Chromosomal polymorphism in the yeast species Debaryomyces hansenii

Pulse field gel electrophoresis karyotypes of 41 strains of the genus Debaryomyces, including 35 strains confirmed as D. hansenii species by D1/D2 ribosomal DNA sequence analysis, were performed. Electrophoretic karyotypes of the 41 strains exhibited 4 to 10 chromosomal bands ranging between 0.7 Mb and 4.2 Mb. Among D. hansenii species, the patterns of strains obtained from the CBS collection and cheese isolates differed strongly from D. hansenii var. hansenii CBS767^T. Both D. hansenii var. hansenii and D. hansenii var. fabryii showed chromosome length polymorphism. Electrophoretic karyotypes of the D. hansenii strains were analyzed by Southern hybridization with various species-specific probes isolated from D. hansenii var. hansenii CBS767^T. Repeated sequences including the F01pro, M18pro, the Ty1-copia retrotransposon Tdh5 and hypothetical telomeric sequence hybridized to several chromosomal bands, while a D1/D2 probe derived from the large ribosomal sub-unit hybridized only to the largest chromosome. Unique probes such as those hybridizing to actin ACT1, glycerol-3-phosphate dehydrogenase GPD1 and β-glucosidase LAC4 encoding genes were assigned to specific chromosomal bands of D. hansenii var. hansenii CBS767^T. These probes failed to hybridize to D. hansenii var. fabryii strongly suggesting that strains of this variety actually represent a different taxon. This revised version was published online in August 2006 with corrections to the Cover Date.     

德巴利汉逊酵母的两个变种及其相关种的脉冲电泳核型比较分析

摘要:用CHEF(钳位均匀电场)脉冲电泳系统分析了德巴利汉逊酵母的两个变种及两个相关种的脉冲电泳核型。对每个分类群的染色体条数,染色体DNA的分子量大小范围及整个基因组大小作出了估算,结果如下:Debaryomyces hansenii(Zopf) Lodder et Kreger-van Rij var. hansenii具有6-7条染色体,分子量范围为1.2-2.6(个别3.5)Mb,整个基因组大小为I 0.6-14.9Mb;D. hansenii var. fabryi (Ota) Nakase et Suzuki具有7条染色体,分子量范围为0.7-2.4M b,整个基因组大小为12.0-12.7Mb;D. nepalensis Goto et Sugiyama具有6-8条染色体,分子量范围为(个别0.2)1.1-2.7Mb,整个基因组大小为10.6-11.0Mb;Candida saitoana Nakase et Suzuki具有10-11条染色体,分子量范围为0.6-3.6Mb,整个基因组大小为18.1-18.9Mb.本研究表明C. saitoana与上述德巴利酵母属的三个分类群在脉冲电泳核型上具有明显差异,而后三者之间在染色体DNA带型上却没有发现有价值的区别之处.     

Pulsed field gel electrophoresis reveals chromosome length differences between strains of Cladosporium fulvum (syn. Fulvia fulva)

Summary Methods are described for the electrophoretic separation of chromosome-sized DNA molecules from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva). Using a hexagonal electrode array and switching times of 75 min at 45 V for 14 days, nine bands could be resolved. By comparison with co-electrophoresed Aspergillus nidulans chromosomal DNA (which was resolved into seven bands), the sizes of the C. fulvum bands are estimated to be between 1.9 Mb and 5.4 Mb. The two largest bands are believed to be doublets, giving a minimum genome size of 44 Mb. Cloned probes for the ribosomal DNA repeat, an anonymous single copy fragment and a newly discovered retrotransposon were hybridized to blots of the pulsed field gels, demonstrating the use of this technique for genomic mapping. Most strains of C. fulvum had an identical pattern of bands. Two strains exhibited two polymorphisms which could be due to a translocation.     

Population Polymorphism of Nuclear Mitochondrial DNA Insertions Reveals Widespread Diploidy Associated with Loss of Heterozygosity in Debaryomyces hansenii

Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767^T, and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.Debaryomyces hansenii is a ubiquist, hemiascomycetous yeast that can be found in soil, fruits, and various manufactured foodstuff in which it participates by contributing to the maturation or as a contaminant. Its ability to grow at low temperatures and in high salinity environments makes it the most common yeast in cheeses, to which it brings a number of proteolytic and lipolytic activities and aromas in the course of maturation. D. hansenii has also been implicated as an emerging pathogen, sometimes under the name of Candida famata var. famata (see reference 17). Taxonomic classification of the species related to D. hansenii has always been subject to debate. Recent analyses have reinstated D. hansenii (previously D. hansenii var. hansenii), Debaryomyces fabryi (previously D. hansenii var. fabryi), and Debaryomyces subglobosus (previously Candida famata var. flareri) (13, 25). Phylogenetic analysis using conserved spliceosomal intron sequence comparison has shown that D. hansenii is a complex of species, which comprises at least four members: D. hansenii, Debaryomyces tyrocola, D. fabryi, and Candida flareri (previously Candida famata var. flareri) (18). In addition, our study has revealed the existence of at least three populations (clades 1 to 3) in D. hansenii, with the first one containing the strain CBS 767^T, which has been entirely sequenced (8), and the last one containing Candida famata var. famata CBS 1795.Most eukaryotic nuclear genomes contain pieces of mitochondrial sequences (designated NUMT [nuclear mitochondrial DNA] for nuclear sequences of mitochondrial origin) that result from the transfer of fragments of mitochondrial DNA (mtDNA) to the chromosomes. The number and size of the NUMTs varies greatly between eukaryotic genomes (33). A recent investigation of six hemiascomycetous yeasts has shown that even within this monophyletic group, the number of NUMTs varies greatly, from 1 in Kluyveromyces thermotolerans CBS 6340^T to 145 in D. hansenii CBS 767^T (36). The mtDNA is thought to invade nuclear genomes during the repair of chromosomal DNA double-strand breaks (DSB) by nonhomologous end joining (NHEJ), as shown experimentally in the yeast Saccharomyces cerevisiae (31, 44). The colonization of nuclear genomes by mtDNA is a dynamic evolutionary process, as observed in yeast and humans (3, 32).D. hansenii harbors the highest number of NUMTs known so far for hemiascomycetous yeasts, making it of particular interest for NUMT studies. Conversely, NUMTs are potentially interesting markers to differentiate strains of this species. The 145 NUMTs of type strain CBS 767^T are distributed in 86 loci (61 single NUMTs and 25 clusters). Most clusters (23, 25) are mosaics of NUMTs formed from noncontiguous mtDNA fragments inserted in random orientation at the same chromosomal locus. In the other two clusters, the NUMTs are all in the same orientation and order, as in the mitochondrial genome. These clusters (designated “processions”) correspond to a single ancient mtDNA insertion, followed by mutational decay, leaving recognizable mtDNA segments separated by more diverged sequences (36).Few studies have attempted to evaluate the variability of NUMTs within the same species (2, 23, 32). Here, we have studied natural isolates to assess the intraspecific variability of the NUMT insertions in the nuclear genome of the yeast species D. hansenii. We were able to structure populations in this species, to determine the chronology of the NUMT insertions, and to correlate this chronology to the taxonomy of the D. hansenii complex species. Moreover, NUMT analysis revealed the existence of both haploid and diploid strains, the latter resulting from crosses between different D. hansenii clades.     

Variation in the electrophoretic karyotype analysed by the assignment of DNA probes in Candida albicans

By using pulsed-field gel electrophoresis, we have separated the entire chromosome bands and examined the electrophoretic karyotypes of 27 strains of Candida albicans. The electrophoretic karyotype varied widely among these strains. Their chromosomal DNAs were resolved into 7-12 bands ranging in size from 0.42 to 3.0 Mb. Most of the separated chromosomal bands were assigned by eight cloned C. albicans DNA probes. These results suggest that the haploid number of C. albicans chromosomes is eight. Each of the probes hybridized specifically to one or two bands of similar size in most strains. With the exception of the MGL1 probe, when two bands were detected by one probe, the size of one of them was very conserved whilst the other was of fairly variable size. The sizes of the chromosome bands assigned by the MGL1 probe were much more variable. As C. albicans is considered to be a diploid organism, it is inferred that the karyotype polymorphism between strains is mainly derived from wide size heterogeneity in one of the homologous chromosomes. Furthermore, we have confirmed species-specific and strain-specific variation in medically important Candida species (C. stellatoidea, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. kefyr and C. glabrata). Electrophoretic karyotype analysis is thus useful for species assignation. The TUB2 probe, encoding C. albicans beta-tubulin, hybridized to the chromosomal DNA of all the Candida species examined, but four C. albicans probes exhibited cross-species hybridization with C. stellatoidea only. The karyotype of C. stellatoidea seems to be within the range of the intraspecies variation observed in C. albicans.     

Electrophoretic karyotypes of Phaffia rhodozyma strains

Abstract Electrophoretic karyotypes of strains from the astaxanthin-producing yeast Phaffia rhodozyma have been established. Intact chromosomal DNA molecules released from protoplasts were separated by orthogonal field alternation gel electrophoresis (OFAGE) and contour clamped homogeneous electric field (CHEF). Both small and large chromosomal DNA molecules were resolved simultaneously by optimizing the running conditions. Electrophoretic karyotypes among the Phaffia isolates examined differed significantly. Seven to thirteen chromosomal bands, ranging in size from 0.83 Mb to 3.50 Mb, were resolved, giving total genome sizes of about 15.4 to 23.2 Mb. Ribosomal DNA has been assigned to chromosomal bands using a heterologous gene probe.     

An electrophoretic karyotype of Aspergillus niger

Summary An electrophoretic karyotype of Aspergillus niger was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Chromosomesized DNA was separated into four bands. Seven of the eight linkage groups could be correlated with specific chromosomal bands. For this purpose DNA preparations from seven transformant strains of A. niger each carrying the heterologous amdS gene of Aspergillus nidulans on a different chromosome were analysed. Some of the assignments were confirmed with linkage groupspecific A. niger probes. The estimated sizes of the A. niger chromosome range from 3.5 to 6.6 Mb, based on gel migration relative to the chromosomes of Schizosaccharomyces pombe strains, Saccharomyces cerevisiae and A. nidulans. The total genome size of A. niger significantly exceeds that of A. nidulans and is estimated to be about 35.5–38.5 Mb. Electrophoretic karyotyping was used to allocate non-mutant rRNA genes and to estimate the number of plasmids integrated in a high copy number transformant.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Electrophoretic karyotypes of the elm tree pathogen Ophiostoma ulmi (sensu lato)

Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato. Twelve wild-type and laboratory strains, representing the less agressive species O. ulmi and both of the biotypes of the more aggressive species O. novo-ulmi were studied and their karyotypes determined. Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception. Strain CESSI6K (O. novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb. This unique band was the smallest O. ulmi s. l. chromosomal DNA observed. Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype. There was no correlation between chromosome profile and species, as some O. novo-ulmi and O. ulmi strains shared common electrophoretic karyotypes.     

Ultrastructural and chemotaxonomic analysis of a xylanolytic strain of Cryptococcus adeliensis isolated from sheep droppings in Spain

Cryptococcus adeliensis was initially described as a psycrophilic species containing a single strain CBS 8351^T isolated from decayed algae in Terre Adelie (Antartida). Later, a second strain of this species was isolated from an immunosuppressed patient affected by leukaemia in Germany and recently several strains from this species have been found in human patients and pigeon droppings of the same country. In this study, we isolated from sheep droppings in Spain a xylanolytic strain named LEVX01 that was phenotypically related to the strain CBS 8351^T and showed a 100% similarity in the D1/D2 domain and 5.8S-ITS region sequences with respect to the remaining described strains of C. adeliensis. These findings suggest that this species has a wide geographical distribution and that the animal faeces are a common habitat for C. adeliensis. The chemotaxonomic analyses showed the absence of detectable amounts of xylose in the cell walls of the strains LEVX01 and CBS8351^T in contrast to other Cryptococcus species. Interestingly, the ultrastructural study showed the presence of fimbriae in these two strains that could be involved in the attachment to the host cells and, as occurs in Candida albicans, they could also be a pathogenicity factor for the man.     

Four novel yeasts from decaying organic matter: Blastobotrys robertii sp. nov., Candida cretensis sp. nov., Candida scorzettiae sp. nov. and Candida vadensis sp. nov

Four novel yeast species are described, two from decaying mushrooms, viz. Candida cretensis and Candida vadensis, and two from rotten wood, viz. Blastobotrys robertii and Candida scorzettiae. Accession numbers for the CBS and ARS Culture Collections, and GenBank accession numbers for the D1/D2 domains of the large subunit of ribosomal DNA are: B. robertii CBS 10106^T, NRRL Y-27775, DQ839395; C. cretensis CBS 9453^T, NRRL Y-27777, AY4998861 and DQ839393; C. scorzettiae CBS 10107^T, NRRL Y-27665, DQ839394; C. vadensis CBS 9454^T, NRRL Y-27778, AY498863 and DQ839396. The GenBank accession number for the ITS region of C. cretensis is AY498862 and that for C. vadensis is AY498864. C. cretensis was the only species of the four that displayed fermentative activity. All four type strains grew on n-hexadecane. C. scorzettiae is the only one of the new species that assimilates some phenolic compounds, viz. 3-hydroxy derivatives of benzoic, phenylacetic and cinnamic acids, but not the corresponding 4-hydroxy acids. This is indicative of an operative gentisate pathway.     

Rhodotorula himalayensis sp. nov., a novel psychrophilic yeast isolated from Roopkund Lake of the Himalayan mountain ranges, India

Twenty-five psychrophilic yeasts were isolated from the soil of Roopkund Lake, Himalayas, India. Two colony morphotypes were identified and representatives of ‘morphotype 1’ were identified as Cryptococcus gastricus. Representatives of ‘morphotype 2’, namely 3A^T, 4A, 4B and Rup4B, showed similar phenotypic properties and are identical with respect to the nucleotide sequence of the ITS1-5.8S rRNA gene-ITS2 region and D1/D2 domain of the 26S rRNA gene. The sequence of D1/D2 domain of 3A^T shows 97.6–98.8% similarity with Rhodotorula psychrophila CBS10440^T, Rhodotorula glacialis CBS10437^T and Rhodotorula psychrophenolica CBS10438^T and in the neighbour-joining phylogenetic tree strains; 3A^T, 4A, 4B and Rup4B form a cluster with Rhodotorula glacialis and Rhodotorula psychrophila. Strains 3A^T, 4A, 4B and Rup4B also differ from their nearest phylogenetic relatives in several biochemical characteristics such as in assimilation of d-galactose, l-sorbose, maltose, citrate, d-glucuronate and creatinine. Thus, based on the phylogenetic analysis and the phenotypic differences 3A^T, 4A, 4B and Rup 4B are assigned the status of a new species of Rhodotorula for which the name Rhodotorula himalayensis sp. nov. is proposed with 3A^T as the type strain (=CBS10539^T =MTCC8336^T). GenBank/EMBL accession numbers for (partial) 18SrRNA gene-ITS1-5.8S rRNA gene-ITS2-26S rRNA gene (partial) sequences of Rhodotorula himalayensis sp. nov. 3A^T is AM410635.     

First report of oral colonization by <Emphasis Type="Italic">Debaryomyces nepalensis</Emphasis> in a dog

A stray, young male, wire-haired pointing griffon dog, found in a street of Perugia (Italy), was examined in order to check his health status. Two oropharyngeal swabs were collected in 24 h and streaked onto Sabouraud agar and after 6 days the yeasts colonies were transferred onto Malt agar. Ascospores were observed on Potato Dextrose Agar medium. The major ubiquinone of an isolated yeast was identified as ubiquinone-9 (Q-9), and genetical analyses were performed together with the type strains of Debaryomyces hansenii (var. hansenii and var. fabry), C. psychrophila and D. nepalensis type strain. The base sequences of ITS1 and ITS2, and D1/D2 domains of LSU rDNA completely coincided with those of D. nepalensis. From these results, the isolated yeast was identified as D. nepalensis. RAPD patterns between the two strains were found to be identical. The results indicate the first colonization of D. nepalensis in a dog.     

Novel class of rDNA repeat units in somatic hybrids between Nicotiana and Atropa

Summary Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to ³²P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled ³²P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.     

Two new species of the genus Candida in the Zygoascus clade, Candida lundiana sp. nov. and Candida suthepensis sp. nov., isolated from raw honey in Thailand

During a survey of yeasts associated with raw honey collected in Thailand, two strains of the Zygoascus clade were isolated from the Asian cavity-nesting honeybee Apis cerana and the stingless bee Homotrigona fimbriata. Phylogeny based on 26S rDNA D1/D2 sequences placed these yeasts as members of a clade including Candida bituminiphila, Candida patagonica and Candida polysorbophila. The strains of the two novel species, CBS 12271^T and CBS 12270^T, respectively, could be unquestionably distinguished from their relatives by rDNA sequences and other taxonomic characteristics. Therefore, the novel anamorphic species, Candida lundiana sp. nov. (type strain CBS 12271^T = JCM 16823^T) and Candida suthepensis sp. nov. (type strain CBS 12270^T = JCM 16822^T) are described.     

DNA probes specific for the yeast species Debaryomyces hansenii: useful tools for rapid identification

We developed a rapid and sensitive identification method for the halotolerant yeast Debaryomyces hansenii, based on the hybridization of species-specific sequences. These sequences were first identified in a survey of D. hansenii strains by random amplification of polymorphic DNA (RAPD) as giving conserved bands in all isolates tested. Two such conserved RAPD products, termed F01pro and M18pro, were cloned from the type strain CBS 767. The specificity of these probes was assessed by hybridizing them to DNA from various species of yeasts commonly found in cheese. F01pro and M18pro hybridized to the DNA of all D. hansenii var. hansenii tested, but not to DNA of other yeast species including the closely related strain of D. hansenii var. fabryii CBS 789. Hybridization patterns of F01pro and M18pro on digested genomic DNA of D. hansenii indicated that the sequences were repeated in the genome of all D. hansenii var. hansenii tested, and gave distinct polymorphic patterns. The single F01pro probe generated 11 different profiles for 24 strains by restriction fragment length polymorphism, using one restriction enzyme. F01pro represents a new type of repeated element found in fungi, useful for both identification and typing of D. hansenii and, together with M18pro, simplifies the study of this species in complex flora.     

Ogataea kanchanaburiensis sp. nov. and Ogataea wangdongensis sp. nov., two novel methylotrophic yeast species from phylloplane in Thailand

Three strains (KM03^T, KM13 ^T and KM15) representing two novel methylotrophic yeast species were isolated from the external surface of plant leaves, which were collected from Kanchanaburi province, Thailand, by three-consecutive enrichments in methanol broth. Strain KM03^T was isolated from phylloplane of a mango tree (Mangifera indica) and two strains, KM13^T and KM15, were obtained from phylloplane of different wine grapes (Vitis vinifera). The sequences of the D1/D2 region of the large subunit (LSU) rRNA gene of the two strains (KM13^T and KM15) were identical and differed markedly from that of strain KM03^T. In terms of pairwise sequence similarity of the D1/D2 region the closest species to the strains KM13^T and KM15 were Candida suzukii (CBS 9253^T) and Candida nitratophila (CBS 2027^T) but with 2.1 % nucleotide substitutions. Strain KM03^T differed from Ogataea wickerhamii (CBS 4307^T), its closest relative, by 2.3 % nucleotide substitutions. Phylogenetic analysis based on the D1/D2 sequences placed the three strains in the Ogataea clade. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analyses of the D1/D2 and the internal transcribed spacer (ITS) regions of the nuclear ribosomal RNA gene (nrRNA) operon, the three strains represent two novel Ogataea species although formation of ascospores was not observed. Ogataea kanchanaburiensis sp. nov. is proposed for strain KM03^T (=BCC 47626^T = NBRC 108603^T = CBS 12673^T). Two strains, KM13^T and KM15, were assigned to Ogataea wangdongensis sp. nov. (type strain KM13^T = BCC 42664^T = NBRC 107778^T = CBS 12674^T). GenBank/EMBL/DDBJ accession numbers for the sequences of the D1/D2 and the ITS regions of O. kanchanaburiensis KM03^T are AB734090 and AB734093, respectively, of O. wangdongensis KM13^T are AB734091 and AB734094, respectively, and of O. wangdongensis KM15 are AB734092 and AB734095, respectively.     

The use of karyotyping in the systematics of yeasts

The use of electrophoretic karyotyping in systematics of yeasts is discussed. New data are provided on the karyotypes of the medically important fungiHortaea werneckii, Filobasidiella (=Cryptococcus)neoformans, andMalassezia species.Hortaea werneckii has twelve to eighteen bands of chromosomal DNA, ranging in size between 500 and 2300 kb. The karyotypes ofFilobasidiella neoformans consist of seven to fourteen bands of chromosomal DNA. The varietiesneoformans andbacillispora cannot be separated by their karyotypes, and no obvious correlation was found with serotypes, geography or habitat. All strains ofMalassezia pachydermatis studied have similar karyotypes consisting of five bands, whereas inM. furfur, four different karyotypes are prevalent. However, each of these karyotypes is stable.     

Identification of Different Agrobacterium Strains Isolated from the Same Forest Nursery

Several Agrobacterium strains isolated from the same forest nursery from 1982 to 1988 were compared by serological, biochemical, and DNA-DNA hybridization methods. Similarities among strains belonging to biovar 2 were observed by indirect immunofluorescence, whereas biovar 1 strains showed serological heterogeneity. Electrophoretic analysis of bacterial envelope-associated proteins showed that few bands appeared in the strains belonging to biovar 1, whereas many proteins appeared in the case of biovar 2 strains. Chromosomal DNA was analyzed with six random C58 chromosomal fragments. None of the six probes hybridized to the DNA of the two biovar 2 strains. One of the probes gave the same hybridization pattern with all biovar 1 strains, whereas the other probes yielded different patterns. The vir regions were closely related in the different pathogenic strains. The T-DNA and replication regions were less conserved and showed some variations among the strains.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.