Amplification Of Internal Transcribed Spacer Region Research Articles

Molecular Identification of Aspergillus flavus isolated from some maize (Zea mays L.) varieties across Abuja, Nigeria

This study aims at isolating and molecularly characterizing Aspergillus flavus from maize (Zea mays L.) varieties (Samaz 15,20, 37, 52, Oba super 6, 9, 39, 98, and DK 777) obtained across Abuja, Nigeria. Maize varieties were obtained across the district areas of Abuja namely; Kuje, Kwali, Gwagalada, AMAC, Abaji, Bwari and UniAbuja Experimental field. The potato dextrose agar as a medium was used in growing the fungi from the maize varieties seeds for 7 days at 25oC and then sub-cultured to obtain an isolate into potato dextrose broth. The mycelial mass produced was processed to extract its DNA using genomic DNA extraction of Zymo, D6005 for fungi and bacteria. Molecular Identification of the Internal transcribed spacer (ITS) region for each A. flavus isolate was carried out with ITS1 and ITS4 primers. anf18S rRNA gene region was also amplified with ITS5 and ITS4 primers. 2.0% agarose gels were electrophoresed for 30 minutes in 0.5× buffer, Dye and 60 mM EDTA at 80 V. A gel documentation system was used in determining PCR product sizes by evaluating with internal Mol. Wt. Standards of Thermo Scientific GeneRuler 100 bp DNA Ladder. Fifteen (15) of the twenty (20) fungal isolates were found to be positive with PCR amplification of ITS regions and amplicon sizes of 600 base pairs (bp) for A. flavus were obtained. All the twenty isolates have the presence of 18S rRNA 300-400bp. Thirteen of the fungal growth from the fourteen maize varieties obtained across Abuja were identified as A. flavus.

Predominant yeasts in the sourdoughs collected from some parts of Turkey.

In the present study, a total of eight sourdough samples were collected from three different bakeries at two different times in Turkey. Also, laboratory-scale sourdough production was conducted by daily back-slopping for 7 days. Microbiological and chemical properties of the sourdoughs were investigated. Yeast species in the sourdoughs were identified by subjecting all presumptive yeast cultures to internal transcribed spacer region amplification of the 5.8S rRNA gene, restriction fragment length polymorphism analysis using Hae III, Hha I, and Hinf I endonucleases, and sequence analysis of the D1/D2 domain of the 26S rDNA gene. A total of seven profiles were determined according to the restriction fragments. Totally, 148 yeast isolates were identified at the species level (≥400 bp, 99% identity) as Saccharomyces cerevisiae (106), Kazachstania bulderi (11), Pichia fermentans (nine), Pichia membranifaciens (eight), Kazachstania servazzii (seven), Kazachstania unispora (four), and Hanseniaspora valbyensis (three). Although collected sourdoughs were produced without using baker's yeast, S. cerevisiae was the most frequently isolated yeast species. This can be related to the contamination of the bakery environment with commercial baker's yeast during the production of other bakery products. The pH and acidity levels of the collected sourdough samples ranged from 3.71 to 3.96 and 6.78 to 23.93 mL 0.1 N NaOH/10 g dough, respectively. Mean values of the content of maltose + sucrose, glucose, fructose, lactic acid, and acetic acid were 2.43, 1.57, 2.67, 7.30, and 1.40 g/kg, respectively. Due to the artisan and region-dependent handling of the sourdough, different biochemical patterns were observed among the collected samples.

DNA-based Simultaneous Identification of Three Terminalia Species Targeting Adulteration.

Background:Various parts of three Terminalia species, namely, Terminalia arjuna (stem bark), Terminalia bellirica (fruit), and Terminalia chebula (fruit) are widely known for their therapeutic principles and other commercial values. However, stem bark of T. bellirica and T. chebula along with Terminalia tomentosa are reported as adulterants of T. arjuna. Correct botanical identification is very critical for safe and effective herbal drugs. DNA-based identification approaches are advancing the conventional methods and sometime proved more beneficial.Objective:The purpose of the study was to develop polymerase chain reaction (PCR) method using internal transcribed spacer (ITS) region to ascertain the identity of T. arjuna herbal material as well as detection of mixing of other three Terminalia species.Materials and Methods:DNA from stem barks samples were isolated and subjected to ITS region amplification and sequencing. Sequences were compared for polymorphic nucleotides determination to develop species-specific primers. Final primers were selected on the basis of in silico analysis and experimentally validated. PCR assays for botanical identification of Terminalia species were developed. Sensitivity testing and assay validation were also performed.Results:The PCR assays developed for Terminalia species were resulted in definite amplicons of the corresponding species. No cross-reactivity of the primers was detected. Sensitivity was found enough to amplify as low as 2 ng of DNA. Mixing of DNA in various concentrations for validation also proved the sensitivity of assay to detect original botanicals in the mixture. The developed methods proved very specific and sensitive to authenticate Arjuna bark to develop evidence-based herbal medicines.SUMMARY Internal transcribed spacer-based species-specific polymerase chain reaction.(PCR) assays were developed to authenticate Terminalia arjuna stem bark and to identify substitution/adulteration of Terminalia bellirica and Terminalia chebula in the genuine starting materialDefinite amplicons were obtained specific to particular species and the assay was found of profound sensitivity to amplify as low as 2 ng of DNAResults of method validation proved that the assay can identify adulterant Terminalia species even when present in lower amountsThe DNA barcodes and PCR methods can also be used to identify Terminalia bellirica and T. chebula related herbal medicinal material. Abbreviations used: ITS: Internal transcribed spacer, BSA: Bovine serum albumin, DMSO: Dimethyl sulfoxide.

Direct Colony Nested-PCR for the Detection of Fusarium oxysporum f. sp. Psidii Causing Wilt Disease in Psidium guajava L.

Guava (Psidium guajava L.) is one of the important fruit crops which are grown in tropical and subtropical countries including India. Wilt is an important disease in guava which is caused by Fusarium oxysporum f. sp. psidii (Fop) as a major obstacle for guava fruit production. It is manifested symptomatically with alterations in the development process such as premature shedding of leaves, pre-maturation of fruits, entire/whole tree defoliation and eventually death of the plant. In this study a colony PCR assay was developed for direct amplification of Internal Transcribed Spacer (ITS) region of fungal rDNA of Fop isolates. For this, two sets of primers were developed for amplification of ITS region. . The sensitivity of both the primer pair was ranged up to 10-6 and 10-7 dilutions of the pure mycelium suspension. This study provides new insight for rapid, sensitive and specific molecular detection of Fop isolates, and also useful for earlier diagnostic for better management of guava wilt disease.

Ectomycorrhizal community structure in chronosequences of Pinus densiflora in eastern China

Ectomycorrhizal (ECM) fungal communities of Pinus densiflora were studied along a chronosequence. Four age classes were sampled: 5, 30, 55 and 80 year-old. Morphotyping (anatomical and morphological identification) and molecular analysis (internal transcribed spacer region amplification and sequencing) were used to identify ECM root tips. A total of 43 ECM morphotypes were distinguished, 38 of which were successfully sequenced. Cenococcum geophilum, Cortinarius sp.1, Rhizopogon sp.1, and Russula densifolia dominated the ectomycorrhiza community of P. densiflora. C. geophilum had its peak of abundance in the 5-year-old stand, R. densifolia in the 30 year-old stand and Cortinarius sp.1 in the 80 year-old stand. However abundance ofRhizopogon sp.1 differed little with stand age. ECM fungus species diversity indices increased with stand age along the chronosequence. ECM fungal species diversity was significantly higher in the 5 year-old than in the 30 year-old stand and 55 and 80 year-old stands had similar diversities. Key words: Diversity, ectomycorrhizal, ITS, Pinus densiflora.

Genetic diversity of ITS sequences of Bursaphelenchus xylophilus

The sequence variation of internal transcribed spacer (ITS) regions of ribosomal DNA has been routinely used for species identification and species-level phylogeny of the pinewood nematode, Bursaphelenchus xylophilus. In this study, the intraspecies ITS genetic diversity of B. xylophilus was evaluated. Three pinewood nematode isolates from the United States, Japan, and Portugal were used for polymerase chain reaction (PCR) ITS region amplification and sequencing. Multiple peaks were observed in sequencing chromatograms from ITS regions of American and Japanese isolates, suggesting the presence of more than one ribosomal sequence for each isolate. PCR products were further cloned and 10 clones of each isolate were subsequently sequenced. Additionally, the ITS regions of individual nematodes from each isolate were amplified, cloned and sequenced. Among the 3 B. xylophilus isolates analyzed, an intraspecific and intra-isolate molecular variability was found. The intra-isolate ITS molecular diversity in the American isolate was higher than that in the Japanese and Portuguese isolates. However, the level of sequence variation observed within isolates was about the same as that described among ITS repeats within individuals.

Optimization of ITS rDNA Amplification for Fungi from Black Soil in North China

In order to optimize polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) rDNA region from fungi found in black soil in North China, an orthogonal experimental design [L16(45)] was used to evaluate five factors (template, Mg2+, dNTP, Taq DNA polymerase, and primer) from four levels. Subsequently, the optimal annealing temperature, annealing time, extension time and cycle numbers were evaluated. The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer, 30 ng soil DNA template, 3.0 mmol · L−1 Mg2+, 0.2 mmol · L−1 dNTPs, 0.1 μmol · L−1 each forward and reverse primer, and 2.0 U Taq enzyme in 50 μL reaction volume. The optimal thermal cycling protocol consisted of initial melting at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 90 s, and a final extension of 72°C for 10 min.

Novel morphological and genetic tools to discriminate species among the family Plumatellidae (Phylactolaemata, Bryozoa)

Some species of the freshwater bryozoans (Bryozoa, Phylactolaemata) belonging to the genus Plumatella are remarkably difficult to identify because of the large similarity of superficial architecture of their statoblasts. The examination of statoblasts by scanning electron microscope (SEM) has in fact resolved only some taxonomic questions. In this article, the authors report on novel morphological and molecular traits to discriminate among ten species of Plumatellidae (P. viganoi, P. repens, P. geimermassardi, P. rugosa, P. reticulata, P. casmiana, P. fungosa, P. emarginata, P. vaihiriae, and Hyalinella punctata). The former traits are based on shape, number, and position of annular chamber pores, whereas the latter reside on amplification and sequence analysis of the Internal Transcribed Spacer (ITS) region of nuclear rDNA. The successful amplification of ITS region from statoblasts and zooidial tubules allowed us to sequence this region on all the species investigated. The ITS sequences showed the presence of sufficient and informative polymorphisms to discriminate among morphologically similar species. It is noteworthy that the resulting ITS phylogenetic tree largely corroborated the distinction of at least two groups of freshwater bryozoans inferred on the basis of the annular chamber pore morphology. This study provides innovative approaches to reliably characterize freshwater bryozoans species and gain more insight into their taxonomy, phylogenetic relationship, and biodiversity.

Morphological and molecular analyses of ectomycorrhizal diversity in Pinus densiflora seedlings

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification) and molecular analysis (internal transcribed spacer region amplification and sequencing), we assessed diversity and composition of the ectomycorrhizal fungal community colonizing P. densiflora seedlings (from 1 to 5 years ) in a naturally regenerating area of a mature forest, in Dongning County of Hei Longjiang Province, China. A total of 22 ectomycorrhizal taxa were determined on the roots of 100 seedlings. The most frequent species was Cenococcum geophilum, which colonized 31.2% of the root tips and occurred in 93.8% of the all samples. It was followed by Clavulina cinerea, Thelephora sp.2, and Russula albidula. They colonized in 22.7%, 17.5%, 13.4% of the root tips and occurred in 63.4%, 42.6%, 29.3% of the samples respectively. These results suggested that ectomycorrhizal diversity increased in P. densiflora seedlings with increasing age.

English

In this study, the taxa which belong to the Jurinea Cass. genus, grown naturally in Turkey and collected from different localities, are compared in terms of their internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA). The DNAs of the taxa were isolated by using a commercial kit (Nucleon Phytopure). Sequences were aligned with Clustal X. Phylogenetic relationships are assessed by sequence analysis, conducted with the DNASTAR software after the amplification of ITS regions with the universally recognized primers, named as 17SE and 26SE. In conclusion, morphologically similar taxa are also clustered together when a phylogenetic analysis is conducted based on molecular data. Jurinea species of the Anatolian peninsula are closely related with the topography of the region and certain level of molecular isolation of the species is evidenced.   Key words: Jurinea, Asteraceae, molecular systematics, phylogeny, internal transcribed spacer, Turkey.

Characterization of juvenile maritime pine (Pinus pinaster Ait.) ectomycorrhizal fungal community using morphotyping, direct sequencing and fruitbodies sampling

Using ectomycorrhizal root tip morphotyping (anatomical and morphological identification), molecular analysis (internal transcribed spacer region amplification and sequencing), and fruitbody sampling, we assessed diversity and composition of the ectomycorrhizal fungal community colonizing juvenile Pinus pinaster Ait. under natural conditions in NW Spain. Overall, we found 15 Basidiomycetes and two Ascomycetes. Members of the family Thelephoraceae represented up to 59.4% of the samples. The most frequent species was Tomentella sublilacina followed by Thelephora terrestris, Russula drimeia, Suillus bovinus, and Paxillus involutus, while the less frequent were Pseudotomentella tristis, Lactarius subdulcis, Russula ochroleuca, and Entoloma conferendum. From October 2007 to June 2008, we sampled 208 sporocarps belonging to seven genera and nine species: Thelephora terrestris, Paxillus involutus, Suillus bovinus, Xerocomus badius, Scleroderma verrucosum, Amanita gemmata, A. rubescens, Amanita sp., and Russula sp. The species belonging to the genus Amanita, X. badius and S. verrucosum were not found on root samples. By comparing our results with a bibliographic review of papers published from 1922 to 2006, we found five genera and six species which have not been previously reported in symbiosis with P. pinaster. This is the first time that the diversity of the ectomycorrhizal fungal community associated with P. pinaster was investigated using molecular techniques. Considering that only 38% of the genera found by sequencing were found as fruitbodies, we conclude that integrating morphotyping and sporocarps surveys with molecular analysis of ectomycorrhizas is important to documenting the ectomycorrhizal fungus community.

Identification of Some Phellinus spp.

Four strains of Phellinus spp. was identified based on internal transcribed spacer (ITS) region of rDNA sequence analysis and morphological characteristics. Basidiocarps of all strains were effused-reflexed and hymenial surface was poroid. Hyphal system was dimitic and basidiospore was globose to ellipsoid. The amplification of ITS regions produced a DNA fragment of 500 to 780 bp in all strains examined. The determined sequences were analyzed for the reconstruction of phylogenetic tree. From these results, Phellinus sp. KM-1, KM-2, and KM-4 was identified as P. hartigii, P. baumii, and P. linteus, respectively.