Isomucor (Mucoromycotina): a new genus from a Cerrado reserve in state of Sao Paulo, Brazil

During a survey of mucoralean fungi from a Cerrado reserve (Brazilian savanna) some isolates of a Mucor-like fungus were isolated from soil plates. Characterization based on morphological, physiological and molecular data from translation elongation factor (EF-1α), 28S (D1/D2) and ITS1-5.8S-ITS2 rDNA sequences was made. The isolates produce lateral branches bearing multispored sporangiola in addition to the multispored sporangia and a uniformly septate mycelium as the main differentiating characteristics. To our surprise this fungus possesses two EF-1α genes 1.4 and 1.5 kb long. Evidence from the analyzed datasets supports the delimitation of a new genus and the inclusion of Mucor fuscus based on 28S and ITS rDNA data.     

Molecular phylogenetic and morphological analyses of Oidium heveae, a powdery mildew of rubber tree

Powdery mildew of rubber tree caused by Oidium heveae is an important disease of rubber plantations worldwide. Identification and classification of this fungus is still uncertain because there is no authoritative report of its morphology and no record of its teleomorphic stage. In this study, we compared five specimens of the rubber powdery mildew fungus collected in Malaysia, Thailand, and Brazil based on morphological and molecular characteristics. Morphological results showed that the fungus on rubber tree belongs to Oidium subgen. Pseudoidium. Nucleotide sequence analysis of the ribosomal DNA internal transcribed spacer (ITS) region and the large subunit rRNA gene (28S rDNA) were conducted to determine the relationships of the rubber powdery mildew fungus and to link this anamorphic fungus with its allied teleomorph. The results showed that the rDNA sequences of the two specimens from Malaysia were identical to a specimen from Thailand, whereas they differed by three bases from the two Brazilian isolates: one nucleotide position in the ITS2 and two positions in the 28S sequences. The ITS sequences of the two Brazilian isolates were identical to sequences of Erysiphe sp. on Quercus phillyraeoides collected in Japan, although the 28S sequences differed at one base from sequences of this fungus. Phylogenetic trees of both rDNA regions constructed by the distance and parsimony methods showed that the rubber powdery mildew fungus grouped with Erysiphe sp. on Q. phillyraeoides with 100% bootstrap support. Comparisons of the anamorph of two isolates of Erysiphe sp. from Q. phillyraeoides with the rubber mildew did not reveal any obvious differences between the two powdery mildew taxa, which suggests that O. heveae may be an anamorph of Erysiphe sp. on Q. phillyraeoides. Cross-inoculation tests are required to substantiate this conclusion.     

Sequence heterogeneity in the internal transcribed spacers of two rat ribosomal DNA clones

The sequence of one cloned rat rDNA segment is determined, encompassing the 3'-terminal part of 18 S rDNA (231 bp), ITS 1 (1072 bp), 5.8 S rDNA (156 bp), ITS 2 (771 bp) and the 5'-terminal part of 28 S rDNA (210 bp). Comparison with a distinct clone of the same rDNA segment reveals the identity of the rRNA gene sequences and considerable heterogeneity in both ITS 1 and ITS 2. The heterogeneities include mainly single base-pair changes (23 deletions or insertions and 14 substitutions) distributed all along the ITS sequences.     

Erysiphe corylopsidis sp. nov., a new powdery mildew fungus found on Corylopsis spicata and C. pauciflora

A powdery mildew fungus occurring on leaves of Corylopsis pauciflora and C. spicata in Japan is described as a new species, Erysiphe corylopsidis. This species is characterized by fewer than 15 appendages on a chasmothecium, primary branches of the appendages occasionally elongated, and a relatively small number (2–5) of ascospores per ascus. Molecular phylogenetic analyses based on rDNA ITS and 28S rDNA sequences indicate that this fungus forms an independent lineage in the genus Erysiphe.     

Kohninia linnaeicola, a new genus and species of the Sclerotiniaceae pathogenic to Linnaea borealis

A new genus and species is described to accommodate a newly discovered fungus pathogenic to Linnaea borealis. The fungus forms true sclerotia on stems and leaves of its host and apothecia arise singly or gregariously on the surface of ripe sclerotia. The new fungus was collected together with a stromatic conidiomal fungus that occurred on the same host. A putative teleomorph-anamorph connection of the observed taxa was ruled out by sequence comparison of the nuclear ribosomal internal transcribed spacer DNA sequences (ITS rDNA). Based on morphology and pathogenicity, the new fungus belongs in the family Sclerotiniaceae, Helotiales, Ascomycota. A phylogenetic analysis of ITS rDNA sequences from 26 taxa of the family Sclerotiniaceae was performed to conclude on the systematic position of the new fungus. The small tuberoid sclerotia, brownish subsessile to substipitate apothecia, four-spored asci, ellipsoid to isthmoid ascospores, inability to grow on PDA culture media and a number of ITS rDNA sequence autapomorphies characterize and distinguish the fungus from other taxa of the Sclerotiniaceae.     

Sequencing and analysis of the internal transcribed spacers (ITSs) and coding regions in the EcoR I fragment of the ribosomal DNA of the Japanese pond frog Rana nigromaculata

The rDNA of eukaryotic organisms is transcribed as the 40S-45S rRNA precursor, and this precursor contains the following segments: 5' - ETS - 18S rRNA - ITS 1 - 5.8S rRNA - ITS 2 - 28S rRNA - 3'. In amphibians, the nucleotide sequences of the rRNA precursor have been completely determined in only two species of Xenopus. In the other amphibian species investigated so far, only the short nucleotide sequences of some rDNA fragments have been reported. We obtained a genomic clone containing the rDNA precursor from the Japanese pond frog Rana nigromaculata and analyzed its nucleotide sequence. The cloned genomic fragment was 4,806 bp long and included the 3'-terminus of 18S rRNA, ITS 1, 5.8S rRNA, ITS 2, and a long portion of 28S rRNA. A comparison of nucleotide sequences among Rana, the two species of Xenopus, and human revealed the following: (1) The 3'-terminus of 18S rRNA and the complete 5.8S rRNA were highly conserved among these four taxa. (2) The regions corresponding to the stem and loop of the secondary structure in 28S rRNA were conserved between Xenopus and Rana, but the rate of substitutions in the loop was higher than that in the stem. Many of the human loop regions had large insertions not seen in amphibians. (3) Two ITS regions had highly diverged sequences that made it difficult to compare the sequences not only between human and frogs, but also between Xenopus and Rana. (4) The short tracts in the ITS regions were strictly conserved between the two Xenopus species, and there was a corresponding sequence for Rana. Our data on the nucleotide sequence of the rRNA precursor from the Japanese pond frog Rana nigromaculata were used to examine the potential usefulness of the rRNA genes and ITS regions for evolutionary studies on frogs, because the rRNA precursor contains both highly conserved regions and rapidly evolving regions.     

Evolution and phylogenetic information content of the ribosomal DNA repeat unit in the Blattodea (Insecta)

The organization, structure, and nucleotide variability of the ribosomal repeat unit was compared among families, genera, and species of cockroaches (Insecta:Blattodea). Sequence comparisons and molecular phylogenetic analyses were used to describe rDNA repeat unit variation at differing taxonomic levels. A reverse similar 1200 bp fragment of the 28S rDNA sequence was assessed for its potential utility in reconstructing higher-level phylogenetic relationships in cockroaches. Parsimony and maximum likelihood analyses of these data strongly support the expected pattern of relationships among cockroach groups. The examined 5' end of the 28S rDNA is shown to be an informative marker for larger studies of cockroach phylogeny. Comparative analysis of the nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among closely related species of Blattella and Periplaneta reveals that ITS sequences can vary widely in primary sequence, length, and folding pattern. Secondary structure estimates for the ITS region of Blattella species indicate that variation in this spacer region can also influence the folding pattern of the 5.8S subunit. These results support the idea that ITS sequences play an important role in the stability and function of the rRNA cluster.     

毛木耳rDNA序列结构及IGS鉴别菌种初探

rDNA序列中的ITS作为DNA barcode广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。有关食用菌rDNA序列的报道较少。本研究对毛木耳Auricularia cornea单核菌株B02进行三代测序与组装,然后用二代测序数据进行校正,得到一个组装效果较好的基因组序列。比对Fibroporia vaillantiir的rDNA序列获得毛木耳rDNA重复单元的完整序列,每个重复单元包含ETS、18S rDNA、ITS1、5.8S rDNA、ITS2、28S rDNA、IGS1、5S rDNA和IGS2,长度分别为398bp、1 790bp、156bp、156bp、206bp、3 432bp、2 247bp、121bp和2 135bp,总长度10 641bp,毛木耳rDNA有310个串联重复单元,转录组和系统发育分析均支持这一结果。与其他已报道的食用菌不同,毛木耳的IGS1、IGS2序列高度保守,其中IGS1的1 400-2 200bp区域在各拷贝之间没有多态性、而在品种之间有较高频率的SNP,这一片段序列有望用于品种鉴别研究。     

Molecular phylogeny and evolution of subsection Magnicellulatae (Erysiphaceae: Podosphaera) with special reference to host plants

The subsection Magnicellulatae of the genus Podosphaera section Sphaerotheca belongs to the tribe Cystotheceae of the Erysiphaceae, which has the characteristic of producing catenate conidia with distinct fibrosin bodies. In this study, we newly determined the nucleotide sequences of the D1/D2 domains of the 28S rDNA region and the sequences of the rDNA internal transcribed spacer (ITS) region to investigate the relationships between the phylogeny of this fungal group and their host plants. The results indicated that the 28S rDNA region is too conservative for phylogenetic analysis of this fungal group. The phylogenetic analysis using 95 ITS sequences demonstrated that two or more Magnicellulatae taxa often infect the same plant genus or species. Although there is a close relationship between Magnicellulatae and asteraceous hosts, this association seems to be not as strict as that between Golovinomyces and the Asteraceae. The difference between the two fungal groups may be explained by their different evolutionary timing.     

从ITS序列探讨猪苓与其伴生菌的亲缘关系

对猪苓菌丝、野生猪苓子实体、野生猪苓菌核和其伴生菌的 5.8SrDNA及其两侧的ITS 1区和ITS2区进行了序列分析。发现猪苓与其伴生菌的ITS序列同源性达 99.36% ,说明猪苓与其伴生菌有着极高的分子亲缘关系。     

Stereumin A-E, sesquiterpenoids from the fungus Stereum sp. CCTCC AF 207024

Five cadinane sesquiterpenoids, named stereumin A (1), B (2), C (3), D (4) and E (5) were isolated from the CHCl(3) extract of the culture broth of the fungal strain CCTCC AF 207024. Based on the sequences at the internal transcribed spacer (ITS) region and partial 28S rDNA, this fungus was identified as a Stereum sp. The structures of the five compounds were elucidated using spectroscopic data from 1D, 2D NMR and HRESIMS experiments, and the structures of 1 and 2 were further confirmed by single-crystal X-ray diffraction analysis. Compounds 1-5 showed nematicidal activities against the nematode Panagrellus redivivus at 400 mg l(-1). Among these five compounds, compounds 3 and 4 killed 84.4% and 94.9% of P. redivivus, respectively in 48 h.     

Phylogenetic analysis of a dataset of fungal 5.8S rDNA sequences shows that highly divergent copies of internal transcribed spacers reported from Scutellospora castanea are of ascomycete origin

Using a dataset comprising 5.8S rDNA sequences from a wide range of fungi, we show that some sequences reported recently from the arbuscular mycorrhizal (AM) fungus Scutellospora castanea most likely originate from Ascomycetes. Other ITS and 5.8S sequences which were previously reported are confirmed as being clearly of mycorrhizal origin and are variable within one isolate of S. castanea. However, these results mean that previous conclusions which were drawn regarding the heterokaryotic status of AM fungal spores remain unproven. We provide an enlarged 5.8S rDNA dataset that can be used to check ITS sequences for conflicts with well-established phylogenies of the organisms that they were obtained from.     

Ribosomal DNA sequence characterization of Maritrema cf. eroliae Yamaguti, 1939 (Digenea: Microphallidae) and its life cycle

The microphallid Maritrema eroliae parasitizes shore birds in marine ecosystems while its larval stages infect mud snails and crustacean hosts. Because it is difficult to morphologically distinguish between larvae of M. eroliae and other microphallids co-occurring in the same habitat, partial nucleotide sequences of the ribosomal DNA (rDNA), including the 28S and 18S in addition to complete sequences of ITS1 and ITS2, were scrutinized. This analysis was used to establish the snail-crab link in the life cycle of M. cf. eroliae . The rDNA 28S, 18S, and ITS sequences of metacercariae from the crab Xantho exaratus and sporocysts from the snail Clypeomorus bifasciata were compared. Sequence alignment demonstrated that the sporocyst and metacercaria may belong to M. eroliae and suggested a new second intermediate host for M. eroliae , the crab X. exaratus . The phylogenetic positions of the larval stages were determined by comparing the 28S, 18S, and ITS sequences with those of other trematodes available in GenBank. The phylogenetic trees confirmed the position of M. cf. eroliae within the Microphallidae and found it to be closely related to Maritrema heardi and Maritrema neomi. The present study represents the first molecular study correlating the larval stages in the life cycle of M. cf. eroliae using partial sequences of 28S and 18S in addition to complete ITS1 and ITS2 sequences. Furthermore, the sequences elucidated the evolutionary relationship of M. cf. eroliae to other microphallids.     

Molecular divergence of the ssu rRNA gene and internal transcribed spacer 1 in Porphyra yezoensis (Rhodophyta)

Nucleotide sequences from the downstream of ssu rDNA to ITS1 region of the individual thalli of both wild-collected Porphyra yezoensis from three different sites and culture strains were determined to obtain the molecular features of strains in the P. yezoensis lineage. Wild-collected thalli identified by morphological systematics, included the individuals that were separate from the P. yezoensis lineage based on ssu rDNA and ITS1 sequence homologies and phylogenetic relationships constructed using ITS1 sequences. Ssu rDNA exon region nucleotide sequences were identical among the wild-collected and clture strains of P. yezoensis. However, all individual wild-collected P. yezoensis thalli had different ITS1 sequences, even among individuals from the same sites. Furthermore, two different ssu rDNA structures with and without an intron were found in individuals from the same site. These results indicated the possibility that the presence and sequence of introns and ITS1 sequences can be used as a characteristic to determine the origin of culture strains. Four of six culture strains examined had an identical sequence from the ssu rDNA to ITS1, while the sequences of another two strains differed. In this study, wild-collected and culture strain thalli sequences were not identical, although similar pairs were identified. This revised version was published online in July 2006 with corrections to the Cover Date.     

The structure of a single unit of ribosomal RNA gene (rDNA) including intergenic subrepeats in the Australian bulldog ant Myrmecia croslandi (Hymenoptera: Formicidae)

A complete single unit of a ribosomal RNA gene (rDNA) of M. croslandi was sequenced. The ends of the 18S, 5.8S and 28S rRNA genes were determined by using the sequences of D. melanogaster rDNAs as references. Each of the tandemly repeated rDNA units consists of coding and non-coding regions whose arrangement is the same as that of D. melanogaster rDNA. The intergenic spacer (IGS) contains, as in other species, a region with subrepeats, of which the sequences are different from those previously reported in other insect species. The length of IGSs was estimated to be 7-12 kb by genomic Southern hybridization, showing that an rDNA repeating unit of M. croslandi is 14-19 kb-long. The sequences of the coding regions are highly conserved, whereas IGS and ITS (internal transcribed spacer) sequences are not. We obtained clones with insertions of various sizes of R2 elements, the target sequence of which was found in the 28S rRNA coding region. A short segment in the IGS that follows the 3' end of the 28S rRNA gene was predicted to form a secondary structure with long stems.     

Phylogenetic Analysis of a Dataset of Fungal 5.8S rDNA Sequences Shows That Highly Divergent Copies of Internal Transcribed Spacers Reported from Scutellospora castanea Are of Ascomycete Origin

Using a dataset comprising 5.8S rDNA sequences from a wide range of fungi, we show that some sequences reported recently from the arbuscular mycorrhizal (AM) fungus Scutellospora castanea most likely originate from Ascomycetes. Other ITS and 5.8S sequences which were previously reported are confirmed as being clearly of mycorrhizal origin and are variable within one isolate of S. castanea. However, these results mean that previous conclusions which were drawn regarding the heterokaryotic status of AM fungal spores remain unproven. We provide an enlarged 5.8S rDNA dataset that can be used to check ITS sequences for conflicts with well-established phylogenies of the organisms that they were obtained from.     

Phyllosticta sp. nov., a New Pathogenic Species from Buxus sincia

In a study of pathogenic fungi infecting Buxus sincia , an interesting Phyllosticta species was isolated. The morphological characteristics of this species, such as slow growing mycelia, large conidia each with a long flagellum tag on diagnostic media, plus phylogenetic analysis based on 16S rDNA ITS sequences indicated that the fungus is a distinct new species which is described.     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

Phylogenetic position of endosymbiotic green algae in Paramecium bursaria Ehrenberg from Japan

Endosymbiotic green algae of Japanese Paramecium bursaria were phylogenetically analyzed based on DNA sequences from the ribosomal DNA operon (18S rDNA, ITS1, 5.8S rDNA, and ITS2). Phylogenetic trees constructed using 18S rDNA sequences showed that the symbionts belong to the Chlorella sensu stricto (Trebouxiophyceae) group. They are genetically closer to the C. vulgaris Beijerinck group than to C. kessleri Fott et Nováková as proposed previously. Branching order in C. vulgaris group was unresolved in 18S rDNA trees. Compared heterogeneities of 18S rDNA, ITS1, 5.8S r, and ITS2 among symbionts and two Chlorella species, indicated that the ITS2 region (and probably also ITS1) is better able to resolve phylogenetic problems in such closely related taxa. All six symbiotic sequences obtained here (approximately 4000-bp sequences of 18S rDNA, ITS1, 5.8S rDNA, and ITS2) were completely identical in each, strongly suggesting a common origin.