D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.     

Characterization of chitinases of polycentric anaerobic rumen fungi

Chitinolytic systems of anaerobic polycentric rumen fungi of genera Orpinomyces and Anaeromyces were investigated in three crude enzyme fractions - extracellular, cytosolic and cell-wall. Endochitinase was found as a dominant enzyme with highest activity in the cytosolic fraction. Endochitinases of both genera were stable at pH 4.5-7.0 with optimum at 6.5. The Orpinomyces endochitinase was stable up to 50 degrees C with an optimum for enzyme activity at 50 degrees C; similarly, Anaeromyces endochitinase was stable up to 40 degrees C with optimum at 40 degrees C. The most suitable substrate for both endochitinases was fungal cell-wall chitin. Enzyme activities were inhibited by Hg(2+) and Mn(2+), and activated by Mg(2+) and Fe(3+). Both endochitinases were inhibited by 10 mmol/L SDS and activated by iodoacetamide.     

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 reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi from phylum to species level

Although the molecular phylogeny, evolution and biodiversity of arbuscular mycorrhizal fungi (AMF) are becoming clearer, phylotaxonomically reliable sequence data are still limited. To fill this gap, a data set allowing resolution and environmental tracing across all taxonomic levels is provided. Two overlapping nuclear DNA regions, totalling c. 3 kb, were analysed: the small subunit (SSU) rRNA gene (up to 1800 bp) and a fragment spanning c. 250 bp of the SSU rDNA, the internal transcribed spacer (ITS) region (c. 475-520 bp) and c. 800 bp of the large subunit (LSU) rRNA gene. Both DNA regions together could be analysed for 35 described species, the SSU rDNA for c. 76 named and 18 as yet undefined species, and the ITS region or LSU rDNA, or a combination of both, for c. 91 named and 16 as yet undefined species. Present phylogenetic analyses, based on the three rDNA markers, provide reliable and robust resolution from phylum to species level. Altogether, 109 named species and 27 cultures representing as yet undefined species were analysed. This study provides a reference data set for molecular systematics and environmental community analyses of AMF, including analyses based on deep sequencing.     

Carbohydrate fermentation by three species of polycentric ruminal fungi from cattle and water buffalo in tropical Australia

Fructose, glucose and xylose were the only monosaccharides to be fermented by the polycentric fungi, Orpinomyces joyonii (three cattle isolates) and O. intercalaris (two cattle isolates) and Anaeromyces spp. (four cattle isolates and two water buffalo isolates). Both Orpinomyces spp. utilised a similar range of oligosaccharides and polysaccharides by fermenting cellobiose, gentiobiose, lactose, maltose, sucrose, cellulose, glycogen, starch and xylan. In contrast, there was considerable variation in carbohydrate fermentation amongst Anaeromyces spp., with only cellobiose, gentiobiose and cellulose being fermented by all strains. Formate, acetate and ethanol were the major fermentation end-products formed from glucose by all polycentric fungi. In addition, Anaeromyces spp. produced considerable amounts of lactate, although only small amounts were formed by Orpinomyces spp. This difference was explained by the low specific activity for lactate dehydrogenase in Orpinomyces spp. Several Anaeromyces spp. also produced malate as a significant end-product of glucose fermentation. Fermentation of specifically-labelled Z14C]glucose molecules by polycentric fungi showed that hexose was catabolised by both polycentric and monocentric fungi via the glycolysis pathway with end-products being derived from the following carbon atoms: lactate and malate (C1-C3; C4-C6), acetate and ethanol (C1-C2; C5-C6), CO2 and formate (C3; C4). The results were compared to those obtained for monocentric and polycentric fungi isolated from temperate climate ruminants.     

Genetic and biochemical diversity among isolates of Paenibacillus alvei cultured from Australian honeybee (Apis mellifera) colonies

Twenty-five unique CfoI-generated whole-cell DNA profiles were identified in a study of 30 Paenibacillus alvei isolates cultured from honey and diseased larvae collected from honeybee (Apis mellifera) colonies in geographically diverse areas in Australia. The fingerprint patterns were highly variable and readily discernible from one another, which highlighted the potential of this method for tracing the movement of isolates in epidemiological studies. 16S rRNA gene fragments (length, 1,416 bp) for all 30 isolates were enzymatically amplified by PCR and subjected to restriction analysis with DraI, HinfI, CfoI, AluI, FokI, and RsaI. With each enzyme the restriction profiles of the 16S rRNA genes from all 30 isolates were identical (one restriction fragment length polymorphism [RFLP] was observed in the HinfI profile of the 16S rRNA gene from isolate 17), which confirmed that the isolates belonged to the same species. The restriction profiles generated by using DraI, FokI, and HinfI differentiated P. alvei from the phylogenetically closely related species Paenibacillus macerans and Paenibacillus macquariensis. Alveolysin gene fragments (length, 1, 555 bp) were enzymatically amplified from some of the P. alvei isolates (19 of 30 isolates), and RFLP were detected by using the enzymes CfoI, Sau3AI, and RsaI. Extrachromosomal DNA ranging in size from 1 to 10 kb was detected in 17 of 30 (57%) P. alvei whole-cell DNA profiles. Extensive biochemical heterogeneity was observed among the 28 P. alvei isolates examined with the API 50CHB system. All of these isolates were catalase, oxidase, and Voges-Proskauer positive and nitrate negative, and all produced acid when glycerol, esculin, and maltose were added. The isolates produced variable results for 16 of the 49 biochemical tests; negative reactions were recorded in the remaining 30 assays. The genetic and biochemical heterogeneity in P. alvei isolates may be a reflection of adaptation to the special habitats in which they originated.     

Phylogenetic analysis of ribosomal RNA operons from uncultivated coastal marine bacterioplankton

Analyses of small subunit ribosomal RNA genes (SSU rDNAs) have significantly influenced our understanding of the composition of aquatic microbial assemblages. Unfortunately, SSU rDNA sequences often do not have sufficient resolving power to differentiate closely related species. To address this general problem for uncultivated bacterioplankton taxa, we analysed and compared sequences of polymerase chain reaction (PCR)-generated and bacterial artificial chromosome (BAC)-derived clones that contained most of the SSU rDNAs, the internal transcribed spacer (ITS) and the large subunit ribosomal RNA gene (LSU rDNA). The phylogenetic representation in the rRNA operon PCR library was similar to that reported previously in coastal bacterioplankton SSU rDNA libraries. We observed good concordance between the phylogenetic relationships among coastal bacterioplankton inferred from SSU or LSU rDNA sequences. ITS sequences confirmed the close intragroup relationships among members of the SAR11, SAR116 and SAR86 clades that were predicted by SSU and LSU rDNA sequence analyses. We also found strong support for homologous recombination between the ITS regions of operons from the SAR11 clade.     

青藏高原食草动物粪栖真菌的多样性

分别利用真菌通用引物和厌氧真菌特异引物构建了西藏地区3种反刍动物和1种单胃动物共8份新鲜粪样的ITS克隆文库,以通过系统发育分析解析其中好氧真菌与厌氧真菌的多样性。通用引物ITS文库测得324条真菌序列,分别属于子囊菌门Ascomycota 3个目、担子菌门Basidiomycota 2个目、接合菌门Zygomycota的1个目和严格厌氧的新丽鞭毛菌门Neocallimastigomycota,共24个OTUs。其中,子囊菌门相对丰度最高,占80.6%;新丽鞭毛菌门相对丰度最低,仅占0.6%。大部分OTUs与已知真菌属、种关系较远。厌氧真菌特异引物文库测得661条序列,全部属于新丽鞭毛菌门,包括所有已知的厌氧霉属Anaeromyces、盲肠菌属Caecomyces、肠霉属Cyllamyces、新丽鞭毛属Neocallimastix、奥式霉属Orpinomyces、胃梨囊霉属Piromyces 6个属和3个未培养的属级类群(NG9、NG10、NG11),共29个OTUs。其中3个已知的单中心属存在于所有反刍动物样品中,并以Piromyces相对丰度最高(37.4%)。单胃动物马粪样中全部为NG9类群。NG9是本研究新发现的属级类群,研究中同时揭示有多个未培养种和潜在的新种。研究结果证明青藏高原反刍动物粪栖真菌多样性较高,并存在丰富的未培养种和潜在的新属及新种。     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

A PCR/RFLP technique to characterize fungal species in <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> Hill ex. Maiden ectomycorrhizas

With the increasing awareness of the significance of mycorrhizas, research is focusing on studies to elucidate the contribution of the symbiosis to ecosystem dynamics. In this sense, molecular biology has acquired great significance. PCR/RFLP techniques were adapted to characterize ectomycorrhizal fungi associated with Eucalyptus grandis.The ITS region of the fungal rDNA from pure cultures and from of mycorrhizas synthesized in vitro was amplified. Primers NSA3/NLC2 were used followed by a nested reaction with primers ITS1F/NLB3. Amplicons were then digested with the enzymes MboI, HinfI and TaqI. Amplification resulted in a 1,000-bp fragment for basidiomycetes and a 1,500 bp fragment for Cenococcum geophillum (an ascomycete). There was no amplification of the plant DNA. The enzymes MboI and HinfI were more effective than TaqI, resulting in patterns of two to five fragments allowing the identification of the isolates both in culture and in mycorrhizas. HinfI allowed greater differentiation among the isolates and a higher number of polymorphisms. Restriction with TaqI resulted in too many fragments. Amplification efficiency for the fungal DNA was 64% in culture and 87% in mycorrhizas. The modified methodology represents a valuable tool to complement traditional approaches in ecosystem studies.     

Phylogenetic relationships among 28 spirotrichous ciliates documented by rDNA

The contiguous sequence of the SSU rDNA, ITS 1, 5.8S, ITS 2, and approximately 1370 bp at the 5(') end of the LSU rDNA was determined in 25 stichotrichs, one oligotrich, and two hypotrichs. Maximum parsimony, neighbor-joining, and quartet-puzzling analyses were used to construct individual phylogenetic trees for SSU rDNA, for LSU rDNA, and ITS 1+5.8S+ITS 2, as well as for all these components combined. All trees were similar, with the greatest resolution obtained with the combined components. Phylogenetic relationships were largely consistent with classical taxonomy, with notable disagreements. DNA sequences indicate that Oxytricha granulifera and Oxytricha longa are rather distantly related. The oligotrich, Halteria grandinella, is placed well within the order Stichotrichida. Uroleptus pisces and Uroleptus gallina probably belong to different genera. Holosticha polystylata (family Holostichidae) and Urostyla grandis (family Urostylidae) are rather closely related. These rDNA sequence analyses imply the need for some modifications of classical taxonomic schemes.     

Inter- and intrasporal nuclear ribosomal gene sequence variation within one isolate of arbuscular mycorrhizal fungus, Diversispora sp.

Most molecular ecological studies of arbuscular mycorrhizal fungi (AMF) have been based on the rRNA gene sequences. However, information about intraspecific nucleotide variation is still limited in these fungi. In this study, we calculated the inter- and intrasporal nucleotide variation of Diversispora sp. EE1 using 78 cloned sequences from four spores within a ca 4960 bp fragment of the nuclear ribosomal operon spanning the near full length small ribosomal subunit (SSU) rRNA gene, the full internal transcribed spacer (ITS: ITS1-5.8S-ITS2) and ca 2740 bp of the large ribosomal subunit (LSU) rRNA gene. Data for each marker region (SSU, ITS and LSU) originated from the very same spores. Sequence variation resulting from point mutations and small indels was recorded in all regions. Highest sequence variation was observed in the ITS region at both the inter- and intrasporal levels. The ITS1 component was more variable than ITS2, whilst the 5.8S gene was the least variable component of the ITS region. Evolutionary divergence of gene copies between spores was intermediate for the LSU and lowest for the SSU. The SSU and the LSU genes had relatively similar evolutionary divergence per spore. Sequence variant richness was not exhaustive for any of the marker regions, indicating that multiple sequences per spore from multiple spores are needed when characterizing a species. This study provides reference sequences for ecological studies, permitting identification of AMF using any of the ribosomal regions or primer systems.     

Seasonal microbial ribotype shifts in the sulfurous karstic lakes Cisó and Vilar, in northeastern Spain.

Spatio-temporal changes in two sulfurous lakes from the karstic area of Banyoles (Girona, Spain), holomictic lake Cisó and meromictic lake Vilar, were studied over one year. Samples were collected at different depths from the two lakes on the same days, during each of the four seasons, and several physico-chemical variables (temperature, light, pH, conductivity, sulfide, oxygen concentration, pigment concentrations, etc.) were measured. To fingerprint bacterial populations from each sample, DNA was extracted, bacterial 16S rRNA genes were amplified by PCR, and restriction fragment length polymorphism (RFLP) analyses of the total bacterial 16S rDNAs were performed. Each 16S rDNA pool was independently digested with three restriction endonucleases (AluI, HinfI, and RsaI) and separated electrophoretically. More restriction fragments were obtained from the Lake Vilar samples than from the Lake Cisó samples. Moreover, intrasample and intersample differences were observed in each lake. RFLP patterns were compared by scoring similarities using the Jaccard coefficient and then building a multidimensional scaling (MDS) map from the resulting similarities matrix. In both lakes, results indicated that seasonality was mostly responsible for the observed fluctuations in the RFLP patterns, while the effect of stratification was less pronounced.     

Cloning and organization of genes for 5S ribosomal RNA in the sea urchin. Lytechinus variegatus

A 1.35-kb EcoRI fragment of Lytechinus variegatus DNA containing a single 5S rRNA gene has been cloned into the plasmid vector pACYC184. Four clones from different transformation experiments contain 5S rDNA inserts of about the same size and have the same restriction enzyme digestion patterns for the enzymes HaeIII, HinfI, HhaI, and AluI. One EcoRI site near the HindIII site of the plasmid vector pACYC184 is missing in all the four clones. By DNA sequencing, the missing EcoRI ws found to be EcoRI site, d(AAATTN)d(TTTAAN) in pLu103, one of the four 5S rDNA clones. The structure of pLu103 was determined by restriction mapping and blot hybridization. Three restriction fragments, 1.0-kb HaeIII/HaeIII, 0.375-kb AluI/AluI and 0.249-kb MboII/MboII, which contain the 5S rRNA coding region, have been subcloned into the EcoRI site of the plasmid pACYC184. The organization of 5S rRNA genes in the sea urchin genome was also investigated. It was found that restriction endonuclease HaeIII has a single recognition site within each 5S rDNA repeat, and yields two fragment lengths, 1.2 and 1.3 kb. The behavior of these 5S rRNA genes when total L. variegatus DNA is partially digested with HaeIII is consistent with an arrangement of 5S rRNA genes in at least two tandemly repeated, non-interspersed families. Both the coding region and spacer region of the 5S rRNA gene in pLu103 hybridize to 1.2 and 1.3-kb rDNA families. This indicates that the cloned EcoRI fragment of 5S rDNA in pLu103 represents one single repeat of 5S rDNA in the genome.     

Independent terrestrial origins of the Halosphaeriales (marine Ascomycota)

A phylogenetic study of marine ascomycetes was initiated to test and refine evolutionary hypotheses of marine-terrestrial transitions among ascomycetes. Taxon sampling focused on the Halosphaeriales, the largest order of marine ascomycetes. Approximately 1050 base pairs (bp) of the gene that codes for the nuclear small subunit (SSU) and 600 bp of the gene that codes for the nuclear large subunit (LSU) ribosomal RNAs (rDNA) were sequenced for 15 halosphaerialean taxa and integrated into a data set of homologous sequences from terrestrial ascomycetes. An initial set of phylogenetic analyses of the SSU rDNA from 38 taxa representing 15 major orders of the phylum Ascomycota confirmed a close phylogenetic relationship of the halosphaerialean species with several other orders of perithecial ascomycetes. A second set of analyses, which involved more intensive taxon sampling of perithecial ascomycetes, was performed using the SSU and LSU rDNA data in combined analyses. These second analyses included 15 halosphaerialean taxa, 26 terrestrial perithecial fungi from eight orders, and five outgroup taxa from the Pezizales. In these analyses the Halosphaeriales were polyphyletic and comprised two distinct lineages. One clade of Halosphaeriales comprised 12 taxa from 11 genera and was most closely related to terrestrial fungi of the Microascales. The second clade of halosphaerialean fungi comprised taxa from the genera Lulworthia and Lindra and was an isolated lineage among the perithecial fungi. Both the main clade of Halosphaeriales and the Lulworthia/Lindra clade are supported by the data as being independently derived from terrestrial ancestors.     

Restriction Analysis of PCR-Amplified Internal Transcribed Spacers of Ribosomal DNA as a Tool for Species Identification in Different Genera of the Order Glomales

A technique combining PCR and restriction fragment length polymorphism analysis was used to generate specific DNA fragment patterns from spore extracts of arbuscular mycorrhizal fungi. With the universal primers ITS1 and ITS4, DNA fragments were amplified from species of Scutellospora and Gigaspora that were approximately 500 bp long. The apparent lengths of the corresponding fragments from Glomus spp. varied between 580 and 600 bp. Within the genus Glomus, the restriction enzymes MboI, HinfI, and TaqI were useful for distinguishing species. Depending on the restriction enzyme used, groups of species with common fragment patterns could be found. Five tropical and subtropical isolates identified as Glomus manihotis and G. clarum could not be distinguished by their restriction patterns, corresponding to the morphological similarity of the spores. The variation of internal transcribed spacer sequences among the Gigaspora species under study was low. Fragment patterns of Scutellospora spp. showed their phylogenetic relationship with Gigaspora and revealed only a slightly higher degree of variation.     

捕食线虫真菌rDNA ITS区间RFLPs分析

利用PCR-RFLP方法对捕食线虫真菌进行了系统发育研究。以ITS1和ITS4为引物对3属14种16个菌株的核糖体DNA转录间区（ITS）进行了PCR扩增,4种内切酶（AluI、HaeIII、HpaII、TaqI）酶切,结果表明不同属的ITS区长度没有明显差异,其长度范围在585～695之间。酶切图谱种间差别明显,种内基本一致,同属菌株图谱没有特异性,暗示传统的分属可能过细,某些属的成立还有待商榷,PCR-RFLP对确定疑难种的地位有重要意义,但不适用于种下水平的系统学研究。