Psychrophilic yeasts in glacial environments of Alpine glaciers

The presence of psychrophilic yeasts in supra- and subglacial sediments, ice and meltwater collected from two glaciers of the Italian Alps (Forni and Sforzellina-Ortles-Cevedale group) was investigated. After incubation at 4 degrees C, subglacial sediments contained from 1.3 x 10(3) to 9.6 x 10(3) CFU of yeasts g(-1). The number of yeast cells in supraglacial sediments was c. 10-100-fold lower. A significant proportion of isolated yeasts exhibited one or more extracellular enzymatic activities (starch-degrading, lipolytic, esterolytic, proteolytic and pectinolytic activity) at 4 degrees C. Selected isolates were able to grow at 2 degrees C under laboratory-simulated in situ conditions. In all, 106 isolated yeasts were identified by MSP-PCR fingerprinting and 26S rRNA gene sequencing of the D1/D2 region as belonging to 10 species: Aureobasidium pullulans, Cryptococcus gilvescens (over 50% of the total), Cryptococcus terricolus, Mrakia gelida, Naganishia globosa, Rhodotorula glacialis, Rhodotorula psychrophenolica, Rhodotorula bacarum, Rhodotorula creatinivora and Rhodotorula laryngis. Four strains, all belonging to a new yeast species, yet to be described, were also isolated.     

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

Among many isolates that resulted from four independent surveys of yeasts associated with plants in Brazil, the USA, Portugal and Taiwan, we have characterized eighteen basidiomycetous strains, two of which were conspecific with the type strain of Rhodotorula acheniorum, whereas the remaining sixteen isolates appeared not to correspond to any previously described species. Microsatellite-PCR fingerprinting with primers M13 and (GTG)5 confirmed that the latter strains formed three genetically distinct groups. Each group was considered to represent a distinct species based on nucleotide sequences of the D1/D2 domains of the 26S rRNA gene and the internal transcribed spacer (ITS) region. Phylogenetic analyses of sequence data placed the putative novel species in a clade with R. acheniorum and the dimorphic smut fungus Farysia chardoniana. A novel anamorphic genus, Farysizyma, is created to accommodate the three undescribed species, which were named Farysizyma itapuensis, Farysizyma setubalensis and Farysizyma taiwaniana. A new combination, Farysizyma acheniorum, is proposed for R. acheniorum, which may represent the yeast-phase anamorph of Farysia thuemenii.     

Characterization of basidiomycetous yeasts in hypersaline soils of the Urmia Lake National Park,Iran

Urmia Lake, located in northwest Iran, is an oligotrophic and extremely hypersaline habitat that supports diverse forms of life. Owing to its unique biodiversity and special environmental conditions, Urmia Lake National Park has been designated as one of the biosphere reserves by UNESCO. This study was aimed to characterize basidiomycetous yeasts in hypersaline soils surrounding the Urmia Lake National Park using a polyphasic combination of molecular and physiological data. Soil samples were collected from eight sites in Lake Basin and six islands insides the lake. Yeast strains were identified by sequencing the D1/D2 domains of the 26S rRNA gene. When D1/D2 domain sequencing did not resolve the identity of the species, strain identification was obtained by ITS 1 & 2 sequencing. Twenty-one species belonging to the genera Cystobasidium, Holtermanniella, Naganishia, Rhodotorula, Saitozyma, Solicoccozyma, Tausonia, Vanrija, and Vishniacozyma were identified. Solicoccozyma aeria represented the dominant species. The ability of isolates to grow at 10 and 15 % of NaCl was checked; about two-thirds of the strains grew at 10 %, while about 13 % of the isolates grew in medium with 15 % NaCl. this study is the first study on the culturable yeast diversity in hypersaline soils surrounding an Asian lake.     

Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river, Portugal

Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.     

Application of temperature gradient gel electrophoresis to the study of yeast diversity in the estuary of the Tagus river,Portugal

Temperature gradient gel electrophoresis (TGGE) was employed for the assessment of yeast diversity in the estuary of the Tagus river (Portugal). The molecular detection of yeasts was carried out directly from water samples and, in parallel, a cultivation approach by means of an enrichment step was employed. A nested PCR was employed to obtain a fungal amplicon containing the D2 domain of the 26S rRNA gene. For identification the TGGE bands were extracted, re-amplified, and sequenced. Fourteen fungal taxa were detected and all except one were yeasts. Most yeast sequences corresponded to members of the Ascomycota and only three belonged to the Basidiomycota. Five yeasts (four ascomycetes and one basidiomycete) could not be identified to the species level due to the uniqueness of their sequences. The number of species detected after enrichment was higher than the number of taxa found using the direct detection method. This suggests that some yeast populations are present in densities that are below the detection threshold of the method. With respect to the analysis of the yeast community structure, our results indicate that the dominant populations belong to Debaryomyces hansenii, Rhodotorula mucilaginosa, Cryptococcus longus, and to an uncultured basidiomycetous yeast phylogenetically close to Cr. longus. The combined analysis of direct detection and cultivation approaches indicates a similar community structure at the two sampled sites since nine species were present at both localities.     

Reinstatement of Rhodotorula colostri (Castelli) Lodder and Rhodotorula crocea Shifrine & Phaff, former synonyms of Rhodotorula aurantiaca (Saito) Lodder

Rhodotorula aurantiaca (Saito) Lodder is an anamorphic basidiomycetous yeast species that belongs to the so-called "Erythrobasidium lineage" of the Urediniomycetes, according to molecular phylogenetic studies based on nucleotide sequence analyses of different ribosomal DNA regions. In the most recent editions of the yeast taxonomy treatises the species Rhodotorula colostri (Castelli) Lodder and Rhodotorula crocea Shifrine & Phaff were listed as synonyms of R. aurantiaca. Taxonomic heterogeneity within R. aurantiaca was demonstrated in a study based on whole-cell protein profiles and is also hinted at by the observed differences in physiological and biochemical characteristics among the different strains under that species name. We determined partial nucleotide sequences of the 26S rRNA gene (D1/D2 domains) of strains maintained in the CBS culture collection under R. aurantiaca, including the type strains of its synonyms. The results showed that R. colostri and R. crocea are clearly distinct from R. aurantiaca and from any other currently recognised basidiomycetous yeast species. Furthermore, phylogenetic analysis of the sequence data placed the former two species in separate lineages of the Microbotryomycetidae: R. colostri in the "ruineniae clade" (Sporidiobolus lineage or Sporidiobolales) and R. crocea loosely linked to Rhodotorula javanica (Microbotryum lineage).     

Molecular identification of yeasts from soils of the alluvial forest national park along the river Danube downstream of Vienna, Austria ("Nationalpark Donauauen")

We analysed the diversity of yeasts from different soils in a river-floodplain landscape at the river Danube downstream of Vienna, Austria ("Nationalpark Donauauen"). 136 strains were isolated, identification of species was done with molecular methods. Partial sequencing of the 26S rRNA gene resulted in 36 different sequences, they could be assigned to 16 genera, apart from two sequence types (from three isolates), which were not clearly assigned to any genus. 18 species were identified and confirmed by means of PCR fingerprinting. The most frequently isolated genus was Cryptococcus (61 isolates and 12 sequence types). Basidiomycetes dominated with about 60% above the members of the Ascomycetes. About half the yeasts was isolated from the litter, the quantity decreased with soil depth.     

Stability of microbial communities in goat milk during a lactation year: molecular approaches

The microbial communities in milks from one herd were evaluated during 1-year of lactation, using molecular methods to evaluate their stability and the effect of breeding conditions on their composition. The diversity of microbial communities was measured using two approaches: molecular identification by 16S and 18S rDNA sequencing of isolates from counting media (two milks), and direct identification using 16S rDNA from clone libraries (six milks). The stability of these communities was evaluated by counting on selective media and by Single Strand Conformation Polymorphism (SSCP) analysis of variable region V3 of the 16S rRNA gene and variable region V4 of the 18S rRNA gene. One hundred and eighteen milk samples taken throughout the year were analyzed. Wide diversity among bacteria and yeasts in the milk was revealed. In addition to species commonly encountered in milk, such as Lactococcus lactis, Lactococcus garvieae, Enterococcus faecalis, Lactobacillus casei, Leuconostoc mesenteroides, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus caprae, Staphylococcus equorum, Micrococcus sp., Kocuria sp., Pantoea agglomerans and Pseudomonas putida, sequences were affiliated to other species only described in cheeses, such as Corynebacterium variabile, Arthrobacter sp., Brachybacterium paraconglomeratum, Clostridium sp. and Rothia sp. Several halophilic species atypical in milk were found, belonging to Jeotgalicoccus psychrophilus, Salinicoccus sp., Dietza maris, Exiguobacterium, Ornithinicoccus sp. and Hahella chejuensis. The yeast community was composed of Debaryomyces hansenii, Kluyveromyces lactis, Trichosporon beigelii, Rhodotorula glutinis, Rhodotorula minuta, Candida pararugosa, Candida intermedia, Candida inconspicua, Cryptococcus curvatus and Cryptococcus magnus. The analyses of microbial counts and microbial SSCP profiles both distinguished four groups of milks corresponding to four periods defined by season and feeding regime. The microbial community was stable within each period. Milks from winter were characterized by Lactococcus and Pseudomonas, those from summer by P. agglomerans and Klebsiella and those from autumn by Chryseobacterium indologenes, Acinetobacter baumanii, Staphylococcus, Corynebacteria and yeasts. However, the composition of the community can vary according to factors other than feeding. This study opens new investigation fields in the field of raw milk microbial ecology.     

Isolation and characterization of ethanol-producing yeasts from fruits and tree barks

Aims:  Isolation and identification of yeasts converting xylose to ethanol.
Methods and Results:  A total of 374 yeasts were isolated from a variety of rotten fruits and barks of trees. Out of these, 27 yeast strains were able to assimilate xylose and produce 0·12–0·38 g of ethanol per gram of xylose. Based on phylogenetic analysis of D1/D2 domain sequence of LSU (Large Subunit) rRNA gene and phenotypic characteristics the ethanol-producing strains were identified as member(s) of the genera Pichia, Candida , Kluyveromyces, Issatchenkia, Zygosacchraomyces , Clavispora, Debaryomyces , Metschnikowia , Rhodotorula and Cryptococcus.
Conclusion:  Yeast strains producing ethanol from xylose have been isolated from a variety of rotten fruits and barks of trees and identified.
Significance and Impact of the Study:  Environmental isolates of yeasts which could convert xylose to ethanol could form the basis for bio-fuel production and proper utilization of xylan rich agricultural and forest wastes.     

Degradation of Toluene Hydrocarbon by Isolated Yeast Strains: Molecular Genetic Approaches for Identification and Characterization

Removal of petroleum benzene, toluene, and xylene compounds from the environment is necessary to ensure quality life. In this research, 41 yeasts were isolated from oily soils. Among them, nine yeasts named KKUs (A5, A6, A12, A20, A23, A24, A26, A29, and A38) were selected based on their use of benzene, toluene, and xylene as a sole carbon and energy source. Based on their growth rates, all selected yeasts displayed a high efficiency for toluene degradation, but had no ability to degrade benzene and a low ability to degrade xylene, except A29 and A38, which could not degrade xylene. HPLC analysis for toluene removal indicated that A6, A12, A20, A23, A24, and A26 almost completely removed the toluene compound after 3 days of incubation (92.74, 94.61, 95.05, 91.74, 91.85, and 97.29%, respectively). In addition, strains A29 and A38 showed moderate degradation (88.29 and 85.30%, respectively), while the ability of A5 was low (39.00%). The isolates were identified based on amplifying and sequencing the D1/D2 domain of the 26S rRNA gene. Alignments and comparisons of the 26S rRNA gene sequences of the isolates with those available in GenBank, plus phylogenetic analysis, proved isolates as Rhodotorula lactose KKU-A5, Rhodotorula nymphaeae KKU-A6, Rhodotorula graminis KKU-A12, Rhodotorula minuta KKU-A20, Exophiala dermatitidis KKU-A23, Candida davisiana KKU-A24, Rhodotorula slooffiae KKU-A26, Rhodotorula mucilaginosa KKU-A29, and Rhodosporidium diobovatum KKU-A38. Random amplified polymorphic DNA-PCR fingerprinting was accomplished within seven toluene-degrading red yeasts (A5, A6, A12, A20, A26, A29, and A38). The results indicated no correlation between the random amplified polymorphic DNA profile and the geographic origin of the isolates.     

Yeasts in an industrial malting ecosystem

The malting ecosystem consists of two components: the germinating cereal grains and the complex microbial community. Yeasts and yeast-like fungi are an important part of this ecosystem, but the composition and the effects of this microbial group have been largely unknown. In this study we surveyed the development of yeasts and yeast-like fungi in four industrial scale malting processes. A total of 136 malting process samples were collected and examined for the presence of yeasts growing at 15, 25 and 37°C. More than 700 colonies were isolated and characterized. The isolates were discriminated by PCR-fingerprinting with microsatellite primer (M13). Yeasts representing different fingerprint types were identified by sequence analysis of the D1/D2 domain of the 26S rRNA gene. Furthermore, identified yeasts were screened for the production of α-amylase, β-glucanase, cellulase and xylanase. A numerous and diverse yeast community consisting of both ascomycetous (25) and basidiomycetous (18) species was detected in the various stages of the malting process. The most frequently isolated ascomycetous yeasts belonged to the genera Candida, Clavispora, Galactomyces, Hanseniaspora, Issatchenkia, Pichia, Saccharomyces and Williopsis and the basidiomycetous yeasts to Bulleromyces, Filobasidium, Cryptococcus, Rhodotorula, Sporobolomyces and Trichosporon. In addition, two ascomycetous yeast-like fungi (black yeasts) belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Knowledge of the microbial diversity provides a basis for microflora management and understanding of the role of microbes in the cereal germination process.     

Isolation and identification of crude oil-degrading yeast strains from Khafji oil field,saud Arabia

Twenty five yeasts isolated were isolated from Khurais oil field in Saudi Arabia and assayed to evaluate their biodegradability. Only five isolates (namely, A1, A2, A3, A4 and A5) showed potential use of oil as sole carbon source. During incubation period, highest growth rate were recorded for A1, A2 and A3 isolates. Low growth distinguished A4 isolate; A5 isolate could not degrade oil.Spectrophotometrical analysis for four yeast isolates biodegradation activities indicated that, A1 isolate was superior for oil degradation (61%) comparing with A4 isolate which reflected lowest degradation % (33%). A2 and A3 isolates showed moderate biodegradation activity (56 and 51% respectively).D1/D2 domain of the 26S rRNA gene sequence was used as molecular marker to identify five yeast isolates. After comparing 26S rRNA gene sequences of five yeast isolates with highly similarity isolates, five yeast isolates (A1, A2, A3, A4 and A5)were submitted to database as Candida tropicalis (MW488263), Candida tropicalis (MW488264), Rhodotorula mucilaginosa (MW488265) and Rhodosporidium toruloides (MW488266) respectively. Using OXF1/ACR1 primer, specific lipase gene amplicon with 250 bp were detected with in all four yeast isolates.     

New lipid-dependent Malassezia species from parrots

BackgroundAll the currently recognized Malassezia species have been isolated from mammals. However, only a few of them have been isolated from birds. In fact, birds have been less frequently studied as carriers of Malassezia yeasts than mammals.AimIn this study we describe two new taxa, Malassezia brasiliensis sp. nov. and Malassezia psittaci sp. nov.MethodsThe isolates studied in this publication were isolated from pet parrots from Brazil. They were characterized using the current morphological and physiological identification scheme. DNA sequencing and analysis of the D1/D2 regions of the 26S rRNA gene, the ITS-5.8S rRNA gene sequences and the β-tubulin gene were also performed.ResultsThe strains proposed as new species did not completely fit the phenotypic profiles of any the described species. The validation of these new species was supported by analysis of the genes studied. The multilocus sequence analysis of the three loci provides robust support to delineate these species.ConclusionsThese studies confirm the separation of these two new species from the other species of the genus Malassezia, as well as the presence of lipid-dependent Malassezia yeasts on parrots.     

The yeast flora of some decaying mushrooms on trunks of living trees

Several ascomycetous and basidiomycetous yeasts were isolated from rotten mushrooms on the trunks of beech and tamarisk trees. One strain, identified as the novel species Cryptococcus allantoinivorans, assimilated allantoin as the sole carbon source. Phylogenetically it belongs to the C. laurentii complex, Papiliotrema bandonii being the closest relative. Some ascomycetous strains could not be distinguished from Pichia guillermondii, but deviated considerably in rDNA sequences. In addition to these species, both decaying mushrooms were inhabited by more common species, viz. Candida albicans, C. saitoana, Rhodotorula mucilaginosa, Trichosporon asahii, T. multisporum and T. porosum. The basidiomycetous yeasts, except R. mucilaginosa, assimilated some polysaccharides of plant origin.     

Assessment of yeast diversity in a marine environment in the south of portugal by microsatellite-primed PCR

The occurrence and diversity of yeasts in seawater was investigated in a study site located 20 Km off Faro, Portugal, above the álvares Cabral Trench. A total of 43 water samples from different layers (above the permanent thermocline, under the thermocline and near the bottom) and directly from the surface, originated 234 isolates. All the isolates were identified using a molecular approach that included, in a first stage, MSP-PCR fingerprinting. A total of 31 MSP-PCR classes were formed, 8 for the pigmented yeasts and 23 for the non-pigmented yeasts. The pink coloured isolates were identified by direct comparison of the new fingerprints with those obtained for representative strains of the various species. For identification of the non-pigmented yeasts, a representative isolate of each MSP-PCR class was selected for sequence analysis and compared with reference sequences. The five most abundant yeast species were Sakaguchia dacryoidea, Pseudozyma aphidis, Rhodosporidium babjevae, R. diobovatum and Debaryomyces hansenii. The distribution of isolates and species in the major taxonomic groups indicated that the number of basidiomycetous yeasts and their diversity are prevalent in relation to their ascomycetous counterpart. Diversity indices were determined and superficial water and water near the bottom had the highest diversity. The sampling effort effectiveness was estimated, and found to correspond to approximately 60% of the species present. MSP-PCR identification proved suitable for pigmented basidiomycetous yeasts and, when used in conjunction with sequence analysis, was effective for the characterization of non-pigmented populations. Our results indicate that the MSP-PCR fingerprinting method is appropriate for the characterization of large groups of isolates due to its simplicity and good reproducibility. This revised version was published online in August 2006 with corrections to the Cover Date.     

我国各类基物上的酵母菌分布及其尿素酶活性

本文讨论了2300株由我国各地分离到的酵母菌和类酵母菌与各类基物的关系。共述及23属,占Lodder(1970)系统的半数以上。在十几类基物中酵母菌的分布各有不同,叶子上有大量掷孢酵母,果实上比其它基物更能获得克勒克酵母属(Kloeckera),在咸腌食品中相对地有较多耐高渗透压的酵母如球拟酵母属(Torulopsis)与德巴利酵母属(Debaryomyces)。对部分属的定种工作也已进行,说明某些属的种在我国均有发现而且为数不少。此外还研究了600余株代表各类酵母的尿素酶活性,结果说明担子菌酵母或系统上接近担子菌的酵母都有尿素酶活性,而子囊菌酵母则无。根据作者的观察子囊菌酵母中裂殖酵母属(Schizosaccharomyces)则常常例外地有尿素酶反应,且常有辅酶Q_(10),从而可见裂殖酵母属有其特有的生物学特性,在系统上很可能与其它子囊菌酵母不同。对属于不完全菌的无孢子酵母也进行了尿素酶活性测定,结果表明它们部分与子囊菌有关,部分与担子菌有关。     

Myo-inositol assimilating new species of Rhodotorula Harrison

A new species of anamorphic basidiomycetous yeasts, Rhodotorula pinalis, was isolated from needle litter of Pinus sylvestris L. collected in Moscow oblast (Russia). The cultures are nonpigmented; nitrate- and myo-inositol-positive, forming no ballistoconidia.     

Matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry- and MALDI biotyper-based identification of cultured biphenyl-metabolizing bacteria from contaminated horseradish rhizosphere soil

Bacteria that are able to utilize biphenyl as a sole source of carbon were extracted and isolated from polychlorinated biphenyl (PCB)-contaminated soil vegetated by horseradish. Isolates were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The usage of MALDI Biotyper for the classification of isolates was evaluated and compared to 16S rRNA gene sequence analysis. A wide spectrum of bacteria was isolated, with Arthrobacter, Serratia, Rhodococcus, and Rhizobium being predominant. Arthrobacter isolates also represented the most diverse group. The use of MALDI Biotyper in many cases permitted the identification at the level of species, which was not achieved by 16S rRNA gene sequence analyses. However, some isolates had to be identified by 16S rRNA gene analyses if MALDI Biotyper-based identification was at the level of probable or not reliable identification, usually due to a lack of reference spectra included in the database. Overall, this study shows the possibility of using MALDI-TOF MS and MALDI Biotyper for the fast and relatively nonlaborious identification/classification of soil isolates. At the same time, it demonstrates the dominant role of employing 16S rRNA gene analyses for the identification of recently isolated strains that can later fill the gaps in the protein-based identification databases.     

Identification of Clinically Relevant Fungi and Prototheca Species by rRNA Gene Sequencing and Multilocus PCR Coupled with Electrospray Ionization Mass Spectrometry

Background

Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse.

Methods

One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5′ end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS.

Results

For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively.

Conclusions

rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.