distilled water. Inoculated plants were kept in a transparent moist chamber with a constant humidity near 100% (1). Symptoms appeared within 7 days when the temperature was over 32°C. When the temperature was below 24°C, symptoms were delayed. The fungus was reisolated from the inoculated plants, fulfilling Koch's postulates. To our knowledge, this is the first record of anthracnose on Taxus mairei (2), and anthracnose appears to be the most important disease on this plant in Taiwan. Voucher specimens have been deposited at the Division of Forest Protection, Taiwan Forest Research Institute as TFRIFCH Herbarium specimen 137. References: (1) W. W. Hsiao et al. Taiwan J. For. Sci.17:119, 2002. (2) Y. P. Tsai, ed. List of plant diseases in Taiwan. The Plant Protection Society of the Republic of China and The Phytopathological Society of the Republic of China, 1991.

First Report of Zonate Leaf Spot of Artocarpus altilis Caused by Cristulariella moricola in Taiwan.

Breadfruit (Artocarpus altilis (Parkinson) Fosberg) is an important landscape and garden tree in Taiwan. During the spring of 2002, zonate leaf spots of breadfruit were observed at a Taipei nursery in northern Taiwan. Initially, several small, brown, zonate lesions developed on leaves. As lesions enlarged, they coalesced, leading to blighting of leaves and premature defoliation. Sporophores on the host were generally hypophyllous but sometimes amphigenous, solitary, erect, easily detachable, and as much as 850 µm long. The upper portion of the sporophore is considered an individual conidium and consisted of a pyramidal head that was fusiform to ventricose and cristulate, 495 to 534 µm long and 210 to 290 µm wide at the broadest point. Branches within the pyramidal head were short and compact, and dichotomously or trichotomously branched. The conidia were hyaline, broad, septate, tapering toward an acute apex, and sometimes constricted at the basal septum. Conidiophores were 400 to 680 × 20 to 100 µm. The fungus was isolated from infected tissue and maintained on potato dextrose agar (PDA). Sclerotia were produced on PDA after 4 to 5 weeks at 20°C without light, but conidia were not observed in culture. The fungus was identified as Cristulariella moricola (Hino) Redhead based on morphological characteristics (1,2). To complete Koch's postulates, three sporophores from infected leaves or three sclerotia from cultures were placed individually on each of 10 breadfruit leaves. The plants were placed in plastic bags and incubated at 16 to 20°C. Symptoms were observed after 2 to 3 days on 100% of plants inoculated with sporophores and after 6 days on 50% of plants inoculated with sclerotia. The pathogen was reisolated from lesions on plants inoculated with sporophores and sclerotia. No symptoms were observed on the control plants. C. moricola has been known to cause a bull's eye or zonate leaf spot and defoliation on woody and annual plants, including at least 51 host species and 36 families distributed in the central and eastern United States (1). To our knowledge, this is the first report of zonate leaf spot and defoliation of breadfruit caused by C. moricola. References: (1) T. T. Chang. Bull. Taiwan For. Res. Inst. New Ser. 10 (2):235, 1995. (2) S. A. Redhead. Can. J. Bot. 53:700, 1975.

Cycloartane glycosides from Cimicifuga dahurica.

A new cycloartane bisdesmoside and two new trinorcycloartane glycosides, along with four known cycloartane compounds, were isolated from the rhizomes of Cimicifuga dahurica (Ranunculaceae). The structures of the new compounds were elucidated as 3-O-alpha-L-arabinopyranosyl cimigenol 15-O-beta-D-glucopyranoside, 24-hydroxy-12beta-acetoxy-25,26,27-trinorcycloartan-16,23-dione 3beta-O-alpha-L-arabinopyranoside, and 16alpha,24alpha-dihydroxy-12beta acetoxy-25,26,27-trinor-16,24-cyclocycloartan-23-one 3beta-O-alpha-L-arabinopyranoside by extensive NMR methods, FAB-MS, and hydrolysis.

Expression of four protein kinase C isoforms in rat fibroblasts. Differential alterations in ras-, src-, and fos-transformed cells.

In the accompanying study (Borner, C.B., Guadagno, S. N., and Weinstein, I. B. (1992) J. Biol. Chem. 267, 12892-12899) we found that R6 embryo fibroblasts express four isoforms of PKC, cPKC alpha, nPKC epsilon, nPKC delta, and nPKC zeta whose subcellular distribution, activation, and down-regulation are differentially regulated. Furthermore, we demonstrated that overproduction of an exogenous cPKC beta I isoform in these cells (R6-PKC3) altered the TPA-induced down-regulation of nPKC delta and nPKC epsilon. In this paper we show that transformation of R6 or R6-PKC3 cells with a variety of different oncogenes results in differential alterations in expression of individual PKC isoforms. R6 or R6-PKC3 cells transformed by an activated c-H-ras oncogene displayed a marked increase in the expression of both cPKC alpha and nPKC delta, decreased expression of nPKC epsilon, and no change in the expression of nPKC zeta. These alterations occurred at both the mRNA and protein levels but did not significantly affect the subcellular distribution of any of the four isoforms. Studies using actinomycin D and nuclear run-off assays indicated that the increased expression of cPKC alpha in ras-transformed cells was due to increased de novo transcription rather than increased mRNA stability. Qualitatively similar, but less extensive changes in the expression of the four PKC isoforms were seen in v-fos-transformed R6-PKC3 cells. Decreased expression of nPKC epsilon was also seen in the v-src-transformed R6- and R6-PKC3 lines; however, the cellular level of cPKC beta I appeared to be a limiting factor in mediating the effects of v-src on the increased expression of cPKC alpha and nPKC delta. Interestingly, no major changes in the levels of expression of any of the four PKC isoforms were found when R6 cells were transformed by myc, neu/erb-B2, or mos oncogenes. These results demonstrate that transformation of R6 cells by the oncogenes ras, src, and fos differentially alter the expression of three isoforms of PKC in the same host cell, and they suggest that individual isoforms may play distinct roles in mediating cellular transformation by specific oncogenes.