Partial nucleotide sequence of a single ribosomal RNA coding region and secondary structure of the large subunit 25 s rRNA of Candida albicans

A rDNA cistron of Candida albicans strain WO-1 was cloned and the ITS1, ITS2, 5.8 s rDNA and 25 s rDNA coding regions sequenced in their entirety. These sequences were compared to those of three related yeast species (Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Thermomyces lanuginosus), and the 5.8 s rDNA was compared to seven additional 5.8 s rDNAs from organisms ranging in complexity from D. discoideum to H. sapiens. The C. albicans ITS regions are shorter than those of most other eukaryotes. The 25 s and 5.8 s rDNA sequences were folded into a secondary structure model based on comparative methods. In a comparison of regional similarities between the large subunit rDNAs of C. albicans, the three related yeasts and other eukaryotes, it is demonstrated that the additional sequences not present in the E. coli 23 s rDNA are more variable than the regions present in both prokaryotes and eukaryotes.     

Electrophoretic karyotyping of Leptosphaeria maculans differentiates highly virulent from weakly virulent isolates

Summary Whole chromosomes from the fungal phytopathogen Leptosphaeria maculans were separated by transverse alternating field electrophoresis. The chromosome complements from several isolates of both highly and weakly virulent strains were compared. Small variations in chromosome size and apparent number were detected among isolates of the same strain. However, dramatic differences in both chromosome number and size were found when isolates of the highly virulen strain were compared to those of the weakly virulent. Highly virulent isolates had 6–8 distinct bands whereas weakly virulent isolates had 12–14. The genome sizes were estimated to be at least 8.6x10⁶ base pairs for the virulent strain and 1.6x10⁷ base pairs for the weakly virulent strain. The major differences found in the chromosome complements of the two strains, in combination with results of other biochemical, morphological, and genetic studies, indicate that they are distinct species.     

Heterobasidion annosum 5.8s ribosomal DNA and internal transcribed spacer sequence: rapid identification of European intersterility groups by ribosomal DNA restriction polymorphism

Using conserved fungal ribosomal gene sequences the internal transcribed spacer (ITS) regions one and two (ITS1, ITS2) and the 5.8s ribosomal RNA gene (rRNA) of Heterobasidion annosum were amplified by the polymerase chain reaction (PCR). The nucleotide sequence was determined in three European intersterility groups (ISG-S,-F and-P). Three sequence variants of the ITS were found in ISG-S isolates. The sequence of the ITS of ISG-F differed by two residues from the major ISG-S sequence variant. The ISG-P sequence differed from ISG-S and ISG-F at 15–16 and 16 residues, respectively. Amplified intergenic spacer elements were informative for ISG fingerprinting following digestion with various 4-cutter restriction endonucleases. All differences in the restriction fragments between the ISGs were because of sequence differences in the ITS regions. The fingerprint patterns of isolates from the same intersterility group but from different European localities were identical. These results show that ribosomal DNA finger-printing is a rapid technique to identify ISGs in Heterobasidion annosum.     

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani

Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66 – 100% for isolates of different subgroups within an AG, and 55 – 96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance. Received: 11 February /16 May 1997     

Characterization of Cryptocaryon irritans isolates from marine fishes in Mainland China by ITS ribosomal DNA sequences

Seven isolates of Cryptocaryon irritans from different host species and geographical locations in Mainland China were characterized by the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA) using two isolates of Ichthyophthirius multifiliis for comparative purposes. The rDNA region including the ITS-1, 5.8S, ITS-2, and flanking 18S and 28S sequences were amplified by polymerase chain reaction and the amplicons were sequenced directly. The ITS-1, 5.8S, and ITS-2 sequences were 129, 160, and 190 bp in length, respectively, for all seven C. irritans isolates, whereas the corresponding sequences for the two I. multifiliis isolates were 142, 153, and 194 bp, respectively. While sequence variation among the seven C. irritans isolates ranged from 0 to 1.6% in both the ITS-1 and ITS-2, and the two I. multifiliis isolates differed by 1.4% in the ITS-1 and 1.0% in the ITS-2; C. irritans differed from I. multifiliis by 57.1–60.9% in the ITS-1 and 79.4–83.0% in the ITS-2, indicating that ITS sequences provide reliable genetic markers for the identification and differentiation of the two species. Phylogenetic analysis using the sequence pairwise-distance data using the neighbor-joining method inferred that the seven C. irritans isolates from Mainland China and two other isolates (T.A and Aus.C) from other countries clustered together to show monophyly, which could be readily distinguished from the other monophyletic group all from other regions. Therefore, ITS sequence data and phylogenetic analysis provided strong support that C. irritans isolates from Mainland China represent a single species. The definition of genetic markers in the ITS rDNA provide opportunities for studying the ecology and population genetic structures of the C. irritans from Mainland China and elsewhere and is also relevant to the diagnosis and control of fish diseases they cause.     

Identification and characterization of polymorphic minisatellites in the phytopathogenic ascomycete Leptosphaeria maculans

Leptosphaeria maculans causes phoma stem canker, the most serious disease of oilseed rape world-wide. Sexual recombination is important in the pathogen life cycle and increases the risk of plant resistance genes being overcome rapidly. Thus, there is a need to develop easy-to-use molecular markers suitable for large-scale population genetic studies. The minisatellite MinLm1, showing six alleles in natural populations, has previously been used as a marker to survey populations. Here, we report the characterization of five new minisatellites (MinLm2–MinLm6), of which four were identified by a systematic search for tandemly repeated polymorphic regions in BAC-end sequencing data from L. maculans. Of 782 BAC-end sequences analysed, 43 possessed putative minisatellite-type repeats and four of these (MinLm3–MinLm6) displayed both consistent PCR amplification and size polymorphism in a collection of L. maculans isolates of diverse origins. Cloning and sequencing of each allele confirmed that polymorphism was due to variation in the repeat number of a core motif ranging from 11 bp (MinLm3) to 51 bp (MinLm4). The number of alleles found for each minisatellite ranged from three (MinLm4) to nine (MinLm2), with eight, five and six for MinLm3, MinLm5 and MinLm6, respectively. MinLm2–MinLm6 are all single locus markers specific to L. maculans and share some common features, such as conservation of core motifs and incomplete direct repeats in the flanking regions. To our knowledge, L. maculans is the first fungal species for which six polymorphic single locus minisatellite markers have been reported.Electronic Supplementary Material Supplementary material is available for this article at     

Molecular characterization of <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> isolates from the Philippines

Trichomonas vaginalis is a human urogenital pathogen that causes trichomoniasis, the most common nonviral parasitic sexually transmitted infection in the world. Presently, there are no reports on comparative sequence analysis as well as on the identification of phylogenetic positions of T. vaginalis isolates from the Philippines relative to known trichomonads. In this study, 5.8S rDNA and the flanking internal transcribed spacer (ITS) regions of 57 T. vaginalis isolates were sequenced. The phylogenetic positions of the isolates relative to known trichomonads were determined using the model-based (GTR+Γ+I) neighbor-joining, maximum likelihood, and Bayesian-inference analyses and the nonmodel-based maximum parsimony analysis. Construction of a phylogenetic tree showed the clustering of all the sequences in one branch together with other T. vaginalis strains obtained through basic local alignment search tool search. Sequencing of the 5.8S rDNA gene and the flanking ITS1and ITS2 regions of T. vaginalis isolates from the Philippines demonstrated low genetic polymorphism. However, comparison of the ribosomal DNA sequences may have implications on some phenotypic characteristics of T. vaginalis.     

Ribosomal DNA internal transcribed spacer sequences do not support the species status of Ampelomyces quisqualis, a hyperparasite of powdery mildew fungi

Phylogenetic relationships among Ampelomyces isolates, pycnidial hyperparasites and biological control agents of powdery mildews, were inferred from internal transcribed spacer (ITS) sequences of the ribosomal DNA (rDNA). Currently, these hyperparasites are considered to be a single species, A. quisqualis, despite observed morphological and cultural differences. Ten Ampelomyces isolates, representing seven previously defined ITS RFLP groups, were sequenced and analyzed. Sequence-divergence values among isolates belonging to different RFLP groups ranged from 4.3 to 22.4%, suggesting that these isolates may represent different taxa. When Ampelomyces ITS sequences were analyzed by cladistic methods with the sequences of other ascomycetous fungi, they formed two lineages in the Dothideales. Slow-growing Ampelomyces isolates formed a clade with Leptosphaeria microscopica and L. nodorum, whereas fast-growing Ampelomyces isolates formed a clade with Epicoccum nigrum. Sequence-divergence values between these two clades ranged from 17.3 to 22.4%, suggesting that the taxa in the two clades are not closely related and possibly not congeneric. The data presented here indicate that the identification of `A. quisqualis' isolates used in biological control experiments should be re-evaluated. Received: 10 March 1997 / Accepted: 13 February 1998     

Heterogeneity in intergenic regions of the ribosomal repeat of the pine-blister rustsCronartium flaccidum andPeridermium pini

Mixed aeciospore isolates ofCronartium flaccidum andPeridermium pini were obtained from single-tree infections in Britain, Italy and Greece. The 5.8s ribosomal RNA gene and flanking intergenic transcribed spacer regions ITS 1 and ITS2 were found to be highly similar betweenC. flaccidum andP. pini. Within samples heterogeneity was detected at three nucleotide loci in the ITS1 and at four loci in the ITS2 suggesting that several fungal genotypes may occur at a single infection court. The heterogeneity was confirmed by heteroduplex polymorphism analysis of mixed aeciospore products. RFLP of the ribosomal intergenic spacer region 1 (IGSI) amplified from the same templates indicated limited sequence polymorphism in some copies of this repeated locus. Both the sexual and asexual forms ofC. flaccidum show evidence of sequence polymorphism in two independent, non-coding regions of the ribosomal gene array. Variation appears to be greater in the sexual formC. flaccidum, than in the monoaecious formP. pini.     

Identification at strain level ofRhizoctonia solani AG4 isolates by direct sequence of asymmetric PCR products of the ITS regions

The relatedness of nine isolates ofRhizoctonia solani, belonging to anastomosis group (AG) 4, and one isolate of AG1 was determined by comparative sequence analysis based on direct sequencing of PCR-amplified ribosomal DNA [the internal transcribed spacer (ITS) region and the 5.8 s ribosomal DNA]. The 5.8s rDNA is completely conserved, but both ITS regions show variation among strains. AG1 was an outgroup based on anastomosis ability and RFLP analyses. Phylogenetic analyses based on the ITS sequences suggest that the analyzed AG4 strains can be divided into three groups that correlate with habitat and virulence.     

Pathotyping of Australian isolates of Marek's disease virus and association of pathogenicity with meq gene polymorphism

We report the pathotyping of six Australian isolates of Marek's disease virus-1 (MDV1) isolated between 1992 and 2004 and association of virulence with meq gene polymorphism. Unvaccinated and herpesvirus of turkeys (HVT)-vaccinated specific pathogen free chickens were challenged at day 5 with 500 plaque forming units of Marek's disease virus. The isolates induced gross Marek's disease lesions in 53 to 94% of unvaccinated chickens, and HVT induced a protective index ranging from 38 to 100% by 56 days post challenge. This experiment provides evidence that current Australian isolates of MDV1 vary significantly in pathogenicity. However, there was no clear evidence that the most virulent recent isolates were more pathogenic than isolates from the 1980s or that any of the isolates belong to the highest pathotype category of very virulent plus. Evidence is presented that virulence can be predicted by measurements taken as early as 13 days post challenge. The meq gene sequences of five of the isolates used in the experiment were determined. When compared with the very virulent US isolate Md5, there was a 177 base-pair insertion and distinct point mutations in each of the five isolates. There were no individual mutations in the meq sequences that correlated with levels of virulence. However, amino acid alignment of the five Australian and 14 international isolates revealed that the number of repeat sequences of four prolines (PPPP repeats) in the meq gene (overall range 2 to 8) was strongly associated with virulence across all isolates, with the most pathogenic isolates having the fewest number of repeats. The results suggest that the presence of the 177 base-pair insertion alone is not an indicator of attenuation. Rather, the number of PPPP repeats, independent of the presence of the insertion, is a better indicator of pathogenicity.     

Meiotic behaviour of the minichromosome in the phytopathogenic ascomycete Leptosphaeria maculans

All Leptosphaeria maculans field isolates displayed a minichromosome (MC) clearly separated from the overall electrokaryotype following pulsed-field gel electrophoresis. MCs exhibited a length polymorphism ranging from 650 to 950 kb. Tetrad analyses revealed the parental inheritance of MC length polymorphism (50% of the tetrads) or else the generation of novel-sized MCs (27%), which suggested that recombination occurred between MCs. Nineteen percent of the tetrads displayed a lack of the MC band in the electrokaryotype for one or two of the four resulting genotypes. Crosses between isolates carrying or lacking MCs revealed non-Mendelian segregation and suggested that some isolates could display at least two copies of the MC. Only repeated sequences hybridising to all chromosomes were isolated from the MC. Finally, saprophytic or parasitic fitness was not modified when isolates apparently lacked the MC. All these data suggested that the L. maculans MC behaves like a `B' chromosome. Received: 26 February 1996 / 2 August 1996     

Molecular phylogeny of the fungi of the Iceman's grass clothing

To investigate the origin of the fungal hyphae that cover the grass clothing (cloak, boots) found near the neolithic mummy known as the Tyrolean Iceman, two radiocarbon-dated samples of grass were submitted to DNA extraction. The DNA was then PCR amplified using, respectively, primers specific for the region containing the internal transcribed spacers and the 5.8s rDNA (ITS), and primers specific for an approximately 600-bp long fragment of the nuclear small-subunit ribosomal DNA (SSU rDNA) repeat units of eukaryotes. The amplification products were cloned and sequenced. Sequence analysis of 20 individual ITS clones and of ten SSU rDNA clones indicated that three types of fungal DNA can be extracted from the grass. Phylogenetic analyses, using 5.8s and SSU rDNA fungal reference sequences from EMBL and GenBank databases, suggest that the DNAs come, respectively, from a psychrophilic basidiomycetous yeast, phylogenetically close to Leucosporidium scottii, and from two ascomycetes, one of which is possibly related to the Eurotiales     

Nucleotide sequence of the Schizosaccharomyces japonicus var. versatilis ribosomal RNA gene cluster and its phylogenetic implications

Fission yeasts form a small but heterogeneous group of ascomycetes and it is still unclear whether they should be subdivided into three genera (Schizosaccharomyces, Octosporomyces, Hasegawaea) or remain a single genus (Schizosaccharomyces). In order to decide whether a new genus Hasegawaea should be established for the species Schizosaccharomyces japonicus and Schizosaccharomyces versatilis, we have characterized the entire rDNA cluster in Schizosaccharomyces japonicus var. versatilis and compared it with the homologous region from Schizosaccharomyces pombe and with complete rRNA gene sequences from other yeast genera. From a phage genomic library a recombinant lambda phage containing the entire rDNA repeat unit was isolated. In this paper we report the primary sequence of the 18s, 5.8s and 25s rRNA coding regions. The S. japonicus var. versatilis rRNA genes are 1823 (18s), 158 (5.8s) and 3422 (25s) nucleotides long. The two sequences of the larger rRNA genes exhibit 95.7% (18s) and 93% (25s) similarity with the homologous genes from S. pombe. The differences between the rRNA genes of S. japonicus and S. pombe, however, are much smaller than the intrageneric differences within the rDNA sequences of other yeast genera. Therefore, subdivision of fission yeasts into the genera Schizosaccharomyces and Hasegawaea does not to seem to be justified. The sequence has been deposited in the EMBL data bank under the accession number Z 32848.     

Genetic structure of a population of the fungus Leptosphaeria maculans in a disease nursery of Brassica napus in Australia

Microsatellite, minisatellite and mating type markers were used to determine the genetic structure of the fungus Leptosphaeria maculans within a disease nursery, where Brassica napus lines were screened for resistance to blackleg disease under high inoculum pressure. Fungal isolates were collected from pseudothecia in infected stubble and pycnidia within cotyledon lesions on seedlings within the nursery. Genetic diversity was high with gene diversity at H=0.700 across four polymorphic loci, and genotypic diversity at D=0.993. Among the 159 isolates analysed, 102 multilocus genotypes were identified. The even distribution of mating type idiomorphs MAT1-1 and MAT1-2 and gametic equilibrium within the population provided further evidence of random mating. Genetic diversity was distributed on a very fine scale in the disease nursery. The majority of genetic diversity (67%) was distributed among conidia within a lesion or among ascospores from a piece of stubble, while the remainder (33%) was distributed within lesions on seedlings or different stubble pieces. There were no among-group differences between samples from stubble and seedlings. This is consistent with the low level of genetic differentiation between the ascospore and conidia samples (F _ST=0.017) indicating that all isolates of L. maculans from the disease nursery most likely belong to one population, and that ascospores form the primary inoculum in the disease nursery.     

Discrimination of Babesia major and Babesia ovata based on ITS1–5.8S–ITS2 region sequences of rRNA gene

Babesia ovata and Babesia major are two newly identified large Babesia species infective to cattle in China. There is a demand for specific tools for discrimination between the two species due to the confusion of their classification based on traditionally classification methods, such as tick vector, morphology, and pathogenicity. In this study, the internal transcribed spacers (ITS including ITS1, 5.8S coding region and ITS2) were originated from four isolates of B. ovata and one of B. major from different geographic regions of China, and a phylogenetic tree was inferred. It was demonstrated that all of the four isolates of B. ovata were grouped into one cluster, while B. major isolate was placed in another. The sequence percent identity showed that B. ovata isolates had the minimum 85.5% identity, whereas B. major showed only the maximum 46.6% identity to the four B. ovata isolates. In addition, the identity of ITS1 and ITS2 of these Babesia isolates was discussed. The findings implied that the four B. ovata isolates have a quite close relationship, whereas the B. major isolate showed far relationship with all of these B. ovata isolates. This finding may lead to the conclusion that there is actual existence of two large Babesia infective to cattle in China, one is B. ovata and the another is B. major.     

Characterization and genetic diversity of sugarcane streak mosaic virus causing mosaic in sugarcane

Sixty-three sugarcane leaf samples were collected from fifty-eight sugarcane varieties, evolved from eleven major sugarcane growing states in India, Australia, South Africa and USA. In RT-PCR, using gene specific primers for sugarcane streak mosaic virus (SCSMV)-CP, 58 of 63 sugarcane samples were found positive to the virus infection and rest of the five samples were negative. Partial CP gene sequences of 42 SCSMV isolates including an isolate from aphid colony (Melanaphis indosacchari) infested on sugarcane variety from this study were characterized after cloning and sequencing for selective isolates represented by at least one isolate from each location. The new sequences identified in the study were named as SCSMV-CB isolates. Fifty two sequences including the 10 database sequences (complete CP cds) deposited earlier from this institute were compared with each other as well as GenBank database sequences of Potyviridae members viz., Rymovirus, Potyvirus, Ipomovirus, Tritimovirus and eight sequences of SCSMV reported from elsewhere. Among the SCSMV-CB isolates sequenced in the study, 85.7–100% (nucleotide) and 89.9–100% (amino acid) sequence identities were observed and with the other data base sequences of SCSMV, the respective identities were 82.2–97.5 and 89.7–98.6%. Grouping of the isolates by the maximum likelihood with molecular clock model, distributed 60 SCSMV sequences including the eight database sequences deposited by other SCSMV working groups from India and USA in 16 different phylogenetic groups. Although the isolates of SCSMV were relatively close to Ipomovirus and Tritimovirus, they were sandwiched between Rymovirus and Ipomovirus. The sequence comparison and phylogenetic studies revealed that the relatedness of SCSMV with the potyviral related genera was comparatively low to consider it as a member of earlier described potyviral genera, hence the genus “Susmovirus” (sugarcane streak mosaic virus) has been proposed, with SCSMV as the sole species to be included. The 52 SCSMV-CB isolates from this institute were distributed in 14 phylogenetic groups and the grouping pattern revealed that the virus isolates could not be grouped based on geographical origin of the host varieties or longevity of the host variety.     

The phylogeny of the tomato leaf mould fungus Cladosporium fulvum syn. Fulvia fulva by analysis of rDNA sequences

The nucleotide sequence of part of the ribosomal DNA from races of the fungal tomato pathogen Cladosporium fulvum and other Cladosporium species have been determined. Comparisons of the internal transcribed spacer regions (ITS1 and ITS2) of several C. fulvum races showed complete sequence homology suggesting a recent evolutionary divergence. Comparisons of these nucleotide sequences in the ITS region with those of other Cladosporium species showed the close relationship within the Cladosporium genus. Using the nucleotide sequence of part of the 18s ribosomal subunit from these isolates and comparing them with sequences of some Ascomycetes, Basidiomycetes and Chytridiomycetes, obtained from GenBank, we infer the phylogeny of the Cladosporium species studied here. Our analysis shows that the Cladosporia form a monophyletic group which falls within the order Ascomycotina.     

Major chromosomal length polymorphisms are evident after meiosis in the phytopathogenic fungus Leptosphaeria maculans

Chromosomal DNA of Australian field-isolates of the phytopathogenic ascomycete Leptosphaeria maculans was resolved by pulsed-field gel electrophoresis. All isolates examined had highly variable karyotypes. Ascospores (sexual spores) derived from single pseudothecia (sexual fruiting bodies) isolated from Brassica napus (oilseed rape) stubble were analyzed. In two tetrads four distinct karyotypes were observed, with only one chromosomal DNA band in common to all the members of each tetrad. Although isolates had highly variable karyotypes, two overall patterns were present. In one pattern there were at least 12 chromosomal DNA bands, the largest being greater than 2.2 Mb in size; in the other there were more than 15 chromosomal DNA bands, the largest being about 2.0 Mb. The chromosomal DNA preparations included mitochondrial DNA which migrated as a diffuse band between 0.10 and 0.15 Mb in size, and DNA molecules of 8 and 9 kb in size.