Genetic Diversity in the Microsporidian Encephalitozoon hellem Demonstrated by Pulsed-Field Gel Electrophoresis

Encephalitozoon hellem is a microsporidian species responsible for opportunistic infections in AIDS patients. Use of a novel chitinase-based method allowed unsheared chromosomal DNA to be recovered from eleven E. hellem isolates derived from three geographic regions. All isolates were typed by 18S rDNA sequencing, which showed that they belonged to intemal transcribed spacer type 1. After ethidium bromide staining, pulsed-field gel electrophoresis (PFGE) analysis discriminated two new karyotypes comprising 7 and 8 chromosomal bands respectively, ranging in size from 205- to 272-kb pairs. Genomic size was estimated to be 2.39 Mb. Our data indicate PFGE is useful for typing E. hellem and confirms genetic diversity among E. hellem genotypes.     

Detection of new Enterocytozoon genotypes in faecal samples of farm dogs and a cat.

The microsporidian species Enterocytozoon bieneusi had emerged as opportunistic pathogen in AIDS patients causing chronic diarrhoea and was found with high prevalences in faeces of asymptomatic pigs. Analysis of the ribosomal RNA gene internal transcribed spacer (rDNA ITS) had revealed that nine distinct but closely related genotypes occur in humans and in swine. Using primers that were designed to be specific for E. bieneusi, we obtained amplicons from the faecal samples of one from twelve cats and from three out of 36 farm dogs. Sequence analysis of the rDNA ITS, which is part of the diagnostic PCR product, revealed that the isolate from the cat is very closely related to the E. bieneusi genotypes of human or swine origin. The corresponding sequence of all three dog-derived isolates were identical among each other and had a sequence similarity to known sequences of only 47.6-48.2%. In addition, part of the small subunit rRNA gene was amplified and sequenced from one dog-derived isolate revealing a similarity to known sequences of human-derived E. bieneusi of 96-98%. Enterocytozoon-like spores could be detected by light microscopy in one canine sample. Together with recent reports of detection of Enterocytozoon in environmental samples, our findings suggest that microsporidia of the genus. Enterocytozoon seem to be ubiquitous and consist of many genotypes in various naturally infected animal species.     

Molecular tools for isolate and community studies of Pyrenomycete fungi

The Pyrenomycetes, defined physiologically by the formation of a flask-shaped fruiting body present in the sexual form, are a monophyletic group of fungi that consist of a wide diversity of populations including human and plant pathogens. Based on sequence analysis of 18S ribosomal DNA (rDNA), rDNA regions conserved among the Pyrenomycetes but divergent among other organisms were identified and used to develop selective PCR primers and a highly specific primer set. The primers presented here were used to amplify large portions of the 18S rDNA as well as the entire internal transcribed spacer (ITS) region (ITS 1, 5.8S rDNA, and ITS 2). In addition to database searches, the specificity of the primers was verified by PCR amplification of DNA extracted from pure culture isolates and by sequence analysis of fungal rDNA PCR-amplified from environmental samples. In addition, denaturing gradient gel electrophoresis (DGGE) analyses were performed on closely related Colletotrichum isolates serving as a model pathogenic genus of the Pyrenomycetes. Although both ITS and 18S rDNA DGGE analyses of Colletotrichum were consistent with a phylogeny established from sequence analysis of the ITS region, DGGE analysis of the ITS region was found to be more sensitive than DGGE analysis of the 18S rDNA. This study introduces molecular tools for the study of Pyrenomycete fungi by the development of two specific primers, demonstration of the enhanced sensitivity of ITS-DGGE for typing of closely related isolates and application of these tools to environmental samples.     

DNA detection and genotypic identification of potentially human-pathogenic microsporidia from asymptomatic pet parrots in South Korea as a risk factor for zoonotic emergence

We detected and identified genotypes of human-pathogenic microsporidia in fecal samples from 51 asymptomatic captive-bred pet parrots in South Korea. Microsporidia were identified in 8 samples (15.7%); 7 parrots tested positive for Encephalitozoon hellem, and 1 parrot tested positive for both E. hellem and Encephalitozoon cuniculi. In genotypic identifications, E. hellem was present in genotypes 1A and 2B and E. cuniculi was present in genotype II. Pet parrots might be a source of human microsporidian infection.     

Genetically unique microsporidian Encephalitozoon cuniculi strain type III isolated from squirrel monkeys

Microsporidian spores were isolated from two squirrel monkeys (Saimiri sciureus) that had been bred at an animal-breeding colony in Japan. The spores were identified as Encephalitozoon cuniculi on the basis of nucleotide sequence analysis of the small-subunit (SSU) rRNA gene. The internal transcribed spacer (ITS) gene sequence revealed that these isolates were classified into genotype III because it contained tetrarepeats of 5'-GTTT-3'. However, the sequences of the polar tube protein (PTP) gene of the monkey isolates were not identical to a reported sequence of genotype III but were quite similar to a reported sequence of genotype II. On the other hand, sequence analysis of the spore wall protein 1 (SWP-1) gene revealed that the monkey isolates did not belong to any of genotypes I, II and III. These results suggest that the present E. cuniculi isolates of squirrel monkey origin are a new subtype of E. cuniculi ITS genotype III that can cause a disseminated infection.     

Molecular genetic variation within and among isolates of QPX (Thraustochytridae), a parasite of the hard clam Mercenaria mercenaria

The thraustochytrid known as QPX (Quahog Parasite Unknown) has sporadically caused disease in the hard clam Mercenaria mercenaria along the east coast of North America since the 1960s. We hypothesized that genetically distinct QPX strains might be responsible for outbreaks of QPX disease in different areas and tested this hypothesis by comparing several QPX isolates recovered from the recent outbreak in Raritan Bay, New York with QPX strains isolated from 2 outbreaks in Massachusetts, USA. There was no variation in small subunit rDNA (SSU rDNA), 5.8S rDNA, or 4 mitochondrial gene sequences. In contrast, both of the ribosomal ribonucleic acid (rRNA) operon intergenic spacers, internal transcribed spacers 1 and 2 (ITS1 and ITS2), revealed substantial sequence variation. However, strain-specific sequences were not detected because the ITS sequence variation within QPX isolates was comparable to the variation between isolates. ITS1 sequences recovered from an infected clam by amplification with a QPX ITS2-specific primer were identical to those recovered from the QPX isolates.     

Isolation and characterization of melanized fungi from limestone formations in Mallorca

Melanized fungi were isolated from limestone surfaces in upland and coastal environments in the Mediterranean island of Mallorca. One hundred seventeen isolates were recovered from two topographically distinct sites. Due to the difficulty in distinguishing among isolates based on morphological criteria, microsatellite-primed PCR techniques were used to group isolates into genotypes that were assumed to represent species. Seventeen genotypes were characterized from one site and twenty six from the other, with four genotypes common to both. Classical and molecular methods were used to identify representative strains. Morphological methods rarely provided a reliable identification; only three isolates, Hortaea werneckii, Trimmatostroma abietis and Aureobasidium pullulans were identified with certainty, and the identification was confirmed by molecular data. Morphological characters that were widespread among the isolates included scarce micronematous conidial states or non-sporulating sterile mycelia, mature mycelia with dark olive green or black hyphae, mycelia with torulous hyphae, whose cells developed one or more transverse septa. In many of these fungi, the cells of mature hyphae disarticulated, suggesting asexual reproduction by a thallic micronematous conidiogenesis or by simple propagative fragmentation. Sequencing of the internal transcribed spacers (ITS1, ITS2) and 5.8S ribosomal gene, as well as the 18S rDNA ribosomal gene were employed to investigate the isolates’ phylogenetic affinities. The majority of the isolates could be grouped in two main classes of Ascomycetes, Dothideomycetes and Chaetothyriomycetes, although many others did not correspond with any sequence deposited in public databases, suggesting they could be of unknown genera that did not correspond with any well-defined Ascomycete order.     

CONSPECIFICITY AND INDO-PACIFIC DISTRIBUTION OF SYMBIODINIUM GENOTYPES (DINOPHYCEAE) FROM GIANT CLAMS

We previously reported the occurrence of genetically‐diverse symbiotic dinoflagellates (zooxanthellae) within and between 7 giant clam species (Tridacnidae) from the Philippines based on the algal isolates' allozyme and random amplified polymorphic DNA (RAPD) patterns. We also reported that these isolates all belong to clade A of the Symbiodinium phylogeny with identical 18S rDNA sequences. Here we extend the genetic characterization of Symbiodinium isolates from giant clams and propose that they are conspecific. We used the combined DNA sequences of the internal transcribed spacer (ITS)1, 5.8S rDNA, and ITS2 regions (rDNA‐ITS region) because the ITS1 and ITS2 regions evolve faster than 18S rDNA and have been shown to be useful in distinguishing strains of other dinoflagellates. DGGE of the most variable segment of the rDNA‐ITS region, ITS1, from clonal representatives of clades A, B, and C showed minimal intragenomic variation. The rDNA‐ITS region shows similar phylogenetic relationships between Symbiodinium isolates from symbiotic bivalves and some cnidarians as does 18S rDNA, and that there are not many different clade A species or strains among cultured zooxanthellae (CZ) from giant clams. The CZ from giant clams had virtually identical sequences, with only a single nucleotide difference in the ITS2 region separating two groups of isolates. These data suggest that there is one CZ species and perhaps two CZ strains, each CZ strain containing individuals that have diverse allozyme and RAPD genotypes. The CZ isolated from giant clams from different areas in the Philippines (21 isolates, 7 clam species), the Australian Great Barrier Reef (1 isolate, 1 clam species), Palau (8 isolates, 7 clam species), and Okinawa, Japan (1 isolate, 1 clam species) shared the same rDNA‐ITS sequences. Furthermore, analysis of fresh isolates from giant clams collected from these geographical areas shows that these bivalves also host indistinguishable clade C symbionts. These data demonstrate that conspecific Symbiodinium genotypes, particularly clade A symbionts, are distributed in giant clams throughout the Indo‐Pacific.     

Locally isolated yeasts from Malaysia: identification, phylogenetic study and characterization

Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 μg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 μg/mL and 100 μg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.     

Dynamics of Saccharomyces cerevisiae populations in controlled and spontaneous fermentations for Franciacorta D.O.C.G. base wine production

The evolution of the yeast populations was investigated during controlled and spontaneous fermentations of Chardonnay must in two Franciacorta wineries (A and B) that used the same starter culture. Two hundred and three isolates were collected and identified as Saccharomyces cerevisiae (97.5 %), Pichia membranifaciens (2.0 %) and Hanseniaspora vinae (0.5 %) through the analyses of ITS rDNA region by RFLP, D1/D2 of 26S rDNA partial sequence and scHO gene. A high intraspecific diversity of S. cerevisiae isolates was detected by means of the inter-delta sequence PCR analysis: 117 profiles corresponding to different strains were distinguished (at level of similarity <90.5 %) and monitored to follow the dynamics of cell populations. In winery A, the commercial strain maintained the predominance since its δ-PCR profile constituted most of the colonies recovered at different times of sampling (from 44 to 100 % of plate counts), in this case only 18 different genotypes out of 74 isolates were recognized. In winery B, where spontaneous fermentations were performed in the same environment, the starter culture never took control and a succession of indigenous populations overcame without one prevailed on the others; actually, 40 genotypes out of 53 isolates can be identified. The highest level of biodiversity was observed in spontaneous fermentation (winery B) where 59 genotypes out of 71 S. cerevisiae isolates were discriminated; a continuous change in cell populations was noticed with the simultaneous presence from 6 to 10 different genotypes. The management of the starter culture and the environmental hygiene was shown to be fundamental to control the inoculated fermentations.     

Ribosomal DNA sequence divergence and group I introns within the Leucostoma species L. cinctum, L. persoonii, and L. parapersoonii sp. nov., ascomycetes that cause Cytospora canker of fruit trees

Leucostoma species that are the causal agents of Cytospora canker of stone and pome fruit trees were studied in detail. DNA sequence of the internal transcribed spacer regions and the 5.8S of the nuclear ribosomal DNA operon (ITS rDNA) supplied sufficient characters to assess the phylogenetic relationships among species of Leucostoma, Valsa, Valsella, and related anamorphs in Cytospora. Parsimony analysis of the aligned sequence divided Cytospora isolates from fruit trees into clades that generally agreed with the morphological species concepts, and with some of the phenetic groupings (PG 1-6) identified previously by isozyme analysis and cultural characteristics. Phylogenetic analysis inferred that isolates of L. persoonii formed two well-resolved clades distinct from isolates of L. cinctum. Phylogenetic analysis of the ITS rDNA, isozyme analysis, and cultural characteristics supported the inference that L. persoonii groups PG 2 and PG 3 were populations of a new species apparently more genetically different from L. persoonii PG 1 than from isolates representative of L. massariana, L. niveum, L. translucens, and Valsella melastoma. The new species, L. parapersoonii, was described. A diverse collection of isolates of L. cinctum, L. persoonii, and L. parapersoonii were examined for genetic variation using restriction fragment length polymorphism (RFLP) analysis of the ITS rDNA and the five prime end of the large subunit of the rDNA (LSU rDNA). HinfI and HpaII endonucleases were each useful in dividing the Leucostoma isolates into RFLP profiles corresponding to the isozyme phenetic groups, PG 1-6. RFLP analysis was more effective than isozyme analysis in uncovering variation among isolates of L. persoonii PG 1, but less effective within L. cinctum populations. Isolates representative of seven of the L. persoonii formae speciales proposed by G. Défago in 1935 were found to be genetically diverse isolates of PG 1. Two large insertions, 415 and 309 nucleotides long, in the small subunit (SSU) of the nuclear rDNA of L. cinctum were identified as Group 1 introns; intron 1 at position 943 and intron 2 at position 1199. The two introns were found to be consistently present in isolates of L. cinctum PG 4 and PG 5 and absent from L. cinctum PG 6 isolates, despite the similarity of the ITS sequence and teleomorph morphology. Intron 1 was of subgroup 1C1 whereas intron 2 was of an unknown subgroup. RFLP patterns and presence/absence of introns were useful characters for expediting the identification of cultures of Leucostoma isolated from stone and pome fruit cankers. RFLP patterns from 13 endonucleases provided an effective method for selecting an array of diverse PG 1 isolates useful in screening plant germplasm for disease-resistance.     

Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing

Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease. To assess the genetic variability the 16S ribosomal DNA, the internal transcribed spacer (ITS) and the 23S ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16S rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7% similarity). Two-thirds of the 23S rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16S rDNA, ITS and 23S rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (> 99.4% 16S rDNA similarity, 99.1% to 99.7% ITS and 99.3 to 99.8% 23S rDNA similarities). The sequence of one Chilean isolate, EM-90, was unique, with 16S rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23S rDNA from 97.6 to 98.5%.     

枇杷属内栽培种和部分野生种ITS序列分析

对不同栽培区的25种普通枇杷品种以及7种枇杷属野生种的ITS序列进行扩增并测序,采用邻接法和最大简约法进行系统发育树的构建并对枇杷属内不同种间的遗传关系进行了分析。结果表明:枇杷属植物ITS序列ITS1+5.8S rDNA+ITS2总长度为592 bp或594 bp,长度变化发生在ITS2。所有样本的ITS1和5.8S rDNA长度一样,都是223 bp和168 bp;而ITS2为201 bp或203 bp。5种枇杷属野生种的ITS序列长度为594 bp,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;其余2种枇杷属野生种(麻栗坡枇杷、小叶枇杷)和普通枇杷栽培种的ITS序列长度都为592 bp。所有样本ITS序列的GC含量为64.2%~64.5%,其中ITS1为64.1%~65.5%,ITS2为68.1%~72.6%。对所有样本的ITS序列比对产生44个可变位点,其中38个为简约信息位点,其中11个位于ITS1,5个位于5.8S rDNA,22个位于ITS2。最大的种间序列差异为7.7%,最小的种间差异发生在麻栗坡枇杷和小叶枇杷之间,仅为0.2%。普通枇杷种内的ITS序列差异很低,25种普通枇杷栽培种之间的序列差异为0~1.5%。所研究的枇杷属植物可分为3个分支。分支Ⅰ包括所有普通枇杷品种,分支Ⅱ包含5种野生枇杷种,包括栎叶枇杷、大渡河枇杷、南亚枇杷、南亚枇杷窄叶变种和大瑶山枇杷;分支Ⅲ由2个野生枇杷种(麻栗坡枇杷、小叶枇杷)组成。该研究结果表明ITS序列对枇杷种间鉴定和系统发育分析具有一定意义,但对普通枇杷栽培种间的鉴定作用不大。     

Comparisons of Isolates of Heterodera avenae using 2-D PAGE Protein Patterns and Ribosomal DNA

Six geographic isolates of Heterodera avenae, including two isolates each from Sweden, Australia, and the United States, were compared on the basis of 2-D PAGE protein patterns and the complete DNA sequence for the two internal transcribed ribosomal DNA spacers (rDNA ITS1 and ITS2) and the 5.8S rRNA gene. The protein pattern data and rDNA ITS sequence data both indicated that the Swedish Gotland strain of H. avenae differed markedly from the rest of the isolates. Protein patterns for the Australia isolates differed more from a Swedish strict H. avenae isolate and isolates from Oregon and Idaho, than the two U.S. isolates and the Swedish strict H. avenae isolate differed from each other. Except for the Gotland strain isolate, the rDNA ITS sequences were highly conserved among all of the H. avenae isolates, just as we earlier found them to be conserved among species of the schachtii group of Heterodera.     

Molecular identification of Aspergillus and Eurotium species isolated from rice and their toxin-producing ability

Thirty milled rice samples were collected from retailers in 4 provinces of Malaysia. These samples were evaluated for Aspergillus spp. infection by direct plating on malt extract salt agar (MESA). All Aspergillus holomorphs were isolated and identified using nucleotide sequences of ITS 1 and ITS 2 of rDNA. Five anamorphs (Aspergillus flavus, A. oryzae, A. tamarii, A. fumigatus and A. niger) and 5 teleomorphs (Eurotium rubrum, E. amstelodami, E. chevalieri, E. cristatum and E. tonophilum) were identified. The PCR-sequencing based technique for sequences of ITS 1 and ITS 2 is a fast technique for identification of Aspergillus and Eurotium species, although it doesn't work flawlessly for differentiation of Eurotium species. All Aspergillus and Eurotium isolates were screened for their ability to produce aflatoxin and ochratoxin A (OTA) by HPLC and TLC techniques. Only A. flavus isolate UPM 89 was able to produce aflatoxins B1 and B2.     

Antigenic diversity of Encephalitozoon hellem demonstrated by subspecies-specific monoclonal antibodies

Encephalitozoon hellem is a unicellular, obligate intracellular microsporidian species detected and isolated in HIV-infected patients presenting with keratoconjunctivitis, sinusitis, tracheobronchitis, nephritis, cystitis, and disseminated infection. A total of 24 monoclonal antibodies were produced against E. hellem and characterized. The monoclonal antibodies were of the immunoglobulin (Ig) G and Ig M subclasses, and, when incorporated into indirect immunofluorescence and immunoblotting assays, reacted against 13 isolates of E. hellem originating from three geographic regions. These monoclonal antibodies did not react with one strain each of either Encephalitozoon intestinalis or Encephalitozoon cuniculi, demonstrating their specificity. Two monoclonal antibodies reacted with all karyotype B-E. hellem isolates but did not react with karyotype A-isolates from North America and the Netherlands, thus demonstrating antigenic diversity among E. hellem isolates. These results add to the increasing evidence for diversity among E. hellem, which therefore may be reclassified into subspecies.     

Comparison of the ITS1 and ITS2 rDNA in Eimeria callospermophili (Apicomplexa:Eimeriidae) from sciurid rodents

The taxonomy of the coccidia has historically been morphologically based. The purpose of this study was to establish if conspecificity of isolates of Eimeria callospermophili from 4 ground-dwelling squirrel hosts (Rodentia: Sciuridae) is supported by comparison of rDNA sequence data and to examine how this species relates to eimerian species from other sciurid hosts. Eimeria callospermophili was isolated from 4 wild-caught hosts, i.e., Urocitellus elegans, Cynomys leucurus, Marmota flaviventris , and Cynomys ludovicianus . The ITS1 and ITS2 genomic rDNA sequences were PCR generated, sequenced, and analyzed. The highest intraspecific pairwise distance values of 6.0% in ITS1 and 7.1% in ITS2 were observed in C. leucurus. Interspecific pairwise distance values > 5% do not support E. callospermophili conspecificity. Generated E. callospermophili sequences were compared to Eimeria lancasterensis from Sciurus niger and Sciurus niger cinereus and to Eimeria ontarioensis from S. niger. A single, well-supported clade was formed by E. callospermophili amplicons in neighbor joining and maximum parsimony analyses. However, within the clade, there was little evidence of host or geographic structuring of the species.     

Genotypic Diversity among Brazilian Isolates of Sclerotium rolfsii

Thirty isolates of Sclerotium rolfsii Sacc. from different hosts and regions of Brazil were studied in relation to morphology, mycelial compatibility, analysis of genomic DNA through random amplified polymorphic DNA (RAPD), variation within the nuclear rDNA [internal transcribed spacers (ITS)] and sequencing of ITS fragments. There was considerable variability among isolates in relation to the number, size and location of sclerotia on the medium surface. Thirteen mycelial compatibility groups (MCG) were identified among 23 isolates. Seven isolates were only self‐compatible. With the exception of group 3, where all the isolates came from soybean, there was no apparent correlation between group and isolate origin. On the basis of RAPD profiles, 11 haplotypes (A to K) were identified. There was an association between the RAPD groups and MCG. Haplotypes A, B, D, G, I and K belonged to MCG groups 1, 2, 3, 4, 5 and 6, respectively. All other RAPD haplotypes contained incompatible isolates. Polymerase chain reaction (PCR) amplification with primers 4R and 5F amplified two fragments containing ITS1, ITS2 and 5.8 S rDNA sequences, that were present in all isolates, with molecular sizes of 739 and 715 bp. Restriction analysis of PCR products showed that the two fragments had sequence divergency which is referred to as ‘ITS types’. Four arbitrarily chosen soybean isolates (2, 6, 7 and 23) and two non‐soybean isolates (11 and 22) were used to investigate the variation within the ITS sequence and its role in the phylogeny. The strict consensus of nine most‐parsimonious trees inferred from the data set which included six isolates of S. rolfsii, four of which have two different ‘ITS types’, showed three well‐supported groupings. The neighbour‐joining tree inferred from the data set also showed three major clades as did the parsimony tree. The major difference was that in the neighbour‐joining tree the ‘ITS type’ 11 was resolved and grouped in one clade. These results show that the ‘ITS types’ within isolates are almost always phylogenetically distinct. There was no clear correlation between ITS‐based phylogeny and isolate origin.     

四川省雅江松茸菌的分离与系统发育

对外生菌根真菌-松茸的纯培养条件进行了探讨,并从采集自四川省雅江县的松茸子实体中获得了10株松茸菌的纯培养物;分别以NS1和NS6 ,NS1和NS8,ITS4和ITS5为引物,对分离获得的松茸菌进行了18S r DNA PCR- RFL P和ITS PCR-RFL P分析,结果显示,用Alu I,H ae III,H inf I和Msp I四种限制性内切酶,这些松茸菌株的18S r DNA、ITS片段的酶切图谱完全相同;代表菌E7的ITS序列分析结果表明,本研究分离的松茸菌与Tricholoma matsutake的菌株在系统发育上高度同源,在分类上应属于同一个种     

Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis

We compared the DNA sequence difference of isolates of Clonorchis sinensis from one Korean (Kimhae) and two Chinese areas (Guangxi and Shenyang). The sequences of nuclear rDNA (18S, internal transcribed spacer 1 and 2: ITS1 and ITS2) and mitochondrial DNA (cytochrome c oxidase subunit 1: cox1) were compared. A very few intraspecific nucleotide substitution of the 18S, ITS1, ITS2 and cox1 was found among three isolates of C. sinensis and a few nucleotide insertion and deletion of ITS1 were detected. The 18S, ITS1, ITS2 and cox1 sequences were highly conserved among three isolates. These findings indicated that the Korean and two Chinese isolates are similar at the DNA sequence level.