Molecular subtyping of clinical isolates of <Emphasis Type="Italic">Candida albicans</Emphasis> and identification of <Emphasis Type="Italic">Candida dubliniensis</Emphasis>in Malaysia

The genotypes of 221 recent isolates of Candida albicans from various clinical specimens of 213 patients admitted to the University Malaya Medical Centre, Malaysia was determined based on the amplification of a transposable intron region in the 25 S rRNA gene. The analyses of 178 C. albicansisolated from nonsterile clinical specimens showed that they could be classified into three genotypes: genotype A (138 isolates), genotype B (38 isolates) and genotype C (2 isolates). The genotyping of 43 clinical isolates from sterile specimens showed that they belonged to genotype A (29 isolates), genotype B (10 isolates), genotype C (2 isolates) and genotype D (2 isolates). The overall distribution of C. albicans genotypes in sterile and nonsterile specimens appeared similar, with genotype A being the most predominant type. This study reported the identification of C. dubliniensis (genotype D) in 2 HIV-negative patients with systemic candidiasis, which were missed by the routine mycological procedure. The study demonstrated the genetic diversity of clinical isolates of C. albicans in Malaysia.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Biofilm formation and adhesive/invasive properties of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in comparison with <Emphasis Type="Italic">Candida albicans</Emphasis>

Candida dubliniensis and Candida albicans are closely related spp. exhibiting differences in their virulence potency. This study compared clinical isolates of C. dubliniensis with C. albicans from HIV patients with oropharyngeal candidiasis (OPC) and standard strains in power to form biofilm and their adhesive and invasive properties. Members of both spp. were able to form strong biofilms. However, SEM microscopy confirmed that C. albicans undergoes the more effective yeast-to-hyphae transition than C. dubliniensis with prevalent yeast form and limited ability to form filaments. Kinetic patterns indicated that while the first 30 min are critical for sufficient attachment to a polystyrene surface, adhesion to human carcinoma cell lines (Caco-2 and TR 146) needs additional time with maximal saturation observed at 240 min for both spp. The invasion process was tested on 3D RHE (reconstituted human epithelium) with Caco-2 or TR 146 on the collagen surface. C. albicans rapidly produced hyphae that penetrated the tissue layer, demonstrating substantive invasion within 21 h. In contrast, C. dubliniensis attached to the tissue surface and proliferated, suggesting the formation of a biofilm-like structure. After 21 h, C. dubliniensis was able to penetrate the RHE layer and invade unusually, with a cluster of the yeast cells.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

The first clinical isolates of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> in Slovakia

Candida dubliniensis, yeast closely related to Candida albicans, is a new pathogen associated mainly with infections of immunocompromised hosts. In this study, we report the first isolation of three isolates of C. dubliniensis in Slovakia. The first selection of both C. albicans and C. dubliniensis from the other Candida species was done on the basis of specific green color of primoculture grown on CHROMagar Candida. The presumptive identification was completed by supplemental tests: germ-tube formation, production of chlamydospores, ability or inability to grow at 42 and 45,°C and by commercial set API 20C AUX. Parallely, the discrimination between both species was performed by PCR assay using primers specific for Candida dubliniensis     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Funastrum rupicola</Emphasis> (<Emphasis Type="Italic">Apocynaceae:</Emphasis><Emphasis Type="Italic">Asclepiadoideae</Emphasis>), a new species from Bolivia

Summary   Funastrum rupicola Goyder, a new species of Apocynaceae: Asclepiadoideae from Bolivia, is described and illustrated. The conservation status of this species is assessed.     

<Emphasis Type="Italic">Xanthium italicum</Emphasis>, <Emphasis Type="Italic">Xanthium strumarium</Emphasis> and <Emphasis Type="Italic">Arctium lappa</Emphasis> as new hosts for <Emphasis Type="Italic">Diaporthe helianthi</Emphasis>

Sunflower (Helianthus annuus) stem canker caused by Diaporthe helianthi is one of the most important sunflower diseases in Croatia. Until recently, sunflower was the only known host for D. helianthi. In our research carried out in the area of Eastern Croatia, isolates of Diaporthe/Phomospis were collected from Xanthium italicum, X. strumarium and Arctium lappa. Using morphological, cultural and molecular ITS rDNA data, isolates from these weeds were identified as D. helianthi. The following isolates were used in the pathogenicity test: one isolate originated from sunflower (Su5/04), three from X. italicum (Xa2, Xa3 and Xa5), two from X. strumarium (Xa9 and Xa12), one from Xanthium sp. (Xa13) and one from A. lappa (Ar3). According to the results, it was determined that isolate Xa5 (originated from X. italicum) was the most pathogenic to sunflower stems. The average length of the lesion was 11.3 cm. The lowest level of pathogenicity was found in Xa9 (isolated from X. strumarium). The length of the lesion was 0.1 cm.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Amanita concentrica</Emphasis>: a new species in <Emphasis Type="Italic">Amanita</Emphasis> section <Emphasis Type="Italic">Amanita</Emphasis> from Japan

Amanita concentrica sp. nov. is described from an evergreen broad-leaved forest of Chiba Prefecture with Castanopsis cuspidata var. sieboldii and Quercus glauca. The fungus is a medium to large, white to yellowish-white mushroom characterized by many pyramidal warts on pileus, striate margin of pileus, distinct ascending concen-tric rings at upper part of stipe bulb, and inamyloid basidiospores. It is considered to belong to the section Amanita. Received: March 30, 2001 / Accepted: October 1, 2001     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Elaeocarpus</Emphasis> for <Emphasis Type="Italic">Flora Malesiana</Emphasis>: new taxa and understanding in the <Emphasis Type="Italic">Ganitrus</Emphasis> group

The Ganitrus group of Elaeocarpus L. (incl. Elaeocarpus Sect. Ptilanthus Schltr.) as found in Malesia is examined and some understanding reached. The group is more complex and speciose in eastern Malesia, especially in Papuasia (with the Philippines, Sulawesi and Maluku somewhat less rich) than in western and central Malesia. E. angustifolius Blume (syn. E. ganitrus & E. sphaericus auctt. mult.) — the source of the ornamental beads known as rudraksha of eastern commerce, from which malas or rosaries are made — is considered to be very variable in Malesia and probably varies further in the extremes of its range; outside Malesia the Ganitrus group is represented by ‘E. angustifolius s.l.’ only. The following species are recognised: E. altisectus Schltr. with a new subsp. carrii Coode from New Guinea; E. angustifolius Blume; E. avium Coode sp. nov. from New Guinea; E. buderi Coode; E. dolichostylus Schltr. with E. chloranthus treated as var. chloranthus (A. C. Sm.) Coode from New Guinea, with a new var. elliptifolius Coode from the Talaud/Sangihe Is. and a new var. hentyi Coode (replacing and re-interpreting subsp. collinus Coode) from New Guinea; E. fulvus Elmer; E. kaniensis Schltr.; E. ornatus Coode sp. nov. from New Guinea; E. osiae Coode sp. nov. from Bougainville I.; E. ptilanthus Schltr.; E. ramiflorus Merr. and E. trichopetalus Merr. & Quisumb. — a total of 12 species although the last two are perhaps not truly distinct from E. angustifolius. The stone sculpturing is discussed and some useful characters revealed. Attention is drawn to a collection from mid-altitude in Papua New Guinea, inadequately known but probably a new species in this group.     

A new methanol assimilating yeast, <Emphasis Type="Italic">Ogataea</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>, the ascosporic state of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>

Ogataea parapolymorpha sp. n. (NRRL YB-1982, CBS 12304, type strain), the ascosporic state of Candida parapolymorpha, is described. The species appears homothallic, assimilates methanol as is typical of most Ogataea species and forms hat-shaped ascospores in asci that become deliquescent. O. parapolymorpha is closely related to Ogataea angusta and Ogataea polymorpha. The three species can be resolved from gene sequence analyses but are unresolved from fermentation and growth reactions that are typically used for yeast identification. On the basis of multiple isolates, O. angusta is known only from California, USA, in association with Drosophila and Aulacigaster flies, O. parapolymorpha is predominantly associated with insect frass from trees in the eastern USA but O. polymorpha has been isolated from various substrates in the USA, Brazil, Spain and Costa Rica.     

Comparative Study of the Effects of Fluconazole and Voriconazole on <Emphasis Type="Italic">Candida glabrata</Emphasis>, <Emphasis Type="Italic">Candida parapsilosis</Emphasis> and <Emphasis Type="Italic">Candida rugosa</Emphasis> Biofilms

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC₅₀ of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.     

<Emphasis Type="Italic">Phuphanochloa,</Emphasis> a new bamboo genus (<Emphasis Type="Italic">Poaceae</Emphasis>: <Emphasis Type="Italic">Bambusoideae</Emphasis>) from Thailand

Summary  A new monotypic bamboo genus Phuphanochloa (Poaceae: Bambusoideae) from north-eastern Thailand is described, together with a new species, P. speciosa.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Transformation of Industrialized Strain <Emphasis Type="Italic">Candida glycerinogenes</Emphasis> with Resistant Gene <Emphasis Type="Italic">zeocin</Emphasis> via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Candida glycerinogenes WL2002-5 has a modest sugar tolerance and an extremely high glycerol productivity. Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, into the nuclear genome of a wide variety of host cells. In this study, we constructed the plasmid pZR and transferred it into A. tumefaciens LBA4404 to form the strain LBA4404-ZR. LBA4404-ZR was cocultivated with C. glycerologenesis, and putative transformants were identified by selection for zeocin resistance. Polymerase chain reaction and Southern blot analysis confirmed that the gene zeocin was integrated into the genome of engineered C. glycerologenesis. Optimization of the transformation condition was performed in darkness at 25 degrees C on induction medium for 24 h by cocultivation of C. glycerinogenes and LBA4404-ZR with a cell ratio of 1:500-1000. The transformation efficiency reached 2 transformants per 10(4) C. glycerologenesis cells. Our results demonstrated that A. tumefaciens-mediated transformation can be used for C. glycerinogenes. This transformation system can provide the basis for research of C. glycerologenesis in the future.     

<Emphasis Type="Italic">Pm37</Emphasis>, a new broadly effective powdery mildew resistance gene from <Emphasis Type="Italic">Triticum </Emphasis><Emphasis Type="Italic">timopheevii</Emphasis>

Powdery mildew is an important foliar disease in wheat, especially in areas with a cool or maritime climate. A dominant powdery mildew resistance gene transferred to the hexaploid germplasm line NC99BGTAG11 from T. timopheevii subsp. armeniacum was mapped distally on the long arm of chromosome 7A. Differential reactions were observed between the resistance gene in NC99BGTAG11 and the alleles of the Pm1 locus that is also located on chromosome arm 7AL. Observed segregation in F_2:3 lines from the cross NC99BGTAG11 × Axminster (Pm1a) demonstrate that germplasm line NC99BGTAG11 carries a novel powdery mildew resistance gene, which is now designated as Pm37. This new gene is highly effective against all powdery mildew isolates tested so far. Analyses of the population with molecular markers indicate that Pm37 is located 16 cM proximal to the Pm1 complex. Simple sequence repeat (SSR) markers Xgwm332 and Xwmc790 were located 0.5 cM proximal and distal, respectively, to Pm37. In order to identify new markers in the region, wheat expressed sequence tags (ESTs) located in the distal 10% of 7AL that were orthologous to sequences from chromosome 6 of rice were targeted. The two new EST-derived STS markers were located distal to Pm37 and one marker was closely linked to the Pm1a region. These new markers can be used in marker-assisted selection schemes to develop wheat cultivars with pyramids of powdery mildew resistance genes, including combinations of Pm37 in coupling linkage with alleles of the Pm1 locus.