Specific Internal Transcribed Spacer Research Articles

Genetic Diversity Analysis based on the Virulence, Physiology and Regional Variability in Different Isolates of Powdery Mildew in Pea.

Powdery mildew is an omnipresent disease that reduces the yield and quality of pea crops (Pisum sativum L.). To examine the powdery mildew pathogen’s morphological, molecular, and genetic diversity, we collected samples of powdery mildew-affected pea crops from ten distinct locations in the Nilgiris district of Tamil Nadu, India. The pathogen Erysiphe pisi was identified morphologically based on anamorphic characters. Molecular identification of E. pisi isolates was befitted by targeting the internal transcribed spacer (ITS) region of rDNA and specific primers of powdery mildew fungi. The genetic variation between ten different E. pisi isolates collected from topographically distinct mountainous areas was studied using random amplified polymorphic (RAPD). Based on its morphological characteristics, the powdery mildew fungus presented high similarities to E. pisi. Molecular characterization of the ITS rDNA of E. pisi produced 650 bp nucleotides, PMITS (powdery mildew-internal transcribed region) primers produced 700 bp nucleotides, and an Erysiphe specific ITS primer pair amplified and synthesized 560 bp nucleotides. According to the findings, the collected E. pisi strains exhibited a low level of genetic diversity and only a slight differential in virulence on the host. In the study, E. pisi isolates from Anumapuram, Emerald Valley, Indira Nagar, and Thuneri showed a greater disease incidence in the natural field conditions and shared the same genetic lineage with other isolates in UPGMA hierarchical cluster analysis based on RAPD markers. There was no evidence of a link between the occurrence of the disease and these grouped populations.

PCR detection of Colletotrichum gloeosporioides in Psidium guajava

Colletotrichum gloeosporionides is the causative agent of a major disease affecting Psidium guajava fruits from fructescence to post-harvest. A polymerase chain reaction (PCR)-based method for the detection of C. gloeosporioides was developed in this study. A set of two primers (CGF and CGR) specific for C. gloeosporioides were designed based on the sequences of the ribosomal internal transcribed spacer (ITS) regions. The specificity of primers was analyzed by the absence of amplified product with DNA of tested isolates. The designed primers amplified a single product of ~390 bp with DNA extracted from isolates of C. gloeosporioides, and did not amplify DNA from several Colletotrichum species and other different fungal genera. The sensitivity of conventional PCR was 10 pg purified genomic DNA in 25 μL reaction. A nested PCR system was established to combine specific and universal ITS primers (ITS1/ITS4), and this enhanced the sensitivity to 1 fg of DNA per 25 μL reaction, representing a 10,000-fold improvement over conventional PCR. The optimised reaction system could detect C. gloeosporionides successfully from diseased plant tissue, infected soil and water. The developed system can detect C. gloeosporioides during the early stages of disease onset and provide evidence to guide management and decision-making.

Identification and characterization of Pseudomonas fluorescensstrains effective against Xanthomonasoryzaepv. oryzae causing bacterial blight of rice in Punjab, India

For the control of bacterial blight of rice caused by Xanthomonasoryzaepv. oryzae, sixty four Pseudomonas fluorescens strains were recovered from rice and wheat rhizosphere. These strains were identified on the basis of internal transcribed spacer (ITS) region. It was observed that the strains showing fluorescence in the selective media showed the amplification of the targeted P. fluorescens specific ITS region. The strains were also characterized for the production of the antibiotic 2, 4-diacetylphloroglucinol (DAPG) using phlDlocus. The characteristic 750bp region was amplified in all the DAPG producing strains. These strains were evaluated against X. oryzae in vitro by dual culture method. The P. fluorescens strains found effective in vitro were further tested in field for their antagonistic potentiality and disease suppression ability. P. fluorescens strain number Pf-4-R showed maximum inhibition i.e. of 5.5 mm against the test pathogen X. oryzaepv. oryzae. Talc based powder formulation of the effective strain Pf-4-R used for field evaluation, showed that pre-inoculation foliar sprays were effective in controlling bacterial blight of rice with disease suppression efficiency ranging from 29.6 to 65.6 percent in different treatments.

DNA barcoding for identification of consumer-relevant mushrooms: A partial solution for product certification?

One challenge in the dietary supplement industry is confirmation of species identity for processed raw materials, i.e. those modified by milling, drying, or extraction, which move through a multilevel supply chain before reaching the finished product. This is particularly difficult for samples containing fungal mycelia, where processing removes morphological characteristics, such that they do not present sufficient variation to differentiate species by traditional techniques. To address this issue, we have demonstrated the utility of DNA barcoding to verify the taxonomic identity of fungi found commonly in the food and dietary supplement industry; such data are critical for protecting consumer health, by assuring both safety and quality. By using DNA barcoding of nuclear ribosomal internal transcribed spacer (ITS) of the rRNA gene with fungal specific ITS primers, ITS barcodes were generated for 33 representative fungal samples, all of which could be used by consumers for food and/or dietary supplement purposes. In the majority of cases, we were able to sequence the ITS region from powdered mycelium samples, grocery store mushrooms, and capsules from commercial dietary supplements. After generating ITS barcodes utilizing standard procedures accepted by the Consortium for the Barcode of Life, we tested their utility by performing a BLAST search against authenticate published ITS sequences in GenBank. In some cases, we also downloaded published, homologous sequences of the ITS region of fungi inspected in this study and examined the phylogenetic relationships of barcoded fungal species in light of modern taxonomic and phylogenetic studies. We anticipate that these data will motivate discussions on DNA barcoding based species identification as applied to the verification/certification of mushroom-containing dietary supplements.

DNA barcoding for identification of species in mushrooms: A component of product certification

One challenge in the botanical industry is the confirmation of species identification, and this can be compounded by the processing of the materials before reaching the vendor, such as milling or drying. This can be particularly difficult for samples that contain fungal mycelia, where morphological characteristics do not present sufficient variation to differentiate species. However, monitoring the safety and quality of such products is a requirement for the protection of consumer health. DNA barcoding, a genetic-based species identification system, has become popular in the past decade, and it shows great promise for species level identification of fungi. By using DNA barcoding of nuclear ribosomal internal transcribed spacer (ITS) of the rRNA gene with fungal specific ITS primers, we generated ITS barcodes for two representative mushroom fungi, which are being used by consumers for food and dietary supplement purposes. After generating ITS barcodes utilizing standard procedures accepted by the Consortium for the Barcode of Life, we tested the utility of the ITS by performing a BLAST search against both curated and non-curated public databases. Results demonstrated that the ITS region was able to identify both mushrooms used in the present study to species-level. The BLAST search provided 99% similarity of the query sequence to those available in the public database. We anticipate that this presentation will motivate a discussion on DNA barcoding based species identification, particularly as it applies to the verification/certification of mushrooms containing products.

Multiplex PCR을 이용한 4 종류 목향(木香)의 감별

Objectives : Aucklandiae Radix (Muxiang) one of important herbal medicines in oriental medicine, is defined as the dried root of Aucklandia lappa (Asteraceae). Owing to the similarities in the morphology and name, Inulae Radix (Tu-Muxiang) and Vladimiriae Radix (Chuan-Muxiang) as well as Aristolochiae Radix (Qing-Muxiang) originated from other medicinal plants are often used as substitutes and/or adulterants of Aucklandiae Radix. Therefore, a reliable authentication of these herbal medicines is necessarily for the public health and prevention of misuse. Methods : 32 samples of medicinal plants supplying Aucklandiae Radix, Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix were collected in Korea and China. The ITS (Internal transcribed spacer) nucleotide sequences of samples were determined. The PCR primers to amply DNA marker of each herbal medicine were designed basing on the specific ITS regions showing differences in the sequences among medicinal plants. Results : Primer set Al R/IS F designed in this work amplified 220 bp PCR product only in samples of Aucklandiae Radix. In contrast, primer set Ih F/IS R, Vs R/IS F, and AcR F1/Ac R amplified 250 bp product, 356 bp prouct, and 516 bp product respectively to identify Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix. Conclusions : The primers designed basing on the nucleotide sequences of ITS regions appearing differenced in the sequences among medicinal plants amplified the DNA markers for the identification of Aucklandiae Radix, Inulae Radix, Vladimiriae Radix, and Aristolochiae Radix. These herbal medicines were more efficiently identified by multiplex PCR method using all primers in a single PCR process.

Molecular diversity, phylogeography and genetic relationships of the Silene paradoxa group of section Siphonomorpha (Caryophyllaceae)

We investigate the genetic diversity and demographic patterns of the Silene paradoxa group from section Siphonomorpha, which includes the three species S. paradoxa, S. fruticosa and S. aristidis. Eight additional taxa, belonging to the same section, were also included in this study for comparative purposes: S. nutans, S. viridiflora and six species of the italica complex. The nuclear internal transcribed spacer (ITS) and the two chloroplast spacers trnH–psbA and trnS–trnG show that the paradoxa group constitutes a separate genetic lineage within section Siphonomorpha and includes three well-differentiated species, characterized by different levels of genetic diversity and demographic histories. All three species showed specific haplotypes and ITS variants, intraspecific variation and contrasting demographic histories. S. paradoxa probably followed a demographic expansion scenario, whereas S. fruticosa and S. aristidis most likely went through spatial expansions in the past. Chasmophyte species and species from open rocky habitats are found in both the paradoxa group and the italica complex, suggesting that the switch between the chasmophytic and open habitats occurred at least twice in the Siphonomorpha section.

Characterization of Two Fungal Isolates from Cotton and Evaluation of their Potential for Biocontrol of Verticillium Wilt of Cotton

AbstractTwo isolates (CVd‐WHw and CVn‐WHg) recovered from Verticillium‐wilt‐symptomatic cotton grown in Hubei Province of China were identified based on their morphology, growth characteristics in culture, specific amplification and identification of internal transcribed spacer (ITS) rDNA sequence. According to the morphological characteristics, specific PCR amplification and ITS sequences, CVd‐WHw was identified as V. dahliae and CVn‐WHg as Gibellulopsis nigrescens. In bioassays, the two isolates had significantly lower pathogenicity to cotton plant than V. dahliae isolate CVd‐AYb. Cotton pre‐inoculated with isolate CVn‐WHg or CVd‐WHw exhibited reduced disease indices of Verticillium wilt compared with those inoculated with CVd‐AYb alone. Cotton co‐inoculated with CVn‐WHg or CVd‐WHw and CVd‐AYb provided increased protection from subsequent CVd‐AYb inoculation. These results suggest that the two isolates have the potential to be developed as biocontrol agents for the control of Verticillium wilt in cotton. To our knowledge, this is the first report of a cross‐protection phenomenon using Gibellulopsis nigrescens against Verticillium wilt caused by V. dahliae on cotton.

Accession-Specific Haplotypes of the Internal Transcribed Spacer Region in Arabidopsis thaliana--A Means for Barcoding Populations

Eukaryote genomes contain multiple copies of nuclear ribosomal DNA (nrDNA) harboring both highly conserved and variable regions. This has made nrDNA the most popular genetic marker for phylogenetic studies and the region of choice for barcoding projects. Furthermore, many scientists believe that all copies of nrDNA within one nucleus are practically identical due to concerted evolution. Here, we investigate the model plant species Arabidopsis thaliana for intragenomic variation of the internal transcribed spacer (ITS) region of nrDNA. Based on a modified deep sequencing approach, we provide a comprehensive list of ITS polymorphisms present in the two most widely used accessions of A. thaliana-Col-0 and Ler. Interestingly, we found that some polymorphisms are shared between these genetically very distinct accessions. On the other hand, the high number of accession-specific polymorphisms shows that each accession can be clearly and easily characterized by its specific ITS polymorphism patterns and haplotypes. Network analysis based on the detected haplotypes demonstrates that the study of ITS polymorphism patterns and haplotypes is an extremely powerful tool for population genetics. Using the methods proposed here, it will now be possible to extend the traditionally species-bound barcoding concept to populations.

Analytical characterization and structure elucidation of metabolites from Aspergillus ochraceus MP2 fungi

Objective To isolate and characterize the bioactive secondary metabolites from Aspergillus ochraceus ( A. ochraceus) MP2 fungi. Methods The anti bacterial activity of marine sponge derived fungi A. ochraceus MP2 was thoroughly investigated against antagonistic human pathogens. The optimum inhibitory concentration of the fungi in the elite solvent was also determined. The promising extracts that showed good antimicrobial activity were subjected to further analytical separation to get individual distinct metabolites and the eluants were further identified by GC MS instrumental analysis. The molecular characterization of the elite fungal strains were done by isolating their genomic DNA and amplify the internal transcribed spacer (ITS) region of 5.8s rRNA using specific ITS primer. The novelty of the strain was proved by homology search tools and elite sequences was submitted to GENBANK. Results Three bioactive compounds were characterized to reveal their identity, chemical formula and structure. The first elutant was identified as α – Campholene aldehyde with chemical formula C 10 H 16 O and molecular weight 152 Da. The second elutant was identified as Lucenin-2 and chemical formula C 27 H 30 O 16 and molecular weight 610 Da. The third elutant was identified as 6-Ethyloct- 3-yl- 2- ethylhexyl ester with Chemical formula C 26 H 42 O4with molecular weight 418 Da. Conclusions The isolated compounds showed significant antimicrobial activity against potential human pathogens. Microbial secondary metabolites represent a large source of compounds endowed with ingenious structures and potent biological activities.

Isolation and molecular characterization of bioactive secondary metabolites from Callyspongia spp. associated fungi

Objective To isolate and characterize the bioactive secondary metabolite from Callyspongia spp. associated fungi. Methods In vitro antibacterial screening of fungi associated with Callyspongia species, collected from south east coast of India, against selected clinical isolates of bacteria were conducted in this study. The extracts showing good antimicrobial activity were subjected to further analysis to identify the active constituents sponge associated fungi (both biomass and filtrate) with five different solvents. The compound responsible for bioactivity was characterized using Fouvier-transform infrared (FT-IR) and gas chromatography-mass spectrometry (GC-MS) instrumental analysis to identify the functional group and compound. The molecular characterization of the elite fungal strains were done by isolating their genomic DNA and amplify the internal transcribed spacer (ITS) region of 5.8 sr RNA using specific ITS primer. The novelty of the strain was proved by BlastN analysis against non-redundant (NR) database and hence was submitted to GenBank. Results Active compound was Desmethylnomifensine confirmed by GC-MS and the potent fungi was Aspergillus flavus GU815344. Conclusions The isolate exhibits a marked antagonistic activity against potential bacterial pathogens thus illuminating the advanced researches in this decade to focus on clinical pharmacology to identify novel therapeutic targets. The present study depicts a promising scenario to focus on Aspergillus flavus derived compounds which can be easily scaled up for large biomass production and stable formulation as a drug.

Cryptic species in the Terfezia boudieri complex

Phylogenetic analyses have corroborated the discovery of three internal transcribed spacer (ITS) Types in Terfezia boudieri isolates in the course of earlier studies and have emphasized the divergence of Type 2 from Types 1 and 3. The application of molecular and physiological tools described below, revealed the existence of cryptic species within T. boudieri. The markers used include sequences taken from the 5' end of the ribosomal large subunit gene, a chitin synthase partial sequence, beta-tubulin partial sequence and amplified fragment length polymorphism (AFLP)-based markers. Following initial sequencing of a single PCR amplified sample for each Type, mass analysis of specimens relied on RFLP differences between the Types. Over 100 fruit bodies, 30 or more specimens for each ITS Type, were tested with each of the markers. The markers analysis divided the isolates into three groups, each correlated to a specific ITS Type. Two of the physiological traits examined: mycelial proliferation and mycorrhiza formation, consistently showed responses paralleling the ITS Types; the data presented suggest that T. boudieri is comprised of three cryptic species.

Identification of Medicinal Atractylodes Based on ITS Sequences of nrDNA

Dried rhizomes of five species of Atractylodes (A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana), Compositae, have been used as crude drugs mainly for the treatment of stomach disorders and for their diuretic properties in Chinese and Japanese traditional medicines. The identification of the botanical origins of these crude drugs is generally difficult from their morphological and chemical features only. In this study, for identification with more reliable, nuclear ribosomal DNA (nrDNA), internal transcribed spacer (ITS) regions of five species of medicinal Atractylodes were sequenced. As a result, specific ITS genotypes were recognized by each species. The four species (A. japonica, A. macrocephala, A. lancea, and A. chinensis) prescribed in Chinese and Japanese Pharmacopoeias as botanical origins of crude Atractylodes drugs could be distinguished by their ITS sequences because they had difference genotypes on the ITS sequences. However, the genotype of A. koreana was the same as that of A. chinensis. Additionally, hybrids between A. lancea and A. chinensis were also recognized as nucleotide additives on their ITS sequences. In this study, several morphological characteristics were researched by their genotype, too. As this result, the hybrids recognized from the genetic analysis had intermediate morphological characteristics between A. lancea and A. chinensis. It was also recognized that A. lancea and A. chinensis except for their hybrids were significant differences. It is therefore suggested that ITS sequences of nrDNA would be useful for the identification of the crude drugs derived from Atractylodes species and their interspecific hybridizations.

The Identification of Vahlkampfiid Amoebae by ITS Sequencing

We have determined the internal transcribed spacer (ITS) sequences (including the 5.8S ribosomal DNA) of 30 strains of 14 species belonging to eight vahlkampfiid genera. Each previously described species has a specific ITS sequence, except for Tetramitus aberdonicus, Tetramitus thorntoni, and Tetramitus jugosus, which have identical ITS sequences. The latter three may therefore constitute a single species despite their apparent phenotypic differences. The ITS sequence appears to be conserved within a species. The species Willaertia magna appears to be ubiquitous. The 5.8S rDNA sequences of Singhamoeba horticola and Learamoeba waccamwensis indicate that they do not represent different genera, but both belong to the genus Tetramitus. The ITS sequences of 16 undescribed vahlkampfiid isolates were determined. Based on these sequences, seven isolates were identified as belonging to described species, while nine probably represent seven new species. Five of these presumed new species belong to the genus Tetramitus, and one each to the genera Vahlkampfia and Paravahlkampfia.

Pneumocystis jiroveci in Portuguese immunocompromised patients: association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood

We analyzed the genetic variation among isolates of Pneumocystis jiroveci from Portuguese immunocompromised patients with PCP at the internal transcribed spacer (ITS) regions of the nuclear rRNA operon and at the dihydropteroate synthase (DHPS) gene. Pulmonary secretions from 42 patients with PCP corresponding to 43 episodes were studied. Demographic, immunological, and clinical data were obtained from all patients. By combining the two regions ITS1 and ITS2, we found 17 different ITS types of P. jiroveci, two of them were new types (Pb and Pe). The four most prevalent ITS types were Eg (23.3%), Eb and Ne (11.6% each), and Bi (9.3%). A single type was detected in 95.3% of the samples and 4.7% had mixed infections with three different ITS types. DHPS mutants were present in 17 (46%), and the wildtype was present in 20 (54%) of 37 isolates. No association was found between ITS and DHPS types and between DHPS types and therapy or response to anti-PCP treatment. Type Ne presented an association with negative response to anti-PCP treatment ( P<0.001) and with death before 120 days after PCP diagnosis ( P=0.025). Type Eb was significantly more common in children than in adults ( P=0.001). Our data suggest an association of specific ITS genotypes with treatment failure, bad clinical outcome and childhood.

Primers based on specific ITS sequences of rDNAs for PCR detection of two fairy ring fungi of turfgrass, Vascellum pratense and Lycoperdon pusillum

Vascellum pratense and Lycoperdon pusillum cause fairy ring disease on turfgrass in golf courses. For effective disease control, detection of the mycelia of the two fungi in the soil is important. Comparing the sequence data of the internal transcribed spacer (ITS) regions of these fungi with each other and with those from the database, we designed four pairs of polymerase chain reaction (PCR) primers for each fungus. The primers allowed amplification of the DNA of the objective fungi singly, but of no other DNA from field-collected mushroom-forming fungi or soilborne turfgrass pathogenic fungi.

Heterogeneity and compartmentalization of Pneumocystis carinii f. sp. hominis genotypes in autopsy lungs.

The extent and importance of genotype heterogeneity of Pneumocystis carinii f. sp. hominis within lungs have not previously been investigated. Two hundred forty PCR clones obtained from respiratory specimens and lung segments from three patients with fatal P. carinii pneumonia were investigated to detect genetic diversity in the internal transcribed spacer (ITS) region of the nuclear rRNA operon, the mitochondrial large-subunit (mtLSU) rRNA gene, and the dihydropteroate synthase-encoding gene. For two of the three examined patients, a mixture of different mtLSU rRNA and ITS genotypes was observed. Not all genotypes present in the lungs at autopsy were detected in the diagnostic respiratory samples. Compartmentalization of specific ITS and mtLSU rRNA sequence types was observed in different lung segments. In conclusion, the interpretation of genotype data and in particular ITS sequence types in the assessment of epidemiological questions should be cautious since genotyping done on respiratory samples cannot a priori be assumed to represent all genotypes present within the lung.

Detection of Tricholoma matsutake by specific ITS primers

Sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA of ectomycorrhizal fungi were analysed and a specific PCR primer pair for Tricholoma matsutake was designed. Using the primer pair, part of the ITS region of T. matsutake (400 bp) was amplified, but no amplified fragment was detected for other ectomycorrhizal fungi. PCR was performed on DNA extracted from mycorrhizas of T. matsutake and Shiro soil (extramatrical mycelium of T. matsutake and soil complexes) and the 400 bp fragments were also specifically amplified. This indicates that presence of T. matsutake at any of its life stages can be easily confirmed by PCR typing.