ITS Region Of Ribosomal DNA Research Articles

Phylogenetic Analysis of the Far Eastern Salix (Salicaceae) Based on Sequence Data from Chloroplast DNA Regions and ITS of Nuclear Ribosomal DNA

In order to further elucidate the relationships in the Salix genus, phylogenetic ana lysis was conducted based on comparison of the nucleotide sequences of the chlo roplast DNA regions (petG–trnP, petN–psbM, psaA–ycf3, psbM–trnD, rpoB– trnC, trnC–petN, trnD–trnT, trnS–psbZ, rbcL, matK) and the ITS region (ITS1–5.8S rRNA–ITS2) of nuclear ribosomal DNA of 86 Salix species from three sub­ genera: Salix, Chamaetia, Vetrix, 27 sections and Chosenia arbutifolia. The previous ly recognized genera Chosenia and Toisusu merged with Salix. The divergence of spe­ cies in the Salix genus (according to ITS and cpDNA data) was very low. In most spe cies from Chamaetia and Vetrix subgenera, the sequences differed by several sub­stitutions­or­were­ identical.­Molecular­differences­were­ identified­mainly­ in­ spe cies belonging to the subgenus Salix in its previous broad meaning. ITS­phy­ lo­geny­generally­agrees­with­the­most-recent­infrageneric­classification­of ­Salix: the isolation of subgenera Pleuradenia, Chosenia, Salix (for exception sect. Triand­ rae), Longifoliae has been well supported, but the isolation of subgenera Protitea and Chamaetia is not supported. Relationships between the subgenera Chamaetia and Vetrix remain unresolved. High similarity of the plastid and nuclear genomes re vealed in the majority of Salix species included in the analysis may indicate that they have relatively recently diverged from a common ancestor or there is a result of lineage sorting through hybridization. K e y w o r d s chloroplast DNA, ITS, phylogenetic analysis, Salicaceae, Salix

Steinernema poinari sp. n. (Nematoda: Steinernematidae) a new entomopathogenic nematode from the Czech Republic.

A new steinernematid nematode, herein described as Steinernema poinari sp. n., was recovered by baiting soil samples with the last instar wax moth larvae Galleria mellonella in three localities of southwest Bohemia, Czech Republic. Morphological and molecular data confirmed this nematode as a new species which belongs to the affine/intermedium group. S. poinari sp. n. was characterized by male, female and infective juvenile morphological observations. Male spicules are robust with a pointed tip, SW% = 109 (98-123) and GS% = 70 (58-87). The tail terminus of the first generation have a papillated mucron, whereas in the second generation a short filamentous mucron sometimes develops. Females of the first generation have a moderately protruding vulva, postanal swelling absent or slightly developed. Second generation females have a long filamentous mucron on the tail tip. Infective juvenile have a body length of 768 µm, lateral field formula 2, 5, 6, 5, 2, 1, ratio D% of 46 (40-55), E% of 84 (76-95) and H% of 50 (43-56). The new species was characterized by sequences of ITS and D2D3 regions of ribosomal DNA. S. poinari sp. n. can also be separated from S. affine and S. intermedium by cross-breeding tests.

Morphological and molecular identification of Alternaria alternate: Caraway fruits pathogen in Serbia

During 2013, samples of infected seed grown caraway were collected from commercially grown crops in the experimental field in Mošorin and analyzed for the presence of plant pathogenic fungi. In all samples of seeds, weaker germination and a high seed infection 100%, with phytopathogenic fungi of the genus Alternaria, was found. From the infected caraway seed, monosporial cultures, based on morphology, were identified as Alternaria alternata, and their pathogenicity proved on artificially inoculated caraway and parsley seedlings. Molecular identification was performed utilizing polimerase chain reaction (PCR) with primer pair ITS1/ITS4 and by amplification of the ITS region of ribosomal DNA. Gene sequences of selected isolates CC1 (KP822948), CC2 (KP822949) i CC3 (KP822950) exhibited 100% nucle­otide identity with the sequences of 31 isolates of A. alternata deposited in the GenBank. The presence of a large number of phytopathogenic fungi on caraway seed should be further investigated in order to clarify their relationships and relative significance.

Phylogeny, geographic distribution, and new taxonomic circumscription of the Crocus reticulatus species group (Iridaceae)

Recent phylogenetic analyses proved several infrageneric units within the genus Crocus to be para- or polyphyletic. In an attempt to arrive at a system of Crocus that closely reflects species relationships, we provide here phylogenetic, morphometric, geographic, and nomenclatorial data for the species of a narrower-defined, monophyletic Crocus series Reticulati. We sequenced the ETS and ITS regions of the nuclear ribosomal DNA in 9 Reticulati and 19 outgroup species. Three chloroplast loci (trnL-F, rps16-trnQ, matK-trnK) were sequenced in the newly defined series Reticulati species and 1 outgroup. Data were analyzed with Bayesian and parsimony algorithms. The phylogenies resulted in 2 clearly separated, geographically defined species groups within the series Reticulati. The southern one comprises only the taxa from Turkey, while the species of the second group are distributed from Italy in the west through the areas north of the Black Sea to the Caucasus in the east. To arrive at monophyletic species we describe here C. danubensis sp. nov., C. filis-maculatis sp. nov., and C. orphei sp. nov. as new species, and we define C. reticulatus s.s. to comprise only the populations in the area north and east of the Black Sea.

Polymerase chain reaction (PCR) identification of terverticillate Penicillium species isolated from agricultural soils in eskişehir province

In the present study, nine terverticillate Penicillium isolates (P. griseofulfum, P. puberulum, P. crustosum, P. aurantiogriseum, P. chrysogenum, P. primulinum, P. expansum, P. viridicatum, Eupenicillium egyptiacum) from 56 soil samples were characterized genetically by a PCR method. The DNAs of the strains were isolated using the glass beads and vortexing extraction method and then used for PCR amplification with the internal transcribed spacer 1 (ITS1) and ITS4 universal fungal specific primers. The ITS regions of fungal ribosomal DNA (rDNA) were sequenced through the CEQ 8000 Genetic Analysis System. ITS-5.8S sequences obtained were compared with those deposited in the GenBank Database. The results indicated that the identification of Penicillium species with PCR based methods provided significant information about the solution to taxonomy and improve food safety and to protect the users from harmful contaminants such as mycotoxins, which must be controlled during the production of agricultural materials as well as during the processing of food and feed.

Isolation and Molecular Identification of Keratinophilic Fungi from Public Parks Soil in Shiraz, Iran

Introduction. Keratinophilic fungi are an important group of fungi that live in soil. The aim of this study was to isolate and identify keratinophilic fungi from the soil of different parks in Shiraz. Materials and Methods. A total of 196 soil samples from 43 parks were collected. Isolation of the fungi was performed by hair bait technique. The isolated colonies were identified by morphologic feature of macro- and microconidia and molecular method, using DNA sequence analysis. ITS region of ribosomal DNA was amplified and the PCR products were sequenced. Results. 411 isolates from 22 genera were identified. Fusarium (23.8%), Chrysosporium (13.13%), Acremonium (12.65%), Penicillium (12.39%), Microsporum gypseum (1.94%), Bionectria ochroleuca (1.21%), Bipolaris spicifera (1.21%), Scedosporium apiospermum (0.82%), Phialophora reptans (0.82%), Cephalosporium curtipes (0.49%), Scedosporium dehoogii (0.24%), Ochroconis constricta (0.24%), Nectria mauritiicola (0.49%), Chaetomium (0.49%), Scopulariopsis (0.24%), Malbranchea (0.24%), and Tritirachium (0.24%) were the most important isolates. Most of the fungi were isolated from the soils with the PH range of 7 to 8. Conclusion. Our study results showed that many keratinophilic fungi isolated from the parks soil are important for public health and children are an important group at a high risk of being exposed to these fungi.

Deladenus prorsus n. sp. (Nematoda: Neotylenchidae) in dunnage wood from Malaysia intercepted in Ningbo, China

Deladenus prorsus n. sp., isolated from dunnage wood originating from Malaysia and intercepted in Ningbo port, P.R. China, is described and illustrated. The dunnage wood material appeared to have holes resembling the typical exit holes of wood-boring insects and have associated blue stain. The new species is characterised by the presence of both the mycophagous and infective forms (Deladenus consists of mycetophagous-only forms and those with known female dimorphism), the body length of 814 (670-1147) and 898 (812-979) μm for mycetophagous males and females, respectively, and 1129 (1114-1352) μm for the infective female, and by the very anterior position of the excretory pore which is situated at 30.7 (24.4-33.0), 30.6 (25.4-35.6) and 37.9 (27.3-39.7) μm for the mycetophagous males and females and infective females, respectively, and the distance that the excretory pore is located anterior to the hemizonid which is 82 (77-90), 74 (54-92) and 86 (64-106) μm for the mycetophagous males and females and infective females, respectively. The diagnostic value of the excretory pore and the hemizonid of the species of the genus are discussed. The ribosomal DNA ITS region was sequenced and analysed.

Distribution patterns of Dikarya in arid and semiarid soils of Baja California, Mexico

Abstract Approximately four-fifths of the land area of Baja California (BC) in Mexico are occupied by arid and semiarid soils, the mycobiota of which is virtually uncharacterized. In the first culture-independent study of the mycobiota of BC, we collected soil from five different locations in the region and constructed a Dikarya-specific gene library for the ITS region of nuclear ribosomal DNA. Clones were analyzed by RFLP, were sequenced for phylogenetic analyses, and diversity and similarity indices were calculated. The ascomycete Penicillium dipodomyicola was the most frequent fungus found in soil at the most arid location studied, and the basidiomycete Coprinellus radians was the most frequent at the location receiving the highest rainfall. Other frequent members of the soil mycobiota were identified as Alternaria spp., Ceratobasidium sp., Coniozyma leucospermi, Nematoctonus robustus, Penicillium griseofulvum, Tulostoma kotlabae and uncultured members of the Dikarya. Several sequences were identified as those of uncultured fungi, one of which was previously reported from other hot deserts. Arid soils and the transitional zones between arid and semiarid soils had the most similar fungal diversity, with the former soils having a community from which basidiomycetes were absent, and the soil receiving the highest precipitation having a community dominated by basidiomycetes.

Fungicide spray programs to manage downy mildew (dryberry) of blackberry caused by Peronospora sparsa

Downy mildew (dryberry) of blackberry, caused by Peronospora sparsa, is a major disease of blackberry in Michoacán, México. The objective of this research was to evaluate different fungicide spray programs to manage systemic infections of downy mildew and dryberry disease in two different regions of Michoacan, Mexico in the 2007 and 2008 growing seasons. The presence of asymptomatic infections of P. sparsa in experimental plots was confirmed by nested PCR using published primers amplifying a fragment of 477 bp of the ITS region of ribosomal DNA. In 2007, the experiment was conducted in an 8-year-old commercial planting in Tangancicuaro. Fungicide applications began early in the growing season approximately 10 days after bud brake (July 10). In 2008, the experiment was conducted at the same location, but treatments began later (August 4) approximately 21 days after bud break at which time plants had fully expanded leaves. An additional plot was established in Atapan in 2008. Seven fungicide programs using commercial formulations and recommended rates of mefenoxam, potassium phosphite, azoxystrobin, captan, mancozeb, copper sulfate, Bacillus subtilis, and a sanitizing agent were tested at both locations. Results from both years and locations indicated that all programs beginning with potassium phosphite (3–4 applications) on a 10–14 day schedule after bud brake, significantly reduced the severity and incidence of downy mildew on leaves, and dryberry on fruits compared to the untreated control, regardless of the fungicide (either biological or chemical) that was applied later in the season. Programs that included 1 or 2 applications of mefenoxam, the first application in drench, early in the season also provided good control. Fungicide programs that were initiated with protectant fungicides or used protectant materials only did not provide a satisfactory level of control. Early detection of systemic infections of P. sparsa through the use of PCR and the use of fungicide spray programs based on early season applications of systemic fungicides such as potassium phosphite and mefenoxam provided good to excellent control of foliar downy mildew and dryberry of blackberry in Michoacán, México.

한국산 피막이속(Hydrocotyle L.) 식물의 분자계통학적 연구

Phylogenetic analyses were conducted to evaluate relationships of 5 taxa of Korean Hydrocotyle, H. spp. found in the Ulleung island including one outgroup (Centella asiatica). The molecular phylogenetic methods based on nuclear ribosomal DNA ITS region and cpDNA trnH-psbA region. Centella asiatica was used outgroup for analysis. As the result, genus Korean Hydrocotyle were grouped by 94% bootstrap value. Korean Hydrocotyle was grouped by four clades; Clade Ⅰ-H. maritima, H. sibthorpides and H. yabei clade Clade Ⅱ- H. nepalensis clade clade Ⅲ- H. ramiflora clade clade Ⅳ- H. spp. clade. H. maritima, H. sibthorpides and H. yabei was not distinguished, seperately. H. spp. was distinctly distinguished other Korean Hydrocotyle.

GNOMONIOPSIS CASTANEA sp. nov. (GNOMONIACEAE, DIAPORTHALES) AS THE CAUSAL AGENT OF NUT ROT IN SWEET CHESTNUT

The genus Gnomoniopsis (Gnomoniaceae, Diaporthales) is currently composed of 13 species which are endophytic and/or parasitic to plants in the families Fagaceae, Onagraceae and Rosaceae. Species definition is based on a combination of morphological traits, association with specific plant hosts, and phylogeny. In this paper a new species, Gnomoniopsis castanea sp. nov., is described based on the association with Castanea sativa (a plant species never reported to be infected by fungi the genus Gnomoniopsis), morphology and phylogenetic analysis of the ITS region of ribosomal DNA and on the EF1-a locus. The fungus is consistently associated with nut rot and caused the disease when artificially inoculated to fruits or flowers. Infection incidence varied depending on the orchard and the year and attained up to 83% of the nuts in some areas of north-western Italy. The fungus was also consistently isolated from the bark of symptomless branches in naturally infected chestnut orchards.

Identification of Yeasts Isolated from Varieties of Apples and Citrus Using PCR-Fragment Size Polymorphism and Sequencing of ITS1–5.8S-ITS2 region

Yeasts play a significant role in the nutrition industry, medicine, and biocontrol of plant pathogens. Certain isolates of yeasts act as antagonist agents in postharvest infections on fruits such as apples and citrus. The main goal of this study was identification of yeasts isolated from different types of Iranian apples and citrus using reliable molecular methods. For preliminary identification of the isolates, each of the internal transcribed spacer 1 (ITS1) and ITS2 regions of ribosomal DNA (rDNA) was amplified separately, and the mixture of both amplicons was electrophoresed on agarose gels. For final discrimination of the selected isolates the entire ITS1-ITS2 was sequenced followed by Blast analysis. The most common identified isolates were species belonged to the following genera: Rhodotorula, Torulaspora, Candida, Pichia, Metschnikowia, and Cryptococcus. Based on our results, PCR-FSP coupled to sequencing had important relevance for phylogenetic analysis to differentiate yeasts. Phylogenetic evaluation of yeasts isolated from fruits based on nucleotide sequences of ITS region of rDNA categorized 18 representative species in 10 distinct lineages. It is concluded that the molecular methods can be very efficient, useful, and precise tools for identification of yeasts.

Chronic Disfiguring Facial Lesions in an Immunocompetent Patient Due to Exophiala spinifera: A Case Report and Review of Literature

Exophiala spinifera is a rare fungus causing chromoblastomycosis or different types of phaeohyphomycosis (cutaneous, subcutaneous, disseminated and cyst phaeohyphomycosis). We report a case of a young male with phaeohyphomycosis due to E. spinifera, who had multiple itchy painful papular lesions disfiguring his face for 4years. His diagnosis was delayed and had received antibacterial and antileishmanial therapy elsewhere without any improvement. While he reported to our hospital, the histopathology of the biopsy collected from the lesion demonstrated acute on chronic inflammation with granuloma formation and darkly pigmented fungal elements. The isolate grown on culture was identified as E. spinifera on the basis of morphological characters. The identification of the isolate was further confirmed by sequencing of the ITS region of ribosomal DNA. After treatment with oral itraconazole, he had marked clinical improvement.

Epicoccum nigrum the new pathogen of sorghum seed in Serbia

Sixteen samples of sorghum seed (Sorghum bicolor (L.) Moench.) 'Alba', 'Gold', 'Prima' and 'Reform' were analyzed in the localities of Bački Petrovac and Čantavir in the period 2009-2011. Tipresence of species belonging to the genera Epicoccum, Fusarium, Alternaria, Aspergillus and Penicillium was established in single and mixed infections. From the infected sorghum seed, monosporial cultures identified as Epicoccum nigrum based on morphology, proved their pathogenicity on artificially inoculated sorghum seedlings. Molecular identification was performed by PCR and amplification of the ITS region of ribosomal DNA. Gene sequences of selected isolates 291-09 (JQ619838) and 315-09 (JQ619839) exhibited 99-100% nucleotide identity with the sequences of 31 isolates of E. nigrum deposited in the GenBank. It obtained results represent the first detailed characterization of E. nigrum in Serbia. The presence of a large number of phytopathogenic fungi on sorghum seed should be further investigated in order to clarify their relationships and relative significance.

Detection and molecular characterization of ascarid nematode infection (Toxascaris leonina and Toxocara cati) in captive Asiatic lions (Panthera leo persica)

The objective of this study was to investigate the ascarid infection in Asiatic lions using scat samples, based on microscopic analysis, PCR amplification of the ITS-2 region of ribosomal DNA and sequence analysis of the amplicons. Microscopic analysis indicated the presence of eggs of Toxascaris leonina in eleven of the sixteen scat samples analysed and in one of these eleven scats eggs of Toxocara cati were also detected. In five of the scats eggs were not detectable. The presence of T. leonina in all the infected samples was also confirmed by PCR amplification of the ITS-2 of ribosomal RNA gene and five of these also showed amplicons corresponding to T. cati, respectively. Toxocara canis infection was not observed in any of the scat samples. Nucleotide sequence analysis of the ITS-2 region indicated 97% to 99% similarity with T. leonina and T. cati, respectively. To our knowledge, this is the first molecular characterization of ascarid infection in captive Asiatic lions from a zoological garden of India. This study also indicates that Asiatic lions are more prone to infection either with T. leonina or T. cati and the parasite is not host specific.

Enterocytozoon bieneusiIdentification Using Real-Time Polymerase Chain Reaction and Restriction Fragment Length Polymorphism in HIV-Infected Humans from Kinshasa Province of the Democratic Republic of Congo

Objective. To determine the prevalence and the genotypes of Enterocytozoon bieneusi in stool specimens from HIV patients. Methods. This cross-sectional study was carried out in Kinshasa hospitals between 2009 and 2012. Detection of microsporidia including E. bieneusi and E. intestinalis was performed in 242 HIV-infected patients. Typing was based on DNA polymorphism of the ribosomal DNA ITS region of E. bieneusi. PCRRFLP generated with two restriction enzymes (Nla III and Fnu 4HI) in PCR-amplified ITS products for classifying strains into different lineages. The diagnosis performance of the indirect immune-fluorescence-monoclonal antibody (IFI-AcM) was defined in comparison with real-time PCR as the gold standard. Results. Out of 242 HIV-infected patients, using the real-time PCR, the prevalence of E. bieneusi was 7.9% (n = 19) among the 19 E. bieneusi, one was coinfected with E. intestinalis. In 19 E. bieneusi persons using PCR-RFLP method, 5 type I strains of E. bieneusi (26.3%) and 5 type IV strains of E. bieneusi (26.3%) were identified. The sensitivity of IFI-AcM was poor as estimated 42.1%. Conclusion. Despite different PCR methods, there is possible association between HIVinfection, geographic location (France, Cameroun, Democratic Republic of Congo), and the concurrence of type I and type IV strains.

A New Variety ofMusa itinerans(Musaceae) in Taiwan

Musa itinerans Cheesman var. formosana (Warb.) Häkkinen & C. L. Yeh is one of the three wild bananas in Taiwan and represents the taxon previously recognized as M. formosana (Warb. ex Schum.) Hayata [≡ M. basjoo Siebold & Zucc. ex Iinuma var. formosana (Warb. ex Schum.) S. S. Ying]. The gross morphology of M. itinerans var. formosana is stable. Some populations without variegation on the pericarps and the bracts of male buds were mainly found in a restricted area of northeast Taiwan. The morphological characteristics of the nonvariegated populations are otherwise similar to those of M. itinerans var. formosana. Their principal distinction is based on the absence of the purplish red streaking on both the pericarps and the male, fertile bracts. This character of nonvariegation is stable across the taxon's habitat and as cultivated through a 9-year period of observation. From molecular evidence, the DNA sequence for the ITS region of ribosomal DNA (rDNA) is highly similar in both populations. The nonvariegated population is herein segregated as the new variety, M. itinerans var. kavalanensis H. L. Chiu, C. T. Shii & T. Y. A. Yang. Photos for the three varietal taxa, M. itinerans var. chinensis, variety formosana, and variety kavalanensis, and a key to Taiwanese wild bananas are also provided.

Nuclear ribosomal DNA-ITS region based molecular markers to distinguish botanical entities traded as ‘Vidari’

‘Vidari’ is an Ayurvedic herbal drug used as aphrodisiac and galactagogue. It is also used in the preparation of Chyavanaprash, an Ayurvedic Nutraceutical. Ayurvedic Pharmacopeia of India correlates tubers of Pueraria tuberosa (Roxb. ex Willd.) DC (Fabaceae) as ‘Vidari’ and Ipomoea mauritiana Jacq. (Convolvulaceae) as Kshiravidari, used as a substitute for Vidari in some parts of India. Tubers of Adenia hondala (Gaertn.) de Wilde (Passifloraceae) and pith of Cycas circinalis L. (Cycadaceae) are also traded in the name of Vidari, creating issues of botanical authenticity of the raw drug.DNA markers to distinguish the botanical entities traded as ‘Vidari’ were developed by amplifying and sequencing the complete nuclear ribosomal DNA – internal transcribed spacer (ITS) region (ITS1, 5.8S rRNA and ITS2) from the genomic DNA, using universal primers. Genetic variation in the rDNA-ITS region was identified and exploited to develop species-specific markers. These markers are efficient and reliable in distinguishing P. tuberosa, I. mauritiana, A. hondala and C. circinalis and can be used as molecular pharmacognostic tools.

Mycorrhization of Pecan trees (Carya illinoinensis) with commercial truffle species: Tuber aestivum Vittad. and Tuber borchii Vittad

Pecan (Carya illinoinensis) is an economically important nut tree native to the Mississippi basin and cultivated worldwide. In North America, species of truffles are regularly found fruiting in productive pecan orchards and the truffle genus Tuber appears to be abundant in pecan ectomycorrhizal (EM) communities. As an initial step to determine the feasibility of co-cropping European truffle species with pecan, we evaluated whether mycorrhizae of highly esteemed European truffle species (Tuber aestivum Vittad. T. borchii and T. macrosporum) could be formed on pecan seedlings. Seedlings were inoculated with truffle spores and were grown in a greenhouse for 10months. Levels of EM colonization were estimated visually and quantified by counting EM tips. Ectomycorrhizae were identified both morphologically and molecularly with species-specific amplification and by sequencing of the ITS region of the nuclear ribosomal DNA (nrDNA). Both T. borchii and T. aestivum spores produced well-formed ectomycorrhizae on pecan seedlings with average root colonization levels of about 62% and 42%, respectively, whereas no ectomycorrhizae of T. macrosporum were formed. The anatomy and morphology of these truffle ectomycorrhizae on pecan was characterized. The co-cropping of T. aestivum and T. borchii may hold promise as an additional stream of revenue to pecan growers, although, further studies are needed to assess whether this symbiosis is maintained after planting in the field and whether truffle production can be supported by this host species.

Phylogenetic signal in the evolution of body colour and spicule skeleton in calcareous sponges

Some of the morphological characters used in Porifera taxonomy have often been shown to be inconsistent. In the present study, we tested the phylogenetic coherence of currently used taxonomic characters of the calcarean genus Clathrina. For this, 20 species of Clathrina and three other calcinean genera (Ascandra, Guancha, and Leucetta) were sequenced for the ITS and D2 region of the 28S ribosomal DNA. Maximum-likelihood and maximum-parsimony algorithms were used to reconstruct phylogenetic trees. Deep divergences were observed in our tree and Clathrina was shown to be paraphyletic. The major split in our topology showed a clear-cut distinction between sponges with and without tetractine spicules. Moreover, a group of yellow-coloured Clathrina was clearly separated from the remaining white-coloured species. Our results show that the presence of diactines, water-collecting tubes, the degree of cormus anastomosis, and actine shapes do not correlate with the major clades of the calcinean phylogeny. On the other hand, the presence of tripods, the absence of tetractines, and the presence of spines in the apical actine of tetractines seem to be good synapomorphies for clades in our tree. Our results demonstrate that skeleton characters can be reliably used in higher level taxonomy in Clathrinida.