Application of ITS sequence analysis, RAPD and AFLP fingerprinting in characterising the yeast genus Fellomyces

Three molecular techniques, ITS sequence analysis, random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to study phylogenetic and genotypic relationships among strains of the genus Fellomyces. In the analyses were included strains isolated predominantly from epiphytic lichens collected in Indonesia, China and Mexico. The polyphasic approach indicated that the Fellomyces isolates are genotypically heterogeneous and that lichens represent a specific environment for selection of large number of the sterigmatoconidia producing species. The phylogenetic and genotypic analysis confirmed the existence of 11 currently accepted Fellomyces species and indicated that several species may be the new representatives of the genus. The RAPD and AFLP analyses demonstrated a higher potential in distinguishing the Fellomyces strains than the ITS regions. Since the sequence analysis showed low or no divergence among several strains, both RAPD and AFLP fingerprinting indicated that the strains may be discriminated at the species level.     

Phylogenetic relationships among species of Sterigmatomyces and Fellomyces as determined from partial rRNA sequences

Sequence comparisons of selected regions from small (18S) and large (25S) subunit rRNAs were used to examine species relationships in the anamorphic yeast genera Sterigmatomyces, Fellomyces, Tsuchiyaea, and Kurtzmanomyces. On the basis of sequence similarity, the genus Sterigmatomyces is comprised of Sterigmatomyces halophilus and Sterigmatomyces elviae, while the genus Fellomyces contains three recognized species, Fellomyces fuzhouensis, Fellomyces penicillatus, and Fellomyces polyborus. Tsuchiyaea wingfieldii and Kurtzmanomyces nectairii are well separated from the other species which we examined. Comparison with selected teleomorphs indicated that the genus Fellomyces is closely related to the genus Sterigmatosporidium, whereas the genus Sterigmatomyces exhibited somewhat closer relatedness with the genus Leucosporidium. Impacting on our estimates of relatedness was the finding that nucleotide substitution in the rRNA regions which we examined seems relatively constant only among closely related species.     

Fellomyces horovitziae sp. nov., a new basidiomycetous yeast species isolated from aXenasmatella basidiocarp

A new species of the genusFellomyces, F. horovitziae, was isolated from aXenasmatella basidiocarp. It differs from other accepted species in its carbon assimilation pattern, mol% G+C and low DNA-DNA homology. The delimiting characters are discussed and a key to the genus is provided.     

The cytoskeleton in the unique cell reproduction by conidiogenesis of the long-neck yeast Fellomyces (Sterigmatomyces) fuzhouensis

Summary. The morphology of conidiogenesis and associated changes in microtubules, actin distribution and ultrastructure were studied in the basidiomycetous yeast Fellomyces fuzhouensis by phase-contrast, fluorescence, and electron microscopy. The interphase cell showed a central nucleus with randomly distributed bundles of microtubules and actin, and actin patches in the cortex. The conidiogenous mother cell developed a slender projection, or stalk, that contained cytoplasmic microtubules and actin cables stretched parallel to the longitudinal axis and actin patches accumulated in the tip. The conidium was produced on this stalk. It contained dispersed cytoplasmic microtubules, actin cables, and patches concentrated in the cortex. Before mitosis, the nucleus migrated through the stalk into the conidium and cytoplasmic microtubules were replaced by a spindle. Mitosis started in the conidium, and one daughter nucleus then returned to the mother via an eccentrically elongated spindle. The cytoplasmic microtubules reappeared after mitosis. A strong fluorescence indicating accumulated actin appeared at the base of the conidium, where the cytoplasm cleaved eccentrically. Actin patches then moved from the stalk together with the retracting cytoplasm to the mother and conidium. No septum was detected in the long neck by electron microscopy, only a small amount of fine “wall material” between the conidium and mother cell. Both cells developed a new wall layer, separating them from the empty neck. The mature conidium disconnected from the empty neck at the end-break, which remained on the mother as a tubular outgrowth. Asexual reproduction by conidiogenesis in the long-neck yeast F. fuzhouensis has unique features distinguishing it from known asexual forms of reproduction in the budding and fission yeasts. Fellomyces fuzhouensis develops a unique long and narrow neck during conidiogenesis, through which the nucleus must migrate into the conidium for eccentric mitosis. This is followed by eccentric cytokinesis. We found neither an actin cytokinetic ring nor a septum in the long neck, from which cytoplasm retracted back to mother cell after cytokinesis. Both the conidium and mother were separated from the empty neck by the development of a new lateral wall (initiated as a wall plug). The cytoskeleton is clearly involved in all these processes. Correspondence and reprints: Department of Biology, Faculty of Medicine, Masaryk University, Tomešova 12, 602 00 Brno, Czech Republic.     

Differentiation of Shigella strains by plasmid profile analysis, serotyping and phage typing

Ninety-one Shigella flexneri and 29 Shigella sonnei strains isolated during 1994 from sporadic cases of shigellosis and healthy carriers were analyzed for plasmid profile in order to compare the discriminating ability of this method with that of serotyping and phage typing. Our study revealed 10 plasmid profiles (PP) among S. sonnei strains. A total of 26 out of 29 (89%) S. sonnei isolates could be placed into two phage types (type 1 and 20) comprising four PP for phage type 1 and seven PP for type 20, respectively. Twenty-three different PP were identified among S. flexneri strains. Each serotype was associated with a specific predominant plasmid profile, except serotype 2a. This serotype, the most frequently isolated in Romania, was still rather homogeneous: 33 out of 39 isolates belonged to phage type 125, 27 of which could be placed into two related PP (F10 and F17). Comparison of plasmid patterns of epidemiologically independent S. flexneri serotype 2a isolates with those exhibited by 45 serotype 2a isolates associated to six independent outbreaks revealed the same homogeneity. Thirty-eight strains, representing 4 of 6 outbreaks, had F10 and F17 plasmid patterns. The discrimination indices (D) for plasmid profile analysis alone (D = 0.890) and for the combination of serotyping and phage typing (D = 0.841) indicate that both typing systems have a nearly similar ability of discriminating among S. flexneri strains. By combining the results of the three typing methods, a total of 42 types are distinguished and the D value is 0.942. Our data suggest that plasmid profile analysis can complement phenotyping methods resulting in a degree of discrimination that cannot be achieved by either system alone.     

Interspecific differences in cryoresistance of lichen symbiotic algae of genus Trebouxia assessed by cell viability and chlorophyll fluorescence

Unicellular algae of genus Trebouxia are the most frequent symbiotic photobionts found in lichen species adapted to extreme environments. When lichenised, they cope well with freezing temperature of polar regions, high-mountains environments and were successfully tested in open-space experiments. Trebouxia sp. is considered potential model species for exobiological experiments. The aim of this paper is to evaluate cryoresistence of Trebouxia sp. when isolated from lichen thalli and cultivated on media. In our study, six algal strains were exposed to repeated freezing/thawing cycles. The strains of Trebouxia sp. (freshly isolated from lichen Lasallia pustulata), Trebouxia erici, Trebouxia asymmetrica, Trebouxia glomerata, Trebouxia irregularis, and Trebouxia jamesii from culture collection were cooled from 25 to -40 °C at two different rates. The strains were also shock frozen in liquid nitrogen. After repeated treatment, the strains were inoculated and cultivated on a BBM agar for 7 days. Then, cell viability was assessed as relative share of living cells. Potential quantum yield of photochemical reactions in PS II (F(V)/F(M)), and effective quantum yield of photochemical reactions in PS II (Φ(PSII)) were measured. While the slow cooling rate (0.5 °C min(-1)) did not cause any change in viability, F(V)/F(M), and Φ(PSII), the fast cooling rate (6.0 °C min(-1)) caused species-specific decrease in all parameters. The most pronounced interspecific differences in cryoresistance were found after shock freezing and consequent cultivation. While T. asymmetrica and T. jamesii exhibited low viability of living cells (18.9% and 34.7%) and full suppression of photosynthetic processes, the other strains had viability over 60%, and unaffected values of F(V)/F(M), and Φ(PSII). This indicated a high degree of cryoresistance of T. glomerata, T. erici, T. irregularis and Trebouxia sp. strains. These strains could be used for detailed investigation of underlying physiological mechanisms and as models for astrobiological tests taken in the Earth facilities.     

CULTURAL AND MOLECULAR CHARACTERIZATION OF PHOTOBIONTS OFPELTIGERA MEMBRANACEA

A molecular study was undertaken to clarify the identity of the photobiont in colourmorphs of the lichen,Peltigera membranaceaTwo strains of cyanobacteria, identified asNostocsp. by morphology, were cultivated from each of two lichen specimens. Prokaryotic (16S) ribosomal RNA gene fragments were amplified by the polymerase chain reaction (PCR) from DNA extracted from the isolated strains and the lichens, and sequenced directly. Sequences were 98·1% identical between lichen specimens, TDI#AR94 and TDI#AR95, and highly similar to sequences published, or generated in this study from a type culture, forNostocThe 16S ribosomal RNA gene sequences (‘ 16S rDNA ’) of all four lichen-derived cyanobacteria appeared the same, even though the lichen specimens from which they originated had different sequences. The 16S rDNA from strains 9A and 9B were different from that of specimen TDI#AR94, the thallus from which they were isolated, and instead were the same as that of strains 10A and 10B, and their source, specimen TDI#AR95. When primers selective for the strain 9A sequence were used, however, a small amount of PCR product corresponding to the 16S rDNA of strain 9A was obtained from lichen TDI#AR94. The results confirm that the photobionts ofP. membranaceabelong toNostocand suggest that genetic differences in the photobiont may be a factor in the occurrence of colourmorphs among cyanolichens.     

The Phylogenetic Relationship of the Conidium-forming Anamorphic Yeast Genera Sterigmatomyces,Kurtzmanomyces, Tsuchiyaea and Fellomyces,and the Teleomorphic Yeast Genus Sterigmatosporidium on the Basis of the Partial Sequences of 18S and 26S Ribosomal Ribonucleic Acids

The eleven strains of conidium-forming yeasts classified in the basidiomycetous anamorphic genera Sterigmatomyces, Kurtzmanomyces, Tsuchiyaea and Fellomyces, and the teleomorphic genus Sterigmatosporidium were examined as to the partial sequences of 18S rRNA and 26S rRNA. The positions determined (in Saccharomyces cerevisiae) were 1451 through 1618 of 18S rRNA, and 1618 through 1835 and 470 through 626 of 26S rRNA. The partial sequence determination of positions 470 through 626 of 26S rRNA indicated that T. wingfieldii should be included in the group comprised of Fellomyces and Sterigmatosporidium species. However, the genera Sterigmatomyces, Kurtzmanomyces, Tsuchiyaea and Fellomyces constituted their own separate clusters as to the partial sequences of positions 1451 through 1618 of 18S rRNA and 1618 through 1835 of 26S rRNA. The classification of the conidium-forming yeasts in the four basidiomycetous anamorphic genera mentioned above was proved to be reasonable from the phylogenetic point of view.     

利用26S rDNAD1/D2区序列和微卫星标记分析各种工业酿酒酵母的种内遗传差异

选取30株传统发酵工业中不同来源的酿酒酵母菌株和实验室常用酿酒酵母菌株,利用大亚基(26S)rDNA D1/D2区序列和微卫星标记分析酿酒酵母种内菌株间的遗传多样性和系统发育关系,以揭示酿酒酵母在长期的各种工业发酵环境下发生的遗传变异。结果表明:30株酿酒酵母的26S rDNA D1/D2区序列(591bp)比较保守,与测序菌株S288C相比序列相似度在99.8%–100%,说明种内菌株间存在一定的多态性,但序列差异并不十分明显,其变异情况表现在个别碱基的差异(大多由转换突变引起);通过扩增11个微卫星位点,每个菌株均有其独特的基因型,即30种基因型,共得到188个等位基因,观测杂合度平均值和期望杂合度平均值分别为0.576、0.886,多态信息含量平均值高达0.858,说明酿酒酵母种内菌株间具有较高的遗传多样性,而聚类分析表明30株酿酒酵母可以得到很好地区分,但是没有呈现与其工业来源相关的聚类。     

基于26S rDNAD1/D2区序列分析的15株白地霉分子分类学研究

采用26S rRNA基因D1/D2区系统发育分析的方法对CICC(中国工业微生物菌种保藏管理中心)保藏的15株白地霉(Geotrichum candidum)菌种进行复核鉴定。系统发育分析结果表明15株白地霉属于地霉属的成员,且形成两个系统发育分支,系统发育上最接近Galactomyces geotrichum NRRLY-17569T,与其同源性为96.3%～98.3%。15株白地霉26S rRNA基因D1/D2区序列显著不同于地霉属的模式种及其它种,可能代表地霉属的两个新种,但这一结论尚需进一步的实验去证实。     

对5个S180克隆细胞株RAPD特征的研究

目的运用随机扩增多态性DNA(RAPD)技术建立S180的5个克隆细胞株的特异性图谱。方法运用23条引物对S180-S2D9、S180-S2D7、S180-S1F11、S180-S1H10、S180-S1B115个克隆细胞株的基因组DNA进行RAPD-PCR扩增,以琼脂糖电泳观察结果。结果筛选出3条在各克隆株扩增产物间有差异的引物。结论5个S180克隆细胞株的RAPD特征有明显区别。     

Pichia koratensis sp. nov., a new ascomycetous yeast related to Pichia acaciae isolated from insect frass in Thailand

Two yeast strains (ST-235 and ST-237) isolated from insect frass collected in northeastern Thailand, were identified as Pichia acaciae and a hitherto undescribed species based on the sequence analysis of the D1/D2 domain of 26S rDNA, ITS regions, and DNA-DNA reassociation experiments. The latter strain, ST-237 (=BCC 11769=NBRC 103638=JCM 12576), was described as Pichia koratensis sp. nov. Pichia koratensis is closely related to Pichia acaciae in the D1/D2 domain sequence of 26S rDNA and phenotypical characteristics but differs by 5 nucleotides (0.9%) from the type strain of P. acaciae and is clearly distinguished by the assimilation and fermentation of sucrose.     

Genotyping of a miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii, based on sequence analysis of the partial 26S ribosomal RNA gene and two internal transcribed spacers

We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), the internal transcribed spacer 1, the 5.8S ribosomal RNA gene, and the internal transcribed spacer 2 (the ITS sequence) from 46 strains of miso and soy sauce fermentation yeast, Zygosaccharomyces rouxii and a closely related species, Z. mellis, for typing. Based on the 26S rDNA sequence analysis, the Z. rouxii strains were of two types, and the extent of sequence divergence between them was 2.6%. Based on the ITS sequence analysis, they were divided into seven types (I-VII). Between the type strain (type I) and type VI, in particular, a 12% difference was detected. The occurrence of these nine genotypes with a divergence of more than 1% in these two sequences suggests that Z. rouxii is a species complex including novel species and hybrids. Z. mellis strains were of two types (type alpha and type beta) based on the ITS sequence. Z. rouxii could clearly be distinguished from Z. mellis by 26S rDNA and ITS sequence analyses, but not by the 16% NaCl tolerance, when used as the sole key characteristic for differentiation between the two species.     

葡萄果粒表皮酵母菌多样性研究

从新疆、甘肃、陕西、宁夏、山东主要酿酒葡萄产区收集葡萄果粒并分离得到酵母258株,利用26S rDNA的D1/D2区序列分析并结合形态学、生理学特征对这些菌株进行了分类学研究,探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布.共鉴定出13属26种,其中优势属为汉逊酵母属Hanseniaspora(5种),假丝酵母属Candida(4种),毕赤酵母属Pichia(4种)和伊萨酵母属Issatchenkia(2种).对分离自不同地域的同种内不同菌株进行了D1/D2序列分析比较,以探讨不同地理起源地酵母种内序列稳定性及其变异.     

葡萄果粒表皮酵母菌多样性研究

从新疆、甘肃、陕西、宁夏、山东主要酿酒葡萄产区收集葡萄果粒并分离得到酵母258株, 利用26S rDNA的D1/D2区序列分析并结合形态学、生理学特征对这些菌株进行了分类学研究, 探讨了这些地区葡萄果粒表皮酵母的物种多样性及其分布。共鉴定出13属26种, 其中优势属为汉逊酵母属Hanseniaspora(5种), 假丝酵母属Candida(4种), 毕赤酵母属Pichia(4种)和伊萨酵母属Issatchenkia(2种)。对分离自不同地域的同种内不同菌株进行了D1/D2序列分析比较, 以探讨不同地理起源地酵母种内序列稳定性及其变异。     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

Two new ballistoconidium-forming yeast species, Bullera melastomae and Bullera formosana, found in Taiwan

Two yeast strains, the cells of which contained xylose and Q-10 as the major ubiquinone, were isolated from a plant leaf collected in Taiwan. These yeasts were found to represent two new species of the genus Bullera in the Hymenomycetes. Identification was based on the sequence analysis of the 18S rDNA, the internal transcribed spacer (ITS) regions and the D1/D2 domain of 26S rDNA. The yeasts are named Bullera melastomae sp. nov. and Bullera formosana sp. nov. In the phylogenetic trees based on 18S rDNA and D1/D2 domain of 26S rDNA sequences, these two species constitute a cluster connected with Dioszegia cluster in the Cryptococcus luteolus lineage.     

Evaluation of the usefulness of selected commercial systems for identification of Streptococcus species

The usefulness was assessed of three commercially available systems for rapid identification of streptococcal strains. The studied material comprised 68 strains of streptococci and enterococci (including 24 standard strains) belonging to serological groups: A (14 strains), B (10), C (11), D (10), F (3) and G (10), as well as 10 S. pneumoniae strains. The strains were isolated from throat, nasal, wound swabs, blood, pus of inpatients and throat and nasal swabs of outpatients. For the identification of streptococci 3 commercially available systems were used: API 20 STREP (bioMerieux, France), rapid ID 32 Strep (bioMerieux, France), Streptoplast PPL 18 (HTL, Poland). The determinations were done according to producer's instructions. The highest percent of correctly identified strains was obtained with the rapid ID 32 Strep--80.9%, with the API 20 STREP--76.4% strains were identified correctly and with the PPL 18--61.8%. The study showed that the API 20 STREP and rapid ID 32 STREP are suitable for the identification of streptococcal strains from groups: A, B, C, D, F and enterococci--group D. The proportions of correctly identified strains from these groups with the Streptoplast PPL 18 were lower than those determined with the bioMerieux systems. Using of three identification systems streptococci from group G and S. pneumoniae strains cannot be identified.     

<Emphasis Type="Italic">Metschnikowia viticola</Emphasis> sp. nov., a new yeast species from grape

Two yeast strains, producing needle-shaped ascospores under suitable conditions, were isolated from grapes grown in Hungary. Based on these two strains, Metschnikowia viticola (type strain NCAIM Y.01705, CBS 9950, JCM 12561) is proposed as a new yeast species. Considering its phenotypic features, the restriction fragment patterns of 18S rDNA and the sequence of the D1/D2 domain of 26S rDNA, the proposed new species is closely related to Candida kofuensis.     

Dictyochloropsis splendida (Chlorophyta), the correct phycobiont of Phlyctis argena and the high degree of selectivity or specificity involved

Phlyctis argena is lichenized with Dictyochloropsis splendida and not with D. reticulata, as thought formerly. This results from in-situ investigation of over 150 lichen thalli and more than 70 cultures of the phycobiont. Material studied from a wide range of geographical locations indicates that specificity of the association in this case is highly probable. Compared to the free-living state, the alga as a lichen partner undergoes distinct changes. Differentiation of strains of D. splendida with respect to morphology and development was not observed.