<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus<Emphasis Type="Italic"> Beauveria bassiana</Emphasis>

Agrobacterium tumefaciens was used to stably transform the entomopathogenic deuteromycete Beauveria bassiana to hygromycin B resistance by integration of the hph gene of Escherichia coli into the fungal genome. The transformation protocol was optimized to generate a library of insertion mutants of Beauveria. Transformation frequencies around 10^–4 and suppression of background growth were achieved. Over 90% of the AIM mutants investigated contained single-copy T-DNA integrations at different chromosomal locations. Integrated T-DNAs were re-isolated from ten transformants by a marker rescue approach. When the sequences flanking these T-DNAs were compared with the corresponding locations of the wild-type genome, truncations of T-DNA borders were found to be common, while none of the sites of integration had suffered deletion or rearrangement. Thus, AIM can be considered a promising tool for insertional mutagenesis studies of entomopathogenic filamentous fungi.Communicated by J. HeitmanA. Leclerque and H. Wan contributed equally to this work     

A novel mycovirus identified from the rice false smut fungus <Emphasis Type="Italic">Ustilaginoidea virens</Emphasis>

The complete sequence of a novel mycovirus infecting Ustilaginoidea virens, the causal agent of false smut of rice, is reported here and designated as Ustilaginoidea virens unassigned RNA virus HNND-1 (UvURV-HNND-1). This virus has an undivided dsRNA genome of 2903 nt in length and contains two non-overlapping open reading frames (ORF1 and 2), with the small ORF1 encoding a protein of unknown function that showed sequence similarity to the comparable protein in virus Alternaria longipes dsRNA virus 1(AlRV1) and a larger ORF2 encoded the protein showing identities to the RNA-dependent RNA polymerases of AlRV1 and some other unassigned dsRNA viruses. Phylogenetic analysis showed that UvURV-HNND-1 is more closely related to unclassified viruses such as AlRV1 and distinct from distantly related members of the family Partitiviridae. Here, we propose in accordance with previous reports that UvURV-HNND-1 might belong to a new mycovirus genus together with AlRV1 and other similar viruses.     

Efficacy of female <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis> with entomopathogenic fungus <Emphasis Type="Italic">Fusarium pallidoroseum</Emphasis>

This study was conducted to isolate and identify natural entomopathogenic fungi from female Culex quinquefasciatus and to test their adulticidal activity. Field-collected female C. quinquefasciatus died early and were placed on a Saboraud’s dextrose agar plates for growth and isolation of natural entomopathogenic fungi. The plates were maintained in an incubator at 24 ± 2°C for 3 days. Four fungal species were isolated in two genera namely, Aspergillus and Fusarium. The identified fungal species were A. niger, A. flavus, A. nidulans var acristatus (ITCC-6327.04), and F. pallidoroseum (ITCC-6324.06). Adult bioassays were carried out using spore-impregnated paper in WHO-holding tubes. F. pallidoroseum was found to be more effective than the others. Exposure of C. quinquefasciatus to spores of A. flavus and A. niger for 4 h caused 5.53% and 5.51% mortality in the mosquitoes within a week, respectively. All the female C. quinquefasciatus were killed within 4 days of exposure to F. pallidoroseum at a concentration of 1.11 × 10¹⁰ conidia per m². Significant difference of longevity was observed between the F. pallidoroseum-treated C. quinquefasciatus and control mosquitoes. The LT₅₀ of F. pallidoroseum was 2.08 days for 4 h exposure to C. quinquefasciatus. Results of the present study confirm that F. pallidoroseum is one of the alternative biological control agents of adult mosquitoes.     

Molecular characterisation of two novel double-stranded RNA elements from <Emphasis Type="Italic">Phlebiopsis gigantea</Emphasis>

The incomplete sequences of two large, 10–12 kbp, double-stranded RNAs (dsRNAs) found in the TW-2 isolate of the saprophytic fungus, Phlebiopsis gigantea (Pg) are reported. Both PgV-TW2 dsRNA1 and dsRNA2 potentially encode fusion proteins which are apparently expressed by a translational frameshifting mechanism. The C-terminal region of both predicted proteins was 21% identical and contained the eight motifs conserved in RNA-dependent RNA polymerases of dsRNA mycoviruses and had highest similarity with members of the family Totiviridae, but possibly do not form virions. The remainder of the N-terminal protein sequences predicted from the PgV-TW2 dsRNA1 and dsRNA2 sequences and the 3′-terminal nucleotide sequences of both dsRNAs had no homology with one another or any sequence in the database suggesting that individually both may be members of novel families of mycoviruses. The nucleotide sequence data reported in this article has been assigned the accession numbers AM111O96 and AM111097 for PgV-TW2 dsRNA1 and PgV-TW2 dsRNA2 respectively.     

Identification and characterization of a novel <Emphasis Type="Italic">Tritimovirus</Emphasis> species isolated from wild <Emphasis Type="Italic">Trisetum flavescens</Emphasis> L., family <Emphasis Type="Italic">Poaceae</Emphasis>

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3′ end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).     

Identification and sequence analysis of the <Emphasis Type="Italic">Condylorrhiza vestigialis</Emphasis> MNPV <Emphasis Type="Italic">p74</Emphasis> gene

The baculovirus Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV), isolated from C. vestigialis infected larvae in Paraná (Brazil), was identified in our laboratory. A full-length clone was obtained from the CoveMNPV genome, of the gene that encodes the homolog to baculoviral p74, essential for oral infectivity which was then sequenced and characterized. The CoveMNPV p74 gene (GenBank accession number EU919397) contains an ORF of 1935 bp that encodes a deduced protein of 73.61 kDa. The phylogenetic affiliations of the CoveMNPV gene were determined by a heuristic search of 40 aligned baculovirus p74 nucleotide sequences using maximum parsimony (PAUP 4.0b4a). The phylogenetic analysis placed CoveMNPV within lepidopteran nucleopolyhedrovirus (NPV) Group I, Clade A, as being the closest to Choristoneura fumiferana defective NPV.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Complete nucleotide sequence of double-stranded RNA viruses from <Emphasis Type="Italic">Fusarium graminearum</Emphasis> strain DK3

The complete genomes two different dsRNA mycoviruses, Fusarium graminearum virus 3 (FgV3) and Fusarium graminearum virus 4 (FgV4), was sequenced and analyzed. The viral genome of FgV3 is 9,098 base pairs (bp) long and contains two open reading frames (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) and a protein of unknown function. The FgV4 genome is composed of two dsRNA genome segments of 2,383 bp and 1,739 bp. FgV4 dsRNA-1 contains a single ORF, which has a conserved RdRp motif, while FgV4 dsRNA-2 contains two putative ORFs coding for products of unknown function. Both the genome organization and phylogenetic analysis indicated that FgV3 was closely related to members of the families Totiviriridae and Chrysoviridae, but it was placed outside of their main clusters, whereas FgV4 formed a distinct clade with the family Partitiviridae. This is the first report of the full-length nucleotide sequences of FgV3 and FgV4 infecting Fusarium graminearum.     

Two unrelated double-stranded RNA molecule patterns in <Emphasis Type="Italic">Gremmeniella abietina</Emphasis> type A code for putative viruses of the families <Emphasis Type="Italic">Totiviridae</Emphasis> and <Emphasis Type="Italic">Partitiviridae</Emphasis>

Summary. Two double stranded (ds) RNA molecule patterns, probably of viral origin, were sequenced from Gremmeniella abietina var. abietina type A. The genome of Gremmeniella abietina RNA virus L1 (GaRV-L1) from isolate HR2 was 5133bp and contained two open reading frames (ORFs). The 5-proximal ORF coded for a putative coat protein (CP) and the 3-proximal ORF encoded putative RNA-dependent RNA polymerase (RdRp). GaRV-L1 had sequence similarities with a previously described totivirus (Helminthosporium victoriae 190S virus) and two unclassified members of family Totiviridae (Sphaeropsis sapinea RNA virus 1 and Sphaeropsis sapinea RNA virus 2). The genome of Gremmeniella abietina RNA virus MS1 (GaRV-MS1) from isolate C5 was composed of three dsRNA molecules coding for a putative RdRp (dsRNA1), a putative CP (dsRNA2) and protein of unknown function (dsRNA3). The lengths of these dsRNA molecules were 1782, 1586 and 1181bp, respectively. Sequence comparisons indicated that the GaRV-MS1 dsRNA pattern comprises a putative virus that is highly similar to Discula destructiva virus 1, Discula destructiva virus 2 and Fusarium solani virus 1 of the family Partitiviridae.     

Evaluation of the BactiCard <Emphasis Type="Italic">Neisseria</Emphasis> for Identification of Pathogenic <Emphasis Type="Italic">Neisseria</Emphasis> Species and <Emphasis Type="Italic">Moraxella catarrhalis</Emphasis>

The BactiCard Neisseria (Remel, USA) is a chromogenic enzyme substrate system for identifying Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Moraxella catarrhalis. The identification system consists of a card with four test circles impregnated with chromogenic substrates for indoxyl butyrate esterase (IB), prolyl aminopeptidase (PRO), γ-glutamyl aminopeptidase (GLUT), and ?-galactosidase (BGAL). These substrates permit the identification of Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica, respectively. After hydration of the circles with buffer, colonies from growth on selective media or a subculture are applied to the four circles. IB and BGAL reactions are read for a blue-green color after 2 and 15 min, respectively. PRO and GLUT reactions are read at 15 min for a red color after addition of a developer reagent. Identifications obtained with the BactiCard Neisseria were compared with those obtained using conventional procedures for 558 isolates in a blinded fashion. The BactiCard Neisseria identified 100% of 254 Neisseria gonorrhoeae, 100% of 125 Neisseria meningitidis, 53 (98.2%) of 54 Neisseria lactamica, and 123 (98.4%) of 125 Moraxella catarrhalis isolates. The BactiCard Neisseria is an accurate and rapid system for identification of these microorganisms in the clinical laboratory. Electronic Publication     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.     

<Emphasis Type="Italic">chsZ</Emphasis>, a gene for a novel class of chitin synthase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis>

We cloned and characterized a novel Aspergillus oryzae chitin synthase gene, chsZ, encoding a polypeptide containing a new myosin motor-like domain in its N-terminal half. Alignment analysis revealed that ChsZ was less homologous to known class V enzymes, except for its probable chitin synthase conserved region in the C-terminal half. We also found a chsY gene and found that ChsY showed higher similarity to the class V enzymes than did ChsZ. Phylogenetic analysis clearly demonstrated that the A. oryzae ChsZ, together with Chs4 of Paracoccidioides brasiliensis and Chs6 of Ustilago maydis, formed a new subclass distinct from A. oryzae ChsY and known class V chitin synthases, including A. nidulans CsmA (ChsD) and A. fumigatus ChsE. In conclusion, we propose a new class, class VI chitin synthases, represented by A. oryzae ChsZ, P. brasiliensis Chs4 and U. maydis Chs6. Expression analysis suggested that the regulation of chsZ expression is distinct from that of chsY expression.     

Prevalence of the <Emphasis Type="Italic">ica</Emphasis> operon and insertion sequence IS<Emphasis Type="Italic">256</Emphasis> among <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> prosthetic joint infection isolates

Joint replacement surgery has improved the quality of life for hundreds of thousands of patients. However, the infection of a joint implant is an important and serious complication, though the prevalence is low. Staphylococcus epidermidis is the most important pathogen involved in foreign-body infections. S. epidermidis is also a commensal that comprises a substantial part of the normal skin flora of humans. The possibility to demonstrate potential specific virulence markers may facilitate the interpretation of the bacteriological findings, as well as the clinical decision. The prevalence of the ica locus and insertion sequence IS256 by using polymerase chain reaction (PCR) among 32 clinical S. epidermidis isolates from prosthetic joint infections (PJIs) and 24 commensal isolates from nares and skin was investigated. Sixteen (50%) of the 32 PJI isolates harbored the ica operon compared with one-third of the commensal isolates obtained from the samples of the skin and nares of healthy individuals. The IS256 was demonstrated in 26 (81%) out of 32 PJI isolates. By contrast, IS256 was found in one of 24 commensal isolates. In conclusion, IS256 may be superior to the ica operon as a marker of the invasive capacity of S. epidermidis, since it was found in most of the PJI isolates, but rarely among commensals.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

Comparative analyses among the <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis>, <Emphasis Type="Italic">Trichomonas tenax</Emphasis>, and <Emphasis Type="Italic">Tritrichomonas foetus</Emphasis> 5S ribosomal RNA genes

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers FJ492747, FJ492748, FJ492749, FJ492750, and FJ492751.