Identification in Australia of the quarantine pathogen of sunflower Phoma macdonaldii (Teleomorph : Leptosphaeria lindquistii)

Isolations from black stem lesions of sunflower growing in south-eastern Queensland yielded fungi putatively identified as species of Phoma. Pathogenicity assays showed that these isolates were capable of killing sunflower plants under glasshouse conditions. The isolates were compared with authentic cultures of Phoma macdonaldii and other isolates of Phoma taken from sunflower from around the world. Random amplified polymorphic DNA analysis showed that all the Australian isolates examined were very similar to the holotype culture of Phoma macdonaldii from Canada. Sequencing of the internal transcribed spacer regions also revealed the relatedness of the Australian isolates to the holotype. This is the first official record of P. macdonaldii in Australia.

Intragenomic variation in ITS2 rDNA in the louse of humans, Pediculus humanus: ITS2 is not a suitable marker for population studies in this species

The two internal transcribed spacers (ITS) of ribosomal DNA are often used as markers of populations of insects. We studied the ITS2 of the head lice and body lice of humans, to determine whether this gene is a suitable marker of populations of these insects. ITS2 sequences were amplified by PCR from lice from four different countries: Australia, China, Japan and the USA. Direct cycle-sequencing of some of these PCR products gave equivocal nucleotide chromatograms. This indicated that some lice had more than one ITS2 sequence, so we cloned PCR products from these lice. Temperature gradient gel electrophoresis (TGGE) revealed that 50 of the 67 clones we screened had different nucleotide sequences. All lice had several ITS2 types, including those with unequivocal chromatograms. A phylogenetic tree of 15 different ITS2 sequences showed that the sequences from individual lice were not monophyletic. We conclude that the ITS2 is not a useful marker of populations for Pediculus humanus.

Cryptococcus statzelliae sp nov and three novel strains of Cryptococcus victoriae, yeasts isolated from Antarctic soils

A morphological and physiological characterization of yeast strains CBS 8908, CBS 8915, CBS 8920, CBS 8925(T) and CBS 8926, isolated from Antarctic soils, was performed. Phylogenetic analyses of the sequences of the D1/D2 regions and the adjacent internal transcribed spacer (ITS) regions of the large-subunit rDNA of these strains placed them into the Tremellales clade of the Hymenomycetes. The sequence data identified strains CBS 8908, CBS 8915 and CBS 8920 as belonging to the species Cryptococcus victoriae. Strains CBS 8925(T) and CBS 8926 were found to represent an unique clade within the Hymenomycetes, with Dioszegia crocea CBS 6714(T) being their closest phylogenetic relative. Fatty acid composition and proteome fingerprint data for these novel strains were also obtained. No sexual state was observed. A novel basidiomycetous species, Cryptococcus statzelliae, is proposed for strains CBS 8925(T) and CBS 8926.

Low symbiont diversity in southern Great Barrier Reef corals, relative to those of the Caribbean

The specific identity of endosymbiotic dinoflagellates (Symbiodinium spp.) from most zooxanthellate corals is unknown. In a survey of symbiotic cnidarians from the southern Great Barrier Reef (GBR), 23 symbiont types were identified from 86 host species representing 40 genera. A majority (>85%) of these symbionts belong to a single phylogenetic clade or subgenus (C) composed of closely related (as assessed by sequence data from the internal transcribed spacer region and the ribosomal large subunit gene), yet ecologically and physiologically distinct, types. A few prevalent symbiont types, or generalists, dominate the coral community of the southern GBR, whereas many rare and/or specific symbionts, or specialists, are found uniquely within certain host taxa. The comparison of symbiont diversity between southern GBR and Caribbean reefs shows an inverse relationship between coral diversity and symbiont diversity, perhaps as a consequence of more-rapid diversification of Caribbean symbionts. Among clade C types, generalists C1 and C3 are common to both Caribbean and southern GBR symbiont assemblages, whereas the rest are regionally endemic. Possibly because of environmental changes in the Caribbean after geographic isolation through the Quaternary period, a high proportion of Caribbean fauna associate with symbiont taxa from two other distantly related Symbiodinium clades (A and B) that rarely occur in Pacific hosts. The resilience of Porites spp. and the resistance of Montipora digitata to thermal stress and bleaching are partially explained by their association with a thermally tolerant symbiont type, whereas the indiscriminant widespread bleaching and death among certain Pacific corals, during El Nino Southern Oscillation events, are influenced by associations with symbionts possessing higher sensitivity to thermal stress.

Biofilm microbial communities of denture stomatitis

Introduction: Denture stomatitis is a common lesion that affects denture wearers. Its multifactorial etiology seems to depend on a complex and poorly characterized biofilm. The purpose of this study was to assess the composition of the microbial biofilm obtained from complete denture wearers with and without denture stomatitis using culture-independent methods. Methods: Samples were collected from healthy denture wearers and from patients with denture stomatitis. Libraries comprising about 600 cloned 16S ribosomal DNA (rDNA) bacterial sequences and 192 cloned eukaryotic internal transcribed spacer (ITS) region sequences, obtained by polymerase chain reactions, were analyzed. Results: The partial 16S rDNA sequences revealed a total of 82 bacterial species identified in healthy subjects and patients with denture stomatitis. Twenty-seven bacterial species were detected in both biofilms, 29 species were exclusively present in patients with denture stomatitis, and 26 were found only in healthy subjects. Analysis of the ITS region revealed the presence of Candida sp. in both biofilms. Conclusion: The results revealed the extent of the microbial flora, suggesting the existence of distinct biofilms in healthy subjects and in patients with denture stomatitis.

Identical digeneans in coral reef fishes from French Polynesia and the Great Barrier Reef (Australia) demonstrated by morphology and molecules

Three coral reef fish species, Zanclus cornutus, Chaetodon vagabundus and Naso lituratus, were collected in French Polynesia and on the Great Barrier Reef, Queensland. These fish species were each infected by one morphologically similar digenean species in both localities; Schistorchis Zancli Hanson, 1953 was found in Zanclus cornutus. Preptetos laguncula Bray and Cribb, 1996 in Naso lituratus and Neohypocreadium dorsoporum Machida and Uchida, 1987 in Chaetodon vagabundus. In addition, on the Great Barrier Reef P. laguncula was also found in Naso unicornis and N. dorsoporum in Chaetodon ephippium and Chaetodon flavirostris. Morphometric differences between the species from the two sites were only slight. Sequences from the second internal transcribed spacer of the ribosomal DNA of each worm revealed total homology or negligible divergence between samples from hosts caught in French Polynesia and on the Great Barrier Reef. These results show that across more than 6000 km these digeneans are similar in morphology and genotype. Some species of fishes and molluscs a-re considered to have distributions that encompass the entire tropical Indo-West Pacific. These findings suggest that at least some of their parasites have similarly broad distributions. (C) 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

DNA sequence variation in the ITS-1 rDNA subunit and host relationships in sorghum midge, Stenodiplosis sorghicola (Coquillett) (Diptera : Cecidomyiidae), in Australia

Sequence variation in the internal transcribed spacer (ITS-1) ribosomal DNA subunit was examined for sorghum midge obtained from introduced and native hosts in south-eastern and central Queensland. No variation was observed relative to host plant or geographical distance for midges collected from two introduced hosts, grain sorghum (Sorghum bicolor ) and Johnson grass (S. halepense ); however, sequence differences were observed between midges from introduced and native hosts and among midges from a single native host, slender bluegrass (Dichanthium affine ). No evidence was observed of introduced midges on native hosts, or vice versa. These results agree with previously hypothesised host distributions for native and introduced midges in Australia, and expand the sample of introduced hosts to include Johnson grass. They suggest that Stenodiplosis sorghicola , the principal midge infesting grain sorghum, is also the most common species on Johnson grass. This confirms that Johnson grass plays a role in the population dynamics of S. sorghicola and suggests that midges originating from Johnson grass may influence levels of infestation in grain sorghum.

Testing the applicability of molecular genetic markers to population analyses of scleratinian corals

The abundance of coral reefs worldwide is in decline, and despite the ecological importance of reefs, only a limited number of DNA markers have been identified for scleractinian coral genetic studies. This paper addresses the search for new coral molecular markers and investigates the applicability of the cytochrome c oxidase subunit I (COI), the internal transcribed spacer region 1 (ITS1), and the pocilloporin gene to the question of intraspecific variation in the scleractinian coral Pocillopora verrucosa along the southeast African coastline. The COI fragment was 710 bp long and was identical for P. verrucosa (n = 10) and P. damicornis (n = 3). Only two different ITS1 sequences were found (differing by 13 bp insertion), but more importantly, 24% of the sequences were heterogenous indicating that different multiple copies of the sequence exist. Pocilloporin is an intronless gene that was absolutely conserved throughout all P. verrucosa populations (n = 50). Thus, the three DNA regions studied appear unsuitable for the population genetic analyses of P. verrucosa.

Estudo morfológico e filogenético das subespécies Daucus carota ssp. Azoricus e Daucus carota ssp. Maritimus na ilha de S. Miguel

Dissertação de Mestrado, Biodiversidade e Biotecnologia Vegetal, 17 de Março de 2015, Universidade dos Açores.

Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources

Clinical and environmental samples from Portugal were screened for the presence of Aspergillus and the distributions of the species complexes were determined in order to understand how their distributions differ based on their source. Fifty-seven Aspergillus isolates from clinical samples were collected from 10 health institutions. Six species complexes were detected by internal transcribed spacer sequencing; Fumigati, Flavi, and Nigri were found most frequently (50.9%, 21.0%, and 15.8%, respectively). β-tubulin and calmodulin sequencing resulted in seven cryptic species (A. awamorii, A. brasiliensis, A. fructus, A. lentulus, A. sydowii, A. tubingensis, Emericella echinulata) being identified among the 57 isolates. Thirty-nine isolates of Aspergillus were recovered from beach sand and poultry farms, 31 from swine farms, and 80 from hospital environments, for a total 189 isolates. Eleven species complexes were found in these 189 isolates, and those belonging to the Versicolores species complex were found most frequently (23.8%). There was a significant association between the different environmental sources and distribution of the species complexes; the hospital environment had greater variability of species complexes than other environmental locations. A high prevalence of cryptic species within the Circumdati complex was detected in several environments; from the isolates analyzed, at least four cryptic species were identified, most of them growing at 37ºC. Because Aspergillus species complexes have different susceptibilities to antifungals, knowing the species-complex epidemiology for each setting, as well as the identification of cryptic species among the collected clinical isolates, is important. This may allow preventive and corrective measures to be taken, which may result in decreased exposure to those organisms and a better prognosis.

Molecular epidemiology of Aspergillus collected from cystic fibrosis patients

Background - Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods - Fifty-three Aspergillus isolates recovered from CF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results - Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions - To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogen.

Biogeography, host specificity, and molecular phylogeny of the basidiomycetous yeast phaffia rhodozyma and its sexual form, xanthophyllomyces dendrorhous

Applied and Environmental Microbiology, Vol. 73, No.4

Dioszegia antarctica sp. nov. and Dioszegia cryoxerica sp. nov., psychrophilic basidiomycetous yeasts from polar desert soils in Antarctica

International Journal of Systematic and Evolutionary Microbiology, 60

Farysizyma gen. nov., an anamorphic genus in the Ustilaginales to accommodate three novel epiphytic basidiomycetous yeast species from America, Europe and Asia

FEMS Yeast Research, Vol. 8, Nº 3

Identificação de micobactérias não tuberculosas através de métodos moleculares não comerciais

Embora Mycobacterium tuberculosis, o agente etiológico da tuberculose humana, seja a principal causa de micobacteriose no Homem, outras micobactérias não tuberculosas (MNT), podem também causar infecção em seres humanos, sendo cada vez mais frequentemente isoladas no laboratório de micobacteriologia. Dada a morosidade da identificação de MNT por métodos convencionais, torna-se essencial o desenvolvimento de métodos rápidos e fiáveis para a sua identificação. Neste estudo foram comparados três métodos moleculares para a identificação de MNT, baseados na análise de restrição enzimática após amplificação de: (i) região rRNA 16S-23S “Internal Transcribed Spacer” (ITS), (ii) gene hsp65, e (iii) zona ITS e regiões adjacentes, 16S e 23S rDNA. Para este fim, avaliamos 50 isolados, correspondendo a 47 isolados clínicos de MNT e três estirpes de referência, representando as 11 espécies de MNT mais frequentemente isoladas no laboratório de Micobactérias do Instituto de Higiene e Medicina Tropical (IHMT, UNL) e uma estirpe referência de M. tuberculosis, e identificadas anteriormente utilizando o sistema comercial GenoType® Mycobacterium CM/AS (Hain Lifescience). O PCR-RFLP do gene hsp65 proporcionou os melhores resultados, identificando correctamente 42 dos 49 isolados de MNT, pertencentes a M. avium, M. gordonae, M. intracellulare, M. kansasii, M. chelonae, M. fortuitum, M. abscessus, M. szulgai, M. peregrinum e M. xenopi. O PCR-RFLP da região ITS identificou correctamente 28 dos 49 isolados testados, não distinguindo M. intracellulare/M. scrofulaceum, M. avium/M. bohemicum e M. kansasii/M. szulgai. Finalmente, o PCR-RFLP da região ITS e regiões adjacentes mostrou o pior desempenho, com apenas oito isolados correctamente identificados e falhando na amplificação de diversos isolados. Dos três métodos testados, o PCR-RFLP do gene hsp65 e da região ITS mostraram maior capacidade de identificação e reprodutibilidade. No entanto, a sua aplicação exige uma optimização cuidadosa das condições de análise e a sua aplicabilidade depende, em grande parte, da diversidade xi de MNT em cada laboratório. Verificaram-se também várias dificuldades a nível da interpretação dos resultados. Assim, apesar das vantagens referidas na literatura para estes três métodos, verificou-se que o grau de variabilidade e dificuldade de interpretação associados aos padrões obtidos, limitam a sua implementação no laboratório de diagnóstico de micobacteriologia.