DNA barcoding of invasive plants in China: A resource for identifying invasive plants.

Invasive plants have aroused attention globally for causing ecological damage and having a negative impact on the economy and human health. However, it can be extremely challenging to rapidly and accurately identify invasive plants based on morphology because they are an assemblage of many different families and many plant materials lack sufficient diagnostic characteristics during border inspections. It is therefore urgent to evaluate candidate loci and build a reliable genetic library to prevent invasive plants from entering China. In this study, five common single markers (ITS, ITS2, matK, rbcL and trnH-psbA) were evaluated using 634 species (including 469 invasive plant species in China, 10 new records to China, 16 potentially invasive plant species around the world but not introduced into China yet and 139 plant species native to China) based on three different methods. Our results indicated that ITS2 displayed largest intra- and interspecific divergence (1.72% and 91.46%). Based on NJ tree method, ITS2, ITS, matK, rbcL and trnH-psbA provided 76.84%, 76.5%, 63.21%, 52.86% and 50.68% discrimination rates, respectively. The combination of ITS + matK performed best and provided 91.03% discriminatory power, followed by ITS2 + matK (85.78%). For identifying unknown individuals, ITS + matK had 100% correct identification rate based on our database, followed by ITS/ITS2 (both 93.33%) and ITS2 + matK (91.67%). Thus, we propose ITS/ITS2 + matK as the most suitable barcode for invasive plants in China. This study also demonstrated that DNA barcoding is an efficient tool for identifying invasive species.

Assessment of species richness in Lake Dal, Kashmir, based on classical approach, physiological approach and rDNA ITS sequences from isolates.

As a first description to document the species richness in Dal Lake, a freshwater lake ecosystem in Kashmir valley, an extensive network of sixteen sampling stations with distinguishing features was sampled seasonally for two years. The identification process yielded fifty-one species probably first and new records for this area to date. The taxonomic groups observed were those with species from Ascomycetes (inclusive of yeasts), Basidiomycetes, Blastocladiomycetes, Zygomycetes, and Peronosporomycetes. Each phylum was represented by a single Order, with the exception of the Peronosporomycetes, which was represented by two Orders- Saprolegniales and Pythiales. In the filamentous fungal group, family Trichocomaceae was dominant followed by Saccharomycetaceae, Mucoraceae, Nectriaceae, Tremellaceae and Hypocreaceae. However, in the group of zoosporic & fungal like eukaryotes, family Saprolegniaceae was most dominant followed by Blastocladiaceae and Pythiaceae. A dramatic decrease in fungal load was observed in different seasons with highest colonial load in the summer season and lowest in the winter season. The observed distribution was statistically significant for both the filamentous fungal species (p < 0.01) as well as zoosporic fungi & fungal like eukaryotes (p < 0.05). In order to assess biodiversity patterns of fungi more accurately, it is necessary to repeat such investigations in other areas and to improve the tools for taxonomic identification of these highly diverse but mostly microscopic organisms.

Molecular identification of Ancylostoma species from dogs and an assessment of zoonotic risk in low-income households, São Paulo State, Brazil.

Hookworm infection stands out for its worldwide distribution and for its veterinary and public health relevance. Based on copromicroscopic examinations and polymerase chain reaction (PCR) amplification of the ITS1-5.8S-ITS2 region, we assessed, respectively, the prevalence of intestinal parasites and the identification of canine hookworm species in faeces recovered from 278 dogs living in households of an inland municipality of São Paulo State, Brazil. Intestinal parasites were found in 67.3% of dogs and hookworm infection was found at the highest prevalence rate (56.6%), followed by Toxocara canis (11.9%), Isospora spp. (11.9%), Giardia spp. (5.8%), Sarcocystis spp. (4.0%), 'Hammondia-like' (1.4%), Dipylidium caninum (1.1%) and Trichuris vulpis (0.7%). Of 158 samples positive for hookworm eggs, 106 (67.1%) were amplified by PCR and, of those, 88 (55.7%) were successfully sequenced for species identification. Single infections with Ancylostoma caninum and Ancylostoma braziliense were recorded in 61.4% and 12.5%, respectively, and mixed infections were found in 26.1%. The nucleotide sequences of both species showed high identity rates (98-100%) when compared with reference sequences. Although A. caninum was the most prevalent hookworm in the dogs assessed, the occurrence of both A. caninum and A. braziliense in single and/or mixed infections poses a potential risk for the local population in a low-income area, especially children, to acquire cutaneous larva migrans (CLM).

Phylogenetic assessment of global Suillus ITS sequences supports morphologically defined species and reveals synonymous and undescribed taxa.

The genus Suillus represents one of the most recognizable groups of mushrooms in conifer forests throughout the Northern Hemisphere. Although for decades the genus has been relatively well defined morphologically, previous molecular phylogenetic assessments have provided important yet preliminary insights into its evolutionary history. We present the first large-scale phylogenetic study of the boundaries of each species in the genus Suillus based on the most current internal transcribed spacer (ITS) barcode sequences available inPUBLIC databases, as well as sequencing of 224 vouchered specimens and cultures, 15 of which were type specimens from North America. We found that species boundaries delimited by morphological data are broadly congruent with those based on ITS sequences. However, some species appear to have been described several times under different names, several species groups cannot be resolved by ITS sequences alone, and undescribed taxa are apparent, especially in Asia. Therefore, we elevated S. tomentosus var. discolor to S. discolor; proposed synonymies of S. neoalbidipes with S. glandulosipes, S. borealis with S. brunnescens, Boletus serotinus and B. solidipes with Suillus elbensis, S. lactifluus with S. granulatus, S. himalayensis with S. americanus; and proposed usage of the names S. clintonianus in the place of the North American S. grevillei, S. weaverae for North American S. granulatus, S. ampliporus in the place of the North American S. cavipes, and S. elbensis in place of the North American S. viscidus. We showed that the majority of Suillus species have strong affinities for particular host genera. Although deep node support was low, geographic differentiation was apparent, with species from North America, Eurasia, and Asia often forming their own clades. Collectively, this comprehensive genus-level phylogenetic integration of currently available Suillus ITS molecular data and metadata will aid future taxonomic and ecological work on an important group of ectomycorrhizal fungi.

Yucatán in black and red: Linking edaphic analysis and pyrosequencing-based assessment of bacterial and fungal community structures in the two main kinds of soil of Yucatán State.

Yucatán State is dominated by two kinds of soil, named "Black Leptosol" and "Red Leptosol", which are interwoven across the State. In this work, we analyzed the relation between the edaphic characteristics and the bacterial and fungal community structures in these two kinds of Leptosol. The results revealed that Black Leptosol (BlaS) had a higher content of calcium carbonates, organic matter, nitrogen, and phosphorus than Red Leptosol (RedS). The most outstanding difference in the bacterial community structure between BlaS and RedS was that while in BlaS Actinobacteria was the most abundant phylum (43.7%), followed by Acidobacteria (26.9%) and Proteobacteria (23.6%), in RedS the bacterial community was strongly dominated by Acidobacteria (83%). Two fungal phyla were identified in both kinds of soil; Ascomycota, with 77% in BlaS and 56% in RedS, and Basidiomycota, with 22% in RedS and only 0.67% in BlaS. The most relevant difference between the two fungal communities was that excepting for Fusarium sp., all the species they had were different. Thus, in contrast with bacterial communities, where most of the major OTUs were present in both kinds of soil, fungal communities appeared to be unique to each kind of Leptosol.

Human fascioliasis endemic areas in Argentina: multigene characterisation of the lymnaeid vectors and climatic-environmental assessment of the transmission pattern.

BACKGROUND: In South America, fascioliasis stands out due to the human endemic areas in many countries. In Argentina, human endemic areas have recently been detected. Lymnaeid vectors were studied in two human endemic localities of Catamarca province: Locality A beside Taton and Rio Grande villages; Locality B close to Recreo town. METHODS: Lymnaeids were characterised by the complete sequences of rDNA ITS-2 and ITS-1 and fragments of the mtDNA 16S and cox1. Shell morphometry was studied with the aid of a computer image analysis system. Climate analyses were made by nearest neighbour interpolation from FAO data. Koeppen & Budyko climate classifications were used. De Martonne aridity index and Gorczynski continentality index were obtained. Lymnaeid distribution was assessed in environmental studies. RESULTS: DNA sequences demonstrated the presence of Lymnaea neotropica and L. viator in Locality A and of L. neotropica in Locality B. Two and four new haplotypes were found in L. neotropica and L. viator, respectively. For interspecific differentiation, ITS-1 and 16S showed the highest and lowest resolution, respectively. For intraspecific analyses, cox1 was the best marker and ITS-1 the worst. Shell intraspecific variability overlapped in both species, except maximum length which was greater in L. viator. The desertic-arid conditions surrounding Locality A, the semiaridity-aridity surrounding Locality B, and the very low yearly precipitation in both localities, are very different from the typical fascioliasis transmission foci. Lymnaeids are confined to lateral river side floodings and small man-made irrigation systems. Water availability only depends on the rivers flowing from neighbouring mountains. All disease transmission factors are concentrated in small areas where humans and animals go for water supply, vegetable cultures and livestock farming. CONCLUSIONS: The unusually high number of DNA haplotypes and the extreme climate unsuitable for F. hepatica and lymnaeid development, demonstrate that the transmission foci are isolated. Seasonal transmission may depend on the timely overlap of appropriate temperature and river water availability. Lymnaeids and F. hepatica have probably reached these localities by livestock introduction. DNA differences regarding other populations of L. neotropica and L. viator in Argentina suggest an introduction independent from the spreading movements which allowed these two lymnaeids to expand throughout the country.

Molecular inference, multivariate morphometrics and ecological assessment are applied in concert to delimit species in the Russula clavipes complex.

Species of Russula subsect. Xerampelinae are notoriously difficult to identify and name and have not been subject to molecular study. A group of species, referred to here as the R. clavipes complex, growing in association with Salix, Betula and Populus as well as coniferous tree species from temperate to arctic and alpine habitats, were examined. Analyses of the nuc rDNA internal transcribed spacer (ITS) region and a numerical analysis of morphological characters were used. The R. clavipes complex is a monophyletic group within Russula subsect. Xerampelinae, according to molecular results. The complex includes three species: R. nuoljae is a phylogenetically and morphologically well-supported species while the other two, R. clavipes and R. pascua, are similar based on ITS data and morphology but separate based on their ecology. Russula pseudoolivascens is conspecific with R. clavipes Several combinations of characters traditionally used in the taxonomy of R. subsect. Xerampelinae are inappropriate for species delimitation in this group and the adequacy of the ITS for species identification in this group is discussed. Detailed microscopic observations on the type collection of R. nuoljae are presented and illustrated, along with a key to the European members of R. subsect. Xerampelinae.

Applying DNA barcodes for identification of economically important species in Brassicaceae.

Brassicaceae is a large plant family of special interest; it includes many economically important crops, herbs, and ornamentals, as well as model organisms. The taxonomy of the Brassicaceae has long been controversial because of the poorly delimited generic boundaries and artificially circumscribed tribes. Despite great effort to delimitate species and reconstruct the phylogeny of Brassicaceae, little research has been carried out to investigate the applicability and effectiveness of different DNA regions as barcodes - a recent aid for taxonomic identification - to identify economically important species in Brassicaceae. In this study, we evaluated the feasibility of five intensively recommended regions [rbcL, matK, trnH-psbA, internal transcribed spacer (ITS), ITS2] as candidate DNA barcodes to discriminate economic species of Brassicaceae in China and try to establish a new digital identification method for economic plants of Brassicaceae. All sequences of 58 samples from 27 economic species (Brassicaceae) in China were assessed in the success rates of PCR amplifications, intra- and inter-specific divergence, DNA barcoding gaps, and efficiency of identification. Compared with other markers, ITS showed superiority in species discrimination with an accurate identification of 67.2% at the species level. Consequently, as one of the most popular phylogenetic markers, our study indicated that ITS was a powerful but not perfect barcode for Brassicaceae identification. We further discuss the discrimination power of different loci due to inheritance pattern, polyploidization and hybridization in species-specific evolution. Further screening of other nuclear genes related to species isolation as plant barcode candidates is also proposed.

Assessment of cellulolytic microorganisms in soils of Nevados Park, Colombia

A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP), Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, β-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS) of ribosomal DNA for fungi. Multivariate statistical analysis (MVA) was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.

Secondary structure analyses of the nuclear rRNA internal transcribed spacers and assessment of its phylogenetic utility across the Brassicaceae (mustards).

The internal transcribed spacers of the nuclear ribosomal RNA gene cluster, termed ITS1 and ITS2, are the most frequently used nuclear markers for phylogenetic analyses across many eukaryotic groups including most plant families. The reasons for the popularity of these markers include: 1.) Ease of amplification due to high copy number of the gene clusters, 2.) Available cost-effective methods and highly conserved primers, 3.) Rapidly evolving markers (i.e. variable between closely related species), and 4.) The assumption (and/or treatment) that these sequences are non-functional, neutrally evolving phylogenetic markers. Here, our analyses of ITS1 and ITS2 for 50 species suggest that both sequences are instead under selective constraints to preserve proper secondary structure, likely to maintain complete self-splicing functions, and thus are not neutrally-evolving phylogenetic markers. Our results indicate the majority of sequence sites are co-evolving with other positions to form proper secondary structure, which has implications for phylogenetic inference. We also found that the lowest energy state and total number of possible alternate secondary structures are highly significantly different between ITS regions and random sequences with an identical overall length and Guanine-Cytosine (GC) content. Lastly, we review recent evidence highlighting some additional problematic issues with using these regions as the sole markers for phylogenetic studies, and thus strongly recommend additional markers and cost-effective approaches for future studies to estimate phylogenetic relationships.

Ribosomic DNA intergenic spacer 1 region is useful when identifying Candida parapsilosis spp. complex based on high-resolution melting analysis.

The epidemiology of Candida parapsilosis and the closely related species C. orthopsilosis and C. metapsilosis has changed in recent years, justify the need to identify this complex at the species level. In this study we investigate the intergenic spacer 1 (IGS1) of the ribosomal DNA (rDNA) to evaluate the utility of this gene region as a phylogenetic molecular marker and the suitability of a high-resolution melting (HRM) strategy based on this region for identification of members of the C. parapsilosis spp. complex. We sequenced the IGS1 and the internal transcribed spacer (ITS) regions of the rDNA from 33 C. parapsilosis sensu lato strains. Although both regions are useful in identifying species, comparative sequence analysis showed that the diversity in the IGS1 region was higher than in the ITS sequences. We also developed an HRM analysis that reliably identifies C. parapsilosis spp. complex based on the amplification of 70 bp in the IGS1 region. All isolates were correctly identified with a confidence interval >98%. Our results demonstrate that HRM analysis based on the IGS1 region is a powerful tool for distinguishing C. parapsilosis from cryptic species.

Assessment of four DNA fragments (COI, 16S rDNA, ITS2, 12S rDNA) for species identification of the Ixodida (Acari: Ixodida).

BACKGROUND: The 5' region of cytochrome oxidase I (COI) is the standard marker for DNA barcoding. However, COI has proved to be of limited use in identifying some species, and for some taxa, the coding sequence is not efficiently amplified by PCR. These deficiencies lead to uncertainty as to whether COI is the most suitable barcoding fragment for species identification of ticks. METHODS: In this study, we directly compared the relative effectiveness of COI, 16S ribosomal DNA (rDNA), nuclear ribosomal internal transcribed spacer 2 (ITS2) and 12S rDNA for tick species identification. A total of 307 sequences from 84 specimens representing eight tick species were acquired by PCR. Besides the 1,834 published sequences of 189 tick species from GenBank and the Barcode of Life Database, 430 unpublished sequences representing 59 tick species were also successfully screened by Bayesian analyses. Thereafter, the performance of the four DNA markers to identify tick species was evaluated by identification success rates given by these markers using nearest neighbour (NN), BLASTn, liberal tree-based or liberal tree-based (+threshold) methods. RESULTS: Genetic divergence analyses showed that the intra-specific divergence of each marker was much lower than the inter-specific divergence. Our results indicated that the rates of correct sequence identification for all four markers (COI, 16S rDNA, ITS2, 12S rDNA) were very high (> 96%) when using the NN methodology. We also found that COI was not significantly better than the other markers in terms of its rate of correct sequence identification. Overall, BLASTn and NN methods produced higher rates of correct species identification than that produced by the liberal tree-based methods (+threshold or otherwise). CONCLUSIONS: As the standard DNA barcode, COI should be the first choice for tick species identification, while 16S rDNA, ITS2, and 12S rDNA could be used when COI does not produce reliable results. Besides, NN and BLASTn are efficient methods for species identification of ticks.

Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota).

The internal transcribed spacer (ITS) is a popular barcode marker for fungi and in particular the ITS1 has been widely used for the anaerobic fungi (phylum Neocallimastigomycota). A good number of validated reference sequences of isolates as well as a large number of environmental sequences are available in public databases. Its highly variable nature predisposes the ITS1 for low level phylogenetics; however, it complicates the establishment of reproducible alignments and the reconstruction of stable phylogenetic trees at higher taxonomic levels (genus and above). Here, we overcame these problems by proposing a common core secondary structure of the ITS1 of the anaerobic fungi employing a Hidden Markov Model-based ITS1 sequence annotation and a helix-wise folding approach. We integrated the additional structural information into phylogenetic analyses and present for the first time an automated sequence-structure-based taxonomy of the ITS1 of the anaerobic fungi. The methodology developed is transferable to the ITS1 of other fungal groups, and the robust taxonomy will facilitate and improve high-throughput anaerobic fungal community structure analysis of samples from various environments.

Assessment of ectomycorrhizal biodiversity in Tuber macrosporum productive sites.

Tuber macrosporum Vittad. is a truffle with superb organoleptic properties, whose cultivation is still in its infancy. For the first time we have aimed to provide information on ectomycorrhizal communities in natural and cultivated T. macrosporum sites. Ectomycorrhizal morphotypes were identified using ITS nrDNA sequencing and sorted into molecular operational taxonomic unit (MOTU). We detected 16 MOTUs in the T. macrosporum cultivated plantation. Ascomycota were the most abundant (86.4%) with Helvellaceae, Pyronemataceae and Pezizaceae the most common. Twenty-two MOTUs were collected in the natural T. macrosporum site. Basidiomycota morphotypes were plentiful (70.6%) and Thelephoraceae dominated. Each site had different taxa belowground with only T. macrosporum in common, being more abundant in the natural (18.2%) than in the cultivated (14.4%) site. Species richness, Simpson and Shannon diversity indices, taxonomic diversity, distinctness and variation of taxonomic distinctness were lower in the cultivated than in the natural site.

Assessment of cellulolytic microorganisms in soils of Nevados Park, Colombia.

A systematized survey was conducted to find soil-borne microbes that degrade cellulose in soils from unique ecosystems, such as the Superpáramo, Páramo, and the High Andean Forest in the Nevados National Natural Park (NNNP), Colombia. These high mountain ecosystems represent extreme environments, such as high levels of solar radiation, low atmospheric pressure, and extreme daily changes in temperature. Cellulolytic activity of the microorganisms was evaluated using qualitative tests, such as growth in selective media followed by staining with congo red and iodine, and quantitative tests to determine the activity of endoglucanase, ß-glucosidase, exoglucanase, and total cellulase. Microorganisms were identified using molecular markers, such as the 16S rRNA gene for bacteria and the internal transcribed spacer region (ITS) of ribosomal DNA for fungi. Multivariate statistical analysis (MVA) was used to select microorganisms with high cellulolytic capacity. A total of 108 microorganisms were isolated from the soils and, in general, the enzymatic activities of fungi were higher than those of bacteria. Our results also found that none of the organisms studied were able to degrade all the components of the cellulose and it is therefore suggested that a combination of bacteria and/or fungi with various enzymatic activities be used to obtain high total cellulolytic activity. This study gives an overview of the potential microorganism that could be used for cellulose degradation in various biotechnological applications and for sustainable agricultural waste treatment.

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus.

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH-psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity-based methods and tree-based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH-psbA. The ITS provided better results with 30.61-38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree-based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.

Global assessment of molecularly identified Anisakis Dujardin, 1845 (Nematoda: Anisakidae) in their teleost intermediate hosts.

Here, we present the ITS ribosomal DNA (rDNA) sequence data on 330 larvae of nematodes of the genus Anisakis Dujardin, 1845 collected from 26 different bony fish species from 21 sampling locations and different climatic zones. New host records are provided for Anisakis simplex (Rudolphi, 1809) sensu stricto (s.s.) and A. pegreffli Campana-Rouget et Biocca, 1955 from Anoplopoma fimbria (Pallas) (Santa Barbara, East Pacific), A. typica (Diesing, 1860) from Caesio cuning (Bloch), Lepturacanthus savala (Cuvier) and Katsuwonus pelamis (Linnaeus) (Indonesia, West Pacific), A. simplex s.s. from Cololabis saira (Brevoort) (Hawaii, Central Pacific), A. simplex C of Nascetti et al. (1986) from Sebastolobus alascanus Bean (Santa Barbara, East Pacific) and A. physeteris Baylis, 1923 from Synaphobranchus kaupii Johnson (Namibia, East Atlantic). Comparison with host records from 60 previous molecular studies of Anisakis species reveals the teleost host range so far recorded for the genus. Perciform (57 species) and gadiform (21) fishes were the most frequently infected orders, followed by pleuronectiforms (15) and scorpaeniforms (15). Most commonly infected fish families were Scombridae (12), Gadidae (10), Carangidae (8) and Clupeidae (7), with Merluccius merluccius (Linnaeus) alone harbouring eight Anisakis species. Different intermediate host compositions implicate differing life cycles for the so far molecularly identified Anisakis sibling species.

Assessment of phylogenetic relationship of rare plant species collected from Saudi Arabia using internal transcribed spacer sequences of nuclear ribosomal DNA.

The rare and endangered plants of any country are important genetic resources that often require urgent conservation measures. Assessment of phylogenetic relationships and evaluation of genetic diversity is very important prior to implementation of conservation strategies for saving rare and endangered plant species. We used internal transcribed spacer sequences of nuclear ribosomal DNA for the evaluation of sequence identity from the available taxa in the GenBank database by using the Basic Local Alignment Search Tool (BLAST). Two rare plant species viz, Heliotropium strigosum claded with H. pilosum (98% branch support) and Pancratium tortuosum claded with P. tenuifolium (61% branch support) clearly. However, some species, viz Scadoxus multiflorus, Commiphora myrrha and Senecio hadiensis showed close relationships with more than one species. We conclude that nuclear ribosomal internal transcribed spacer sequences are useful markers for phylogenetic study of these rare plant species in Saudi Arabia.

Evaluation of MALDI-TOF mass spectrometry for the identification of medically-important yeasts in the clinical laboratories of Dijon and Lille hospitals.

Conventional identification (CI) of yeasts is based on morphological, biochemical and/or immunological methods. Matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF or MT-MS) mass spectrometry has been proposed as a new method for the identification of microorganisms. This prospective study compared the performance of MT-MS and CI for the identification of yeasts isolated from clinical samples. Sequencing of the internal transcribed spacer (ITS) regions of ribosomal DNA was used as the reference method in the analysis of a total of 1207 yeast isolates. Concordance between MT-MS and CI was observed for 1105 isolates (91.5%), while 74 isolates (6.1%) were misidentified. Molecular identification revealed that 73 of these 74 isolates were identified correctly by MT-MS and CI correctly identified the last one. Concordance between the two techniques was excellent for the medically-important species (98-100%), including the identification of closely-related species (Candida albicans/C. dubliniensis; C. inconspicua/C. norvegensis; C. parapsilosis/C. metapsilosis/C. orthopsilosis). Only 2.3% of isolates belonging to C. famata, C. lambica and C. magnoliae or to Geotrichum spp. and Trichosporon spp. were not identified by MT-MS. This investigation highlights the potential of MT-MS-based yeast identification as a reliable, time and cost-efficient alternative to CI.

Rapid and accurate identification of isolates of Candida species by melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA).

Rapid identification of clinically important yeasts can facilitate the initiation of anti-fungal therapy, since susceptibility is largely species-dependent. We evaluated melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA) as an identification tool for distinguishing between 16 Candida spp., i.e. Candida albicans, Candida bracarensis, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida inconspicua, Candida kefyr, Candida krusei, Candida lipolytica, Candida lusitaniae, Candida nivariensis, Candida norvegensis, Candida parapsilosis, Candida tropicalis and Candida sojae, and Saccharomyces cerevisiae and one species pair, i.e. Candida metapsilosis/Candida orthopsilosis. Starting from a cultured isolate, ITS2-MCA led to differentiation of these species within 6 h. According to our findings, ITS2-MCA offers a simple, rapid and cost-effective method for identification of cultured isolates of the clinically most relevant and prevalent Candida species. Further studies will be necessary to evaluate how it performs on mixed samples and clinical samples.