Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two Specific Strains of <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis> causing Mucocutaneous Manifestations of Histoplasmosis: Preliminary Analysis of a Frequent Manifestation of Histoplasmosis in Southern Brazil

Objectives  Skin lesions, uncommon in US cases (<10%), occur in 38–85% of cases reported from Latin America. Although these differences may reflect reporting bias, delayed diagnosis, or differences in host immune response among different ethnic groups, they also could result from genetic differences changing the pathobiology of the organism. It is possible that genetic differences among strains of H. capsulatum may influence the pathogenesis and clinical manifestations of histoplasmosis. Methods  We examined the clinical features of patients with mucocutaneous manifestations of histoplasmosis and performed genetic analysis based on nucleotide sequence variations in the internal transcribed spacer regions of rRNA genes of H. capsulatum isolates of patients. Two pairs of PCR primers were designed to develop and amplify the ITS regions of H. capsulatum, 5′-TACCCGGCCACCCTTGTCTA-3′ and 5′-AGCGGGTGGCAAAGCCC-3′. These primers were based on the ITS sequence of Ajellomyces capsulatus, the ascomycetous teleomorph form of H. capsulatum, deposited in the GenBank (accession number U18363). Eight patients attending a tertiary-care hospital in southern Brazil were enrolled into the study. All case patients had skin cultures growing H. capsulatum at the mycology laboratory. Results  Six of eight (75%) patients were HIV-positive and presented involvement of multiples organs by H. capsulatum. Two HIV-negative patients did not present evidence of involvement of other organs besides mucosa and skin. ITS sequencing of a DNA H. capsulatum fragment of 485-bp from isolates of 8 patients revealed two distinct strains. The 2 distinct fragments (Hc1, Hc2) differed from each other at 7 positions in the ITS regions. They were identical to strains of H. capsulatum isolated in patients from Colombia and Argentina, but different from strains isolated in US. Hc1 and Hc2 were isolated in 5 patients and 3 patients, respectively, with mucocutaneous manifestations of histoplasmosis. Both Hc1 and Hc2 strains were isolated in HIV-infected and non-HIV-infected patients. Conclusions  Mucocutaneous manifestations of histoplasmosis, which are frequently seen in Brazilian patients were caused by 2 specific strains in our institution. Those strains have been isolated in patients with these particular clinical features of histoplasmosis in Latin America. Our study suggests that unique pathogenic characteristics among the Latin American species of H. capsulatum might explain its increased dermatotropism.     

Genotyping of Trichophyton rubrum by analysis of ribosomal-DNA intergenic spacer regions

An attempt was made to explore the genotyping of Trichophyton rubrum (T. rubrum) and the relationship between genotype and geographical origin using ribosomal restriction endonuclease polymorphic analysis. The total DNA was extracted by cetyltrimethyl ammonium bromide (CTAB). The probe was amplified from part of the 18S, ITSI, 5.8S, and ITSII region of T. rubrum standard strain with the universal fungal primers NS5 [5′-AACTT AAAGG AATTG ACGGA AG-3′] and ITS4 [5′-TCCTC CGCTT ATTGA TATGC-3′]. The genomic DNA of 49 clinical T. rubrum isolates digested by EcoR1 were hybridized with this probe, and the hybridization patterns were used as the basis of genotyping. Of the data from 49 strains of T. rubrum studied (21 from Nanjing, 26 from Dalian, and two from Beijing), 20 individual patterns (DNA Type A–T) were identified, among which Type A–C accounted for 48.98% of all the strains. The DNA patterns of Nanjing strains were represented by three bands, those of Dalian strains were represented by four bands. The DNA typing of T. rubrum by Southern blotting was highly sensitive and highly distinguishable. The DNA patterns of Nanjing strains were obviously different from those of Dalian strains.     

Diversity of root-associated fungal endophytes in Rhododendron fortunei in subtropical forests of China

To investigate the diversity of root endophytes in Rhododendron fortunei, fungal strains were isolated from the hair roots of plants from four habitats in subtropical forests of China. In total, 220 slow-growing fungal isolates were isolated from the hair roots of R. fortunei. The isolates were initially grouped into 17 types based on the results of internal transcribed spacer-restriction fragment length polymorphism (ITS-RFLP) analysis. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with known sequences of ericoid mycorrhizal endophytes and selected ascomycetes or basidiomycetes. Based on phylogenetic analysis of the ITS sequences in GenBank, 15 RFLP types were confirmed as ascomycetes, and two as basidiomycetes; nine of these were shown to be ericoid mycorrhizal endophytes in experimental cultures. The only common endophytes of R. fortunei were identified as Oidiodendron maius at four sites, although the isolation frequency (3–65%) differed sharply according to habitat. Phialocephala fortinii strains were isolated most abundantly from two habitats which related to the more acidic soil and pine mixed forests. A number of less common mycorrhizal RFLP types were isolated from R. fortunei at three, two, or one of the sites. Most of these appeared to have strong affinities for some unidentified root endophytes from Ericaceae hosts in Australian forests. We concluded that the endophyte population isolated from R. fortunei is composed mainly of ascomycete, as well as a few basidiomycete strains. In addition, one basidiomycete strain was confirmed as a putative ericoid mycorrhizal fungus.     

Screening of antioxidative activity of<Emphasis Type="Italic">Bifidobacterium</Emphasis> species isolated from Korean infant feces and their identification

Among 59 Korean isolated, 20 were confirmed as members of the genusBifidobacterium species based on gram staining, microscopic examination of cell morphology and the TLC method. The oxygen tolerance and antioxidative activities of these 20Bifidobacterium strains and 5 standardBifidobacterium strains were tested. All the strains demonstrated antioxidative activities as regards inhibiting linoleic acid peroxidation. The antioxidative activities of isolated and standard strains were found to range from 10.7–46.4% and from 10.7–22.2%, respectively. In addition, all tested strains exhibited a scavenging ability on DPPH free radicals, range from 15–41% for the isolated strains and 8.3–22% for the standard strain. Accordingly, the isolatedBifidobacterium strains demonstrated higher antioxidative activities than the 5 standardBifidobacterium strains. On the base of grades for each test, HJL 7511 was identified as the best strain, followed by HJL 7501. 2 strains were identified with Polymerase Chain Reaction (PCR) assay using group-specific primers designed from the nucleotide sequences of the 16S rRNA and internal transcribed spacer (ITS) regions of the Bifidobacteria. Based on the sequencing results, HJL 7511 and HJL 7501 were identified asBifidobacterium infantis.     

产黄酮成分的绶草内生真菌的鉴定

利用薄层层析法进行的定性分析表明,分离自绶草Spiranthes sinensis的3株内生真菌产生了黄酮类化合物,在形态学鉴定的同时,扩增、测序了3株内生真菌的rDNA的ITS序列,利用MEGA4.0软件和邻接法（Neighbour-joining methods）进行了聚类分析,菌株S-1鉴定为不等弯孢Curvularia inaequalis,S-2和S-3鉴定为嗜松青霉Penicillium pinophilum。     

Newly Isolated Bacillus gibsonii S-2 Capable of using Sugar Beet Pulp for Alkaline Pectinase Production

Summary A Bacillus strain capable of growing under highly alkaline conditions was isolated from a sample of alkaline soil. The isolate was a Gram-positive, spore-forming, aerobic, alkaliphilic bacterium and was designated as strain S-2. Growth of the strain was observed in the pH range of 7–12 and temperature range of 4–40 °C. Sequence analysis of the 16S rDNA of the strain revealed more than 99% homology with the strains of Bacillus gibsonii. The S-2 strain was confirmed as B. gibsonii by comparing its physiological and biochemical characteristics with the B. gibsonii DSM 8722 strain. The S-2 strain could use sugar beet pulp as the carbon source as well as the pectinase inducer to produce extracellular alkaline pectinase by solid-state fermentation. The maximum polygalacturonase yield of 3600 U/g dry sugar beet pulp was obtained at 35 °C after 48 h of incubation.     

Fusobacterium canifelinum sp. nov., from the oral cavity of cats and dogs

Fourteen strains of Gram-negative, anaerobic, fluoroquinolone-resistant, non-sporulating rods were isolated from various infections in cats and dogs, as well as from wounds in humans after cat- or dog-bites. These strains were characterized by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) regions, 16S rDNA, DNA-DNA hybridization, phylogenetic analysis, and phenotypic tests. The results indicate that the novel strains belong to a distinct species, closely related to Fusobacterium nucleatum. The species Fusobacterium canifelinum sp. nov. is proposed, with strain ATCC BAA 689T as the type strain.     

Molecular characterisation of Hanseniaspora species

The sequences of the internal transcribed spacers (ITS regions) and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 27 strains representative of the six species belonging to the genus Hanseniaspora, were examined. From the analysis of the 5.8S rRNA gene and the ITS regions, the genus Hanseniaspora is monophyletic and can be divided into two subgroups. This subdivision was supported by electrophoretic chromosome patterns. Hanseniaspora guilliermondii, H. uvarum and H. valbyensis show 6–7 bands (8 to 9 chromosomes), while the second group comprises the species H. occidentalis, H. osmophila and H. vineae which have only 5 chromosomes.     

Molecular authentication of ginseng cultivars by comparison of internal transcribed spacer and 5.8S rDNA sequences

Molecular authentication among three Panax species and within cultivars and accessions of P. ginseng was investigated using the DNA sequence in the ribosomal ITS1–5.8S–ITS2 region. Four single-nucleotide polymorphisms were identified between P. ginseng and other Panax species. In the electrophoresis profile, obtained after digestion with the enzyme TaqI, three fingerprinting patterns were obtained from cultivars and accessions of Panax species. Consequently, this authentication procedure based upon the restriction fragment length polymorphism in the ribosomal ITS1–5.8S–ITS2 region can now be utilized to differentiate these Panax species as well as major Korean cultivars such as Gopoong and Kumpoong from other cultivars and accessions in Panax species at the DNA level. O. T. Kim and K. H. Bang contributed equally to this paper.     

Characterization of heat resistant mutant strains of Rhizobium sp. [Cajanus] for growth, survival and symbiotic properties

Fourteen heat resistant mutant strains were isolated from a wild-type strain (PP201, Nod⁺ Fix⁺) of Rhizobium sp. (Cajanus) by giving it a heat shock of 43°C. These mutant strains showed a greater increase in optical density (O.D.) and a higher viable cell count in both rhizospheric and non-rhizospheric soil at high temperature. Symbiotic studies showed that pigeon pea plants inoculated with a few mutant strains had ineffective nodules (Nod⁺ Fix⁻) under controlled temperature (43°C) conditions, but under natural high temperature (40–45°C) conditions, the host plants infected with all the mutant strains showed higher total shoot nitrogen than the plants inoculated with the parent strain. Four mutant strains (HR-3, HR-6, HR-10 and HR-12) were found to be highly efficient for all the symbiotic parameters, and thus have the potential to be used as bioinoculants in the North-Western regions of India during the summer season.     

Biogeography and Degree of Endemicity of Fluorescent Pseudomonas Strains in Soil

Fluorescent Pseudomonas strains were isolated from 38 undisturbed pristine soil samples from 10 sites on four continents. A total of 248 isolates were confirmed as Pseudomonas sensu stricto by fluorescent pigment production and group-specific 16S ribosomal DNA (rDNA) primers. These isolates were analyzed by three molecular typing methods with different levels of resolution: 16S rDNA restriction analysis (ARDRA), 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (ITS-RFLP) analysis, and repetitive extragenic palindromic PCR genomic fingerprinting with a BOX primer set (BOX-PCR). All isolates showed very similar ARDRA patterns, as expected. Some ITS-RFLP types were also found at every geographic scale, although some ITS-RFLP types were unique to the site of origin, indicating weak endemicity at this level of resolution. Using a similarity value of 0.8 or more after cluster analysis of BOX-PCR fingerprinting patterns to define the same genotypes, we identified 85 unique fluorescent Pseudomonas genotypes in our collection. There were no overlapping genotypes between sites as well as continental regions, indicating strict site endemism. The genetic distance between isolates as determined by degree of dissimilarity in BOX-PCR patterns was meaningfully correlated to the geographic distance between the isolates' sites of origin. Also, a significant positive spatial autocorrelation of the distribution of the genotypes was observed among distances of <197 km, and significant negative autocorrelation was observed between regions. Hence, strong endemicity of fluorescent Pseudomonas genotypes was observed, suggesting that these heterotrophic soil bacteria are not globally mixed.     

Intra-specific rDNA-ITS restriction site variation and an improved protocol to distinguish species and hybrids in the Daphnia longispina complex

A collaborative research effort was undertaken to evaluate the robustness of a recently developed genetic tool for species identification of members in the morphologically variable Daphnia longispina species complex. This genetic method, based on restriction fragment length polymorphism (RFLP) of the internal transcribed spacer region (ITS) of nuclear ribosomal DNA (rDNA) with restriction enzymes Mwo I and Sau96 I [Billiones et al., 2004. Hydrobiologia 526: 43–53], was applied to many different European populations. Results were compared with two or more independently obtained characters (morphology, allozymes, mitochondrial DNA (mtDNA), or cloned rDNA-ITS sequences). Individuals of most taxa were readily identified, but unexpected ITS-RFLP patterns were found in many individuals indicated by other markers to be D. galeata or one of its hybrids. Among 43 investigated D. galeata populations (902 specimen analysed by ITS-RFLP), deviant RFLP fragment patterns occurred in 26 (i.e., more than half) of the populations. The deviant patterns could be attributed to the loss of one single restriction site in the ITS2 region. This loss made the distinction of D. galeata from other species unreliable, and F1 hybrids could not be identified. Future users should be aware of this shortcoming of the Billions et al. [2004. Hydrobiologia 526: 43–53] protocol. As a solution to this problem, we present an improved genetic identification protocol based on a simple double digestion of the rDNA-ITS region with the restriction enzymes BsrB I and EagI. Sequence analyses of rDNA-ITS clones and preliminary testing indicate that the new protocol is unaffected by the rDNA variation which troubled the Mwo I/Sau96 I protocol. Further, the new protocol identifies all European species of the D. longispina complex, as well as their F1 hybrids. However, a wider screening is required to verify its general utility for all species, since yet unknown variation may occur. Guest editor: Piet Spaak Cladocera: Proceedings of the 7th International Symposium on Cladocera     

Production of xylanase from a newly isolated <Emphasis Type="Italic">Penicillium</Emphasis> sp. ZH-30

A new strain of Penicillium sp. ZH-30 that produces xylanase was isolated from soil. According to the morphology and comparison of internal transcribed spacer (ITS) rDNA gene sequence, the strain Penicillium sp. ZH-30 was identified as a strain of Penicillium oxalicum. When xylan or wheat bran was used as substrate at 30°C for 3 days under submerged cultivation, xylanase production was 5.3 and 13.3 U ml⁻¹, respectively. The temperature and pH for optimum activity were 50°C and 5.0–6.0, respectively.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

Diversity of Bacillus thuringiensis in the rice field soils of different ecologies in India

Diversity of the Bacillus thuringiensis (Bt) in the rice field soils of different ecologies viz. the island (Port Blair), the Himalayan (Srinagar), brackish water (Mahe) and coastal mesophilic (Mangalore) habitats was analyzed by phenotypic characterization of 5, 66, 14 and 54 Bt isolates, respectively. The Bt isolates produced either monotypic (bipyramidal or spherical) or heterotypic (polymorphic-bipyramidal or bipyramidal-rhomboidal) crystals. The organisms were generally resistant to the penicillin group of antibiotics, tolerated 5–12% NaCl and 0.5M Na-acetate. The Bt isolates contained 1–5 plasmids of 0.89–58.61 kbp sizes. The plasmid profiles had no correlation with crystal morphology or salt tolerance of different bacteria. Each soil was inhabited by different types of Bt. Two Bt strains of Mangalore and one strain each of the other places were phenotypically similar. One Bt strain each of Port Blair and Srinagar was different from all other strains.     

Improvement of natural isolates of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for synthesis of a chiral building block using classic genetics

The asymmetric bio-reduction of 4-chloro-acetoacetic-acid-ethyl-ester to the pharmaceutical building block (S)-4-chloro-3-hydroxybutanoate-ethyl-ester requires the utilization of an enantioselective robust biocatalyst. Some of the natural Saccharomyces cerevisiae strains, isolated from Mount Carmel National Park in Israel, were characterized as resistant to environmental stress. Nevertheless, these strains showed relatively low enantiomeric-excess (ee), while a laboratory strain, Y103, exhibited a selectivity of 98% ee. The enantioselective lab strain was crossed with the multi-stress resistant environmental isolate (93% ee) followed by backcross with Y103, to subsequently obtain a haploid offspring of backcross-1, exhibiting both high multi-stress resistance and high enantioselectivity (98% ee). Introducing osmotic (1 M NaCl), oxidative (0.6 mM H₂O₂) and thermal stress (44°C) to growing cultures of the enantioselective parent, resulted in a decrease of 24–32% in specific activity, while the enantioselectivity of the stress-resistant parent decreased by 4–12% ee. Unlike its original parental strains, the new strain maintained constant specific activity and enantioselectivity when introduced to the various stress factors. This work shows that the classic introgression method, can serve as a viable approach for creating a robust enantioselective biocatalyst, designed for industrial production of chiral compounds.     

Bensingtonia pseudonaganoensis sp. nov., a novel ballistoconidium-forming yeast species isolated from plant leaves

Among the basidiomycetous yeasts isolated from plant leaves collected in different regions of China, two ballistoconidium-forming strains were revealed to represent an undescribed species of the genus Bensingtonia by conventional, chemotaxonomic and molecular phylogenetic characterization. Sequence analysis of the 26S rDNA D1/D2 domains and the internal transcribed spacer (ITS) region indicated that the novel species was located in the Agaricostilbum lineage and closely related to Bensingtonia naganoensis and Bensingtonia ciliata, with the former as its closest relative. The name Bensingtonia pseudonaganoensis sp. nov. is proposed (type strain: AS 2.2601^T = CBS 10121^T)     

Screening for marine nanoplanktic microalgae from Greek coastal lagoons (Ionian Sea) for use in mariculture

Mediterranean mariculture uses imported strains of marine phytoplankton, raising questions of ecological risk and ability to adapt to local conditions for mass culture outdoors. In this context, we report here on the mass-culture potential and chemical composition of six strains of Prasinophyceae (five strains of Tetraselmis sp. and one Pyramimonas sp.) isolated from a Greek coastal lagoon. Proximate composition had a pattern of 10–20% ash, 35–65% protein, 6–10% lipids, and 25–45% other organics including carbohydrates. The amino acid profiles were typical for the marine representatives of the class. All strains had a high PUFA content with dominant the ω3 fraction in four of them. The fatty acid profiles indicated a Tetraselmis strain with high EPA (14%) and a Pyramimonas strain with high DHA (6%). These strains might be a good alternative for the common commercial strains used in Mediterranean aquaculture.     

ITS-RFLP fingerprinting and molecular marker for detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">ciceris</Emphasis>

Genetic diversity of 11 representative isolates of Fusarium oxysporum f.sp. ciceris causing chickpea wilt was determined through internal transcribed spacer (ITS) region of the ribosomal DNA-restriction fragment length polymorphism (ITS-RFLP). ITS1+5.8s+ITS2 regions of the isolates were amplified with a set of primers ITS1 and ITS4 and amplified products were digested with 4 restriction enzymes (AluI, MboI, RsaI, MseI). Six different kinds of ITS-RFLP patterns were obtained. The ITS region of these isolates was sequenced and deposited to NCBI GeneBank. The nucleotide sequence homology of ITS region grouped the isolates into 5 categories. Primers were designed with sequence information using Primer 3 software. F. oxysporum f.sp. ciceris specific markers (FOC F2 and FOC R2) based on ITS region were developed for the first time for detection of the pathogen. The markers produced an amplicon of 292 bp; they were validated against the isolates of the pathogen collected from different locations of India.