Ribosomal RNA modification enzymes stimulate large ribosome subunit assembly in E. coli.

Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.

Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome.

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5' splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 (NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection.

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

Morphological and molecular characteristics of fungal species associated with crown rot of strawberry in South Korea.

BACKGROUND: Crown and root rot is the most important and destructive strawberry diseases in Korea as it causes substantial economic loss. In August 2020, a severe outbreak of crown and root rot on strawberries (Fragaria × ananassa Duch.) was observed in the greenhouse at Sangju, South Korea. Infected plantlets displayed browning rot within the crown and root, stunted growth, and poor rooting. METHODS AND RESULTS: Thirty fungal isolates were obtained from the affected plantlet. Isolates were identified based on morphological characteristics and pathogenicity test as well as sequence data obtained from internal transcribed spacer, large subunit ribosomal ribonucleic acid, translation elongation factor, and RNA polymerase II-second largest subunit. Results showed that the crown and root rot of strawberry in Korea was caused by three distinct fungal species: Fusarium oxysporum f. sp. fragariae, F. solani, and Plectosphaerella cucumerina. To the best of our knowledge, F. solani, and P. cucumerina are reported for the first time as the causal agents of the crown and root rot of strawberry in South Korea. Pathogenicity tests confirmed that these isolates are pathogenic to strawberry. CONCLUSIONS: Understanding the composition and biology of the pathogen population will be helpful to provide effective control strategies for the disease.

Assessing anaerobic gut fungal diversity in herbivores using D1/D2 large ribosomal subunit sequencing and multi-year isolation.

The anaerobic gut fungi (AGF, Neocallimastigomycota) reside in the alimentary tracts of herbivores where they play a central role in the breakdown of plant material. Here, we report on the development of the hypervariable domains D1/D2 of the large ribosomal subunit (D1/D2 LSU) as a barcoding marker for the AGF. We generated a reference D1/D2 LSU database for all cultured AGF genera, as well as the majority of candidate genera encountered in prior internal transcribed spacer 1 (ITS1)-based surveys. Subsequently, a D1/D2 LSU-based diversity survey using long read PacBio SMRT sequencing was conducted on faecal samples from 21 wild and domesticated herbivores. Twenty-eight genera and candidate genera were identified, including multiple novel lineages that were predominantly, but not exclusively, identified in wild herbivores. Association between certain AGF genera and animal lifestyles, or animal host family was observed. Finally, to address the current paucity of AGF isolates, concurrent isolation efforts utilizing multiple approaches to maximize recovery yielded 216 isolates belonging to 12 different genera, several of which have no prior cultured-representatives. Our results establish the utility of D1/D2 LSU and PacBio sequencing for AGF diversity surveys, the culturability of multiple AGF taxa, and demonstrate that wild herbivores represent a yet-untapped reservoir of AGF diversity.

Molecular phylogenetic assessment of the tribal classification of Lamiinae (Coleoptera: Cerambycidae).

Lamiinae is the most diverse subfamily of longhorned beetles, with about 20,000 described species classified into 80 tribes. Most of the tribes of Lamiinae were proposed during the 19th century and the suprageneric classification of the subfamily has never been assessed under phylogenetic criteria. In this study, we present the first tribal-level phylogeny of Lamiinae, inferred from 130 terminals (representing 46 tribes, prioritizing generic type species of the tribes) and fragments of two mitochondrial and three nuclear markers (cox1, rrnL, Wg, CPS and LSU; 5,024 aligned positions in total). Analyses were performed under Maximum Likelihood and Bayesian methods based on two datasets: a dataset including all taxa available for the study, and a reduced dataset with 111 terminals where taxa only contributing with mitochondrial markers were excluded from the matrix. The monophyly of Lamiinae was corroborated in three of the four analyses and 11 of the 35 tribes with more than one species represented in the analyses were consistently recovered as monophyletic. However, 15 tribes were not retrieved as monophyletic, requiring a revision of their boundaries: Acanthocinini, Acanthoderini, Agapanthiini, Apomecynini, Desmiphorini, Dorcaschematini, Enicodini, Hemilophini, Monochamini, Onciderini, Parmenini, Phytoeciini, Pogonocherini, Pteropliini and Saperdini. Based on these results, when strong support values for paraphyly were recovered, we argue a number of tribe synonymies, including Moneilemini as synonym of Acanthocinini; Onocephalini of Onciderini; Dorcadionini, Gnomini, Monochamini and Rhodopinini of Lamiini; and Obereini and Phytoeciini of Saperdini. Other taxonomic changes proposed in this study based on the criterion of monophyly and supported by morphological characters include the transfer of Tricondyloides and Stenellipsis to Enicodini, and of Dylobolus stat. rest., which is removed as subgenus of Mecas and restituted as genus, to Hemilophini. Furthermore, our analyses suggest that Ostedes and Neohoplonotus should be removed from Acanthocinini and Parmenini, respectively, and Colobotheini should be redefined to encompass several genera currently placed in Acanthocinini.

Saccharomycopsis oxydans sp. nov., a new non-fermentative member in the genus Saccharomycopsis isolated from a traditional dairy product of Iran.

A total of 21 yeast isolates were recovered as part of a research project on biodiversity of yeasts in traditional dairy products in Alborz province, Iran. Standard protocols were used to carry out phenotypic, biochemical, physiological characterization and the phylogenetic analysis of combined the D1/D2 domain of the large ribosomal subunit (26S or LSU) and ITS region sequences. Five strains represented a potential new ascomycetous yeast species. Ascospore formation was not observed in these strains, and they did not ferment the examined carbon sources. Phylogenetic analysis placed these isolates in a well-supported sub-clade in the genus Saccharomycopsis. Here, we describe this novel yeast as Saccharomycopsis oxydans sp. nov.

Wickerhamomyces psychrolipolyticus f.a., sp. nov., a novel yeast species producing two kinds of lipases with activity at different temperatures.

Two yeast strains isolated from soil collected in Hokkaido, Japan, were found to secrete two extracellular lipases that exhibited activities at both 25 and 4 °C. Both strains could utilize olive oil, rapeseed oil, lard and fish oil as sole carbon sources. The similarity of the D1/D2 domain of the large subunit ribosomal RNA (LSU rRNA) sequence of these yeast strains to that of other yeasts in the GenBank database was very low (<96 %). The phylogenetic trees based on the LSU rRNA sequences and translation elongation factor-1-α (tef1-α) sequences indicated that both strains represented a member of the Wickerhamomyces /Candida clade. Sexual reproduction was not observed. The name Wickerhamomyces psychrolipolyticus f.a., sp. nov is proposed for this newly described yeast species producing cold-active lipases. This novel species is distinguishable from the type strains of other related species, Wickerhamomyces alni, Candida ulmi and Candida quercuum due to their abilities to grow at 4 to 30 °C, to produce lipase that is active also at 4 °C and to assimilate soluble starch.

Crystal structure of a eukaryotic group II intron lariat.

The formation of branched lariat RNA is an evolutionarily conserved feature of splicing reactions for both group II and spliceosomal introns. The lariat is important for the fidelity of 5' splice-site selection and consists of a 2'-5' phosphodiester bond between a bulged adenosine and the 5' end of the intron. To gain insight into this ubiquitous intramolecular linkage, we determined the crystal structure of a eukaryotic group IIB intron in the lariat form at 3.7 Å. This revealed that two tandem tetraloop-receptor interactions, η-η' and π-π', place domain VI in the core to position the lariat bond in the post-catalytic state. On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role. The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end. Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

The complete structure of the large subunit of the mammalian mitochondrial ribosome.

Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes.

The Rpf84 gene, encoding a ribosomal large subunit protein, RPL22, regulates symbiotic nodulation in Robinia pseudoacacia.

MAIN CONCLUSION: A homologue of the ribosomal protein L22e, Rpf84, regulates root nodule symbiosis by mediating the infection process of rhizobia and preventing bacteroids from degradation in Robinia pseudoacacia. Ribosomal proteins (RPs) are known to have extraribosomal functions, including developmental regulation and stress responses; however, the effects of RPs on symbiotic nodulation of legumes are still unclear. Ribosomal protein 22 of the large 60S subunit (RPL22), a non-typical RP that is only found in eukaryotes, has been shown to function as a tumour suppressor in animals. Here, a homologue of RPL22, Rpf84, was identified from the leguminous tree R. pseudoacacia. Subcellular localization assays showed that Rpf84 was expressed in the cytoplasm and nucleus. Knockdown of Rpf84 by RNA interference (RNAi) technology impaired the infection process and nodule development. Compared with the control, root and stem length, dry weight and nodule number per plant were drastically decreased in Rpf84-RNAi plants. The numbers of root hair curlings, infection threads and nodule primordia were also significantly reduced. Ultrastructure analyses showed that Rpf84-RNAi nodules contained fewer infected cells with fewer bacteria. In particular, remarkable deformation of bacteroids and fusion of multiple symbiosomes occurred in infected cells. By contrast, overexpression of Rpf84 promoted nodulation, and the overexpression nodules maintained a larger infection/differentiation region and had more infected cells filled with bacteroids than the control at 45 days post inoculation, suggesting a retarded ageing process in nodules. These results indicate for the first time that RP regulates the symbiotic nodulation of legumes and that RPL22 may function in initiating the invasion of rhizobia and preventing bacteroids from degradation in R. pseudoacacia.

Genetic diversity of Pneumocystis jirovecii from a cluster of cases of pneumonia in renal transplant patients: Cross-sectional study.

Pneumocystis jirovecii can cause severe potentially life-threatening pneumonia (PCP) in kidney transplant patients. Prophylaxis of patients against PCP in this setting is usually performed during 6 months after transplantation. The aim of this study is to describe the molecular epidemiology of a cluster of PCP in renal transplant recipients in Brazil. Renal transplant patients who developed PCP between May and December 2011 had their formalin-fixed paraffin-embedded (FFPE) lung biopsy samples analysed. Pneumocystis jirovecii 23S mitochondrial large subunit of ribosomal RNA (23S mtLSU-rRNA), 26S rRNA, and dihydropteroate synthase (DHPS) genes were amplified by polymerase chain reaction (PCR), sequenced, and analysed for genetic variation. During the study period, 17 patients developed PCP (only four infections were documented within the first year after transplantation) and six (35.3%) died. Thirty FFPE samples from 11 patients, including one external control HIV-infected patient, had fungal DNA successfully extracted for further amplification and sequencing for all three genes. A total of five genotypes were identified among the 10 infected patients. Of note, four patients were infected by more than one genotype and seven patients were infected by the same genotype. DNA extracted from FFPE samples can be used for genotyping; this approach allowed us to demonstrate that multiple P. jirovecii strains were responsible for this cluster, and one genotype was found infecting seven patients. The knowledge of the causative agents of PCP may help to develop new initiatives for control and prevention of PCP among patients undergoing renal transplant and improve routine PCP prophylaxis.

Sequence variation in nuclear ribosomal small subunit, internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate-dependent.

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra-organism genetic variation. However, information about intra- vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra-isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12-40 clones per isolate. Intra-isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut-off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next-generation sequencing; and its ease of amplification in single-step PCR.

Wickerhamomyces orientalis f.a., sp. nov.: an ascomycetous yeast species belonging to the Wickerhamomyces clade.

Five closely related yeast strains were isolated from soil in Kharg Island, Persian Gulf, Iran, and from fallen fruits in Galle, Sri Lanka, during separate projects. Morphologically, the strains produced white-coloured yeast colonies, with cells that were ovoid to ellipsoidal, making branched, true hyphae and pseudohyphae. Ascospore formation was not observed. Biochemically, the strains were able to ferment d-glucose and weakly ferment d-galactose. The strains could use a wide variety of carbon sources except methanol and hexadecane. Phylogenetic analyses using combined sequences of the small ribosomal subunit and the D1/D2 domains of the LSU, as well as the internal transcribed spacer regions, suggested that these strains belong to the Wickerhamomyces clade and that together they form one strongly supported phylogenetic clade. Differences in their sequences, biochemistry and morphology suggest they are representatives of distinct species of the genus Wickerhamomyces. Therefore, the name Wickerhamomyces orientalis f.a., sp. nov. is proposed to accommodate these novel strains; the type strain is IBRC-M 30103T (=CBS 13306T). The MycoBank number is MB 807323.

Arbuscular mycorrhizal fungal communities and Rhizophagus irregularis populations shift in response to short-term ploughing and fertilisation in a buffer strip.

Short-term effects of soil physical disturbance by ploughing and nitrogen and phosphate fertilisation on arbuscular mycorrhizal fungal (AMF) communities and on intraspecific populations of Rhizophagus irregularis in a buffer strip surrounded by arable fields were studied. Pre-grown Plantago lanceolata plantlets were transplanted into fertilised and/or ploughed experimental plots. After 3 months, the glomeromycotan communities in the roots of these trap plants were analysed using 454 pyrosequencing of a fragment of the RNA polymerase II gene (RPB1). Intraspecific populations of R. irregularis were studied by restriction fragment length polymorphism (RFLP) analysis of the mitochondrial large ribosomal subunit (mtLSU) gene. Soil disturbance significantly increased the diversity of species-level molecular taxa (MTs) and altered community structure, whilst fertilisation alone had no significant effect, unless coupled with ploughing. At the population level, the expected shift from genotypes of R. irregularis typically found in grasslands to those usually found in arable sites was only partially observed. In conclusion, in the short-term, physical soil disturbance, as well as nitrogen fertilisation when coupled with physical soil disturbance, affected AMF community and to a smaller extent population composition.

Assignment of 2'-O-methyltransferases to modification sites on the mammalian mitochondrial large subunit 16 S ribosomal RNA (rRNA).

Advances in proteomics and large scale studies of potential mitochondrial proteins have led to the identification of many novel mitochondrial proteins in need of further characterization. Among these novel proteins are three mammalian rRNA methyltransferase family members RNMTL1, MRM1, and MRM2. MRM1 and MRM2 have bacterial and yeast homologs, whereas RNMTL1 appears to have evolved later in higher eukaryotes. We recently confirmed the localization of the three proteins to mitochondria, specifically in the vicinity of mtDNA nucleoids. In this study, we took advantage of the ability of 2'-O-ribose modification to block site-specific cleavage of RNA by DNAzymes to show that MRM1, MRM2, and RNMTL1 are responsible for modification of human large subunit rRNA at residues G(1145), U(1369), and G(1370), respectively.

Cytonuclear interactions and relaxed selection accelerate sequence evolution in organelle ribosomes.

Many mitochondrial and plastid protein complexes contain subunits that are encoded in different genomes. In animals, nuclear-encoded mitochondrial proteins often exhibit rapid sequence evolution, which has been hypothesized to result from selection for mutations that compensate for changes in interacting subunits encoded in mutation-prone animal mitochondrial DNA. To test this hypothesis, we analyzed nuclear genes encoding cytosolic and organelle ribosomal proteins in flowering plants. The model angiosperm genus Arabidopsis exhibits low organelle mutation rates, typical of most plants. Nevertheless, we found that (nuclear-encoded) subunits of organelle ribosomes in Arabidopsis have higher amino acid sequence polymorphism and divergence than their counterparts in cytosolic ribosomes, suggesting that organelle ribosomes experience relaxed functional constraint. However, the observed difference between organelle and cytosolic ribosomes was smaller than in animals and could be partially attributed to rapid evolution in N-terminal organelle-targeting peptides that are not involved in ribosome function. To test the role of organelle mutation more directly, we used transcriptomic data from an angiosperm genus (Silene) with highly variable rates of organelle genome evolution. We found that Silene species with unusually fast-evolving mitochondrial and plastid DNA exhibited increased amino acid sequence divergence in ribosomal proteins targeted to the organelles but not in those that function in cytosolic ribosomes. Overall, these findings support the hypothesis that rapid organelle genome evolution has selected for compensatory mutations in nuclear-encoded proteins. We conclude that coevolution between interacting subunits encoded in different genomic compartments within the eukaryotic cell is an important determinant of variation in rates of protein sequence evolution.

Clinical characteristics of hospital-onset Pneumocystis pneumonia and genotypes of Pneumocystis jirovecii in a single tertiary centre in Korea.

BACKGROUND: Pneumocystis pneumonia (PCP) may develop as a clinical manifestation of nosocomial pneumonia by means of either reactivation of resident P. jirovecii or de novo infection. However, there have been no studies describing the clinical characteristics of hospital-onset PCP. METHODS: A retrospective review of medical records was performed to identify episodes of hospital-onset PCP in a tertiary care centre in Korea between May 2007 and January 2013. We investigated whether human-to-human contact during hospitalisation contributed to PCP development by molecular analysis of the genes encoding mitochondrial large ribosomal subunit (mtLSU) rRNA and dihydropteroate synthase (DHPS) and a review of hospitalisation history. RESULTS: During the study period, 129 patients (130 episodes) were diagnosed with PCP. Of these, respiratory specimens from 94 patients during 95 PCP episodes were available for analysis. Sixteen episodes (16.8%) were categorised as hospital-onset PCP. There was a trend toward a higher proportion of haematological malignancy (43.8% [7/16] vs. 20.3% [16/79]; P = 0.058) in patients with hospital-onset PCP compared to patients with community-onset PCP. mtLSU genotype 1 was the most common, occurring in 41 (43.2%) patients. There were four possible cases of nosocomial transmission. Mutation in DHPS was not observed in any PCP episode. CONCLUSIONS: PCP can be one of the causes of nosocomial pneumonia, although the mode of acquisition and transmission of P. jirovecii remains uncertain. mtLSU genotype 1 is the predominant P. jirovecii strain in Korea.

Is the extraction by Whatman FTA filter matrix technology and sequencing of large ribosomal subunit D1-D2 region sufficient for identification of clinical fungi?

Although conventional identification of pathogenic fungi is based on the combination of tests evaluating their morphological and biochemical characteristics, they can fail to identify the less common species or the differentiation of closely related species. In addition these tests are time consuming, labour-intensive and require experienced personnel. We evaluated the feasibility and sufficiency of DNA extraction by Whatman FTA filter matrix technology and DNA sequencing of D1-D2 region of the large ribosomal subunit gene for identification of clinical isolates of 21 yeast and 160 moulds in our clinical mycology laboratory. While the yeast isolates were identified at species level with 100% homology, 102 (63.75%) clinically important mould isolates were identified at species level, 56 (35%) isolates at genus level against fungal sequences existing in DNA databases and two (1.25%) isolates could not be identified. Consequently, Whatman FTA filter matrix technology was a useful method for extraction of fungal DNA; extremely rapid, practical and successful. Sequence analysis strategy of D1-D2 region of the large ribosomal subunit gene was found considerably sufficient in identification to genus level for the most clinical fungi. However, the identification to species level and especially discrimination of closely related species may require additional analysis.

Multigene phylogeny of the Phallales (Phallomycetidae, Agaricomycetes) focusing on some previously unrepresented genera.

Phylogenetic relationships within the Phallales were estimated via combined sequences: nuclear ribosomal large subunit (LSU), second largest subunit of RNA polymerase (rpb2), and mitochondrial ATPase subunit 6 (atp6). The ingroup is represented by 62 taxa comprising 18 genera and 44 species, including members of the Clathraceae, Claustulaceae, Gastrosporiaceae, Lysuraceae, Phallaceae and Protophallaceae. Sixty-one new sequences were generated, including tropical and subtropical taxa. This is one of the first studies discussing the phylogenetic placement of Abrachium, Aseroë, Blumenavia, Gastrosporium, Jansia and Xylophallus. Gastrosporiaceae was demonstrated to be sister to Phallaceae and an emended description of the order is presented. Aseroë was demonstrated to be polyphyletic and as a result, A. arachnoidea is transferred to Lysurus.