Analysis Of Internal Transcribed Spacer Research Articles

Identification and characterization of novel transglycosylating α-glucosidase from Aspergillus neoniger.

Aspergillus niger is well established for secreting α-glucosidase having transglycosylation activity, which is used as processing aid for synthesis of isomaltooligosaccharides. The present study focuses on identification and characterization of a non-niger Aspergillus isolate and its gene conferring strong transglycosylation activity. The soil isolate was identified as Aspergillus neoniger belonging to Aspergillus section Nigri using ITS (internal transcribed spacer) and β-tubulin analysis. The sequence analysis of gene responsible for α-glucosidase synthesis revealed significant nucleotide variations when compared to other Aspergillus species. Molecular docking studies using the homology model revealed the presence of threonine at 694 subsite position instead of asparagine as in case of A. niger's α-glucosidase. The enzyme was purified to several fold using DEAE Sepharose-CL6B column and on SDS-PAGE analysis, it was found to be 145kDa. MS/MS analysis of the purified enzyme validated the presence of threonine at 694 position. Commercial α-glucosidase (Transglucosidase L 'Amano') derived from A. niger and the α-glucosidase from isolate were compared for transglycosylation activity using constant test conditions. α-glucosidase from the isolate produced 27·4% higher panose when compared to that of commercial enzyme. Moreover, the rate of secondary hydrolysis of panose is much lower in case of the isolate's enzyme. Fungal isolate A. neoniger was characterized, and its gene conferring α-glucosidase activity was established for strong transglycosylation activity having higher panose yields. To the best of our knowledge, this is the first report to establish a variant of α-glucosidase having strong transglycosylation activity from A. neoniger strain. We have demonstrated that this enzyme when used as processing aid could improve panose significantly, which is a potential prebiotic. Also, the sequence analysis established in our studies could provide pointers for directed evolution of this enzyme to further improve transglycosylation activity.

Problems, Limitations, and Challenges in Species Identification of Ascomycota Members on the Basis of ITS Regions

The internal transcribed spacer (ITS) region is regarded as a formal fungal primary barcode with a high probability of the correct identification for a broad group of fungi. ITS sequences have been widely used to determine many fungal species and analysis of rDNA ITS is still one of the most popular tools used in mycology. However, this region is not equally variable in all groups of fungi; therefore, identification may be problematic and result in ambiguous data, especially in some species-rich genera of Ascomycota. For these reasons, identification based on rDNA ITS is usually complemented by morphological observations and analysis of additional genes. Reliable species identification of Ascomycota members is essential in diagnosing plant diseases, verifying air quality and the effectiveness of agronomic practices, or analyzing relationships between microorganisms. Therefore, the present study aimed to verify, using specific examples, the extent to which ITS sequence analysis is useful in species identification of pathogens and saprobionts from Ascomycota and demonstrate problems related to such identification in practice. We analyzed 105 ITS sequences of isolates originating from air and plant material. Basic local alignment search tool (BLASTn) significantly contributed to the reliable species identification of nearly 80% of isolates such as <em>Arthrinium arundinis</em>, <em>Beauveria bassiana</em>, <em>Boeremia exigua</em>, <em>Cladosporium cladosporioides</em>, <em>Epicoccum nigrum</em>, <em>Nigrospora oryzae</em>, <em>Sclerotinia sclerotiorum</em>, or <em>Sordaria fimicola </em>and members of the genera <em>Alternaria </em>and <em>Trichoderma</em>. However, for most isolates, additional morphological observations, information regarding the isolate origin and, where possible, a PCR with species-specific primers were helpful and complementary. Using our practical approach, we determined that ITS-based species identification and comparative analysis with GenBank sequences significantly helps identifying Ascomycota members. However, in many cases, this should be regarded as suggestive of a taxon because the data usually require the use of additional tools to verify the results of such analysis.

Phylogenetic placement of Tritirachium strains from the URM culture collection originally founded by Augusto Chaves Batista (1916-1967) in Brazil, and the description of T. batistae sp. nov.

Twenty-seven Tritirachium strains were present in the URM culture collection originally founded in Brazil by Augusto Chaves Batista. Fifteen freshly-prepared cultures were obtained from these original strains preserved under mineral oil. DNA was extracted for analysing phylogenetic relationships using the sequence information available from Tritirachium type materials and reference strains. Phylogenetic analysis using the internal transcribed spacer (ITS) ribosomal DNA sequences revealed that eight of these strains belong to the same clade as of T. oryzae and that the four other strains belong to the same clade as of T. candoliense. The strain URM 38, which was previously identified as “T. brumptii”, appeared to exist as a single lineage, related to T. roseum and T. candoliense. Based on morphological features and multi-locus phylogenetic analysis, including the analyses of ITS and LSU rDNA, and rpb2 sequences, we propose that URM 38 belongs to the new species T. batistae. This novel species exhibited velutinous to cottony colonies of varying colour, septate hyphae without clamp connections, conidiophores reduced to conidiogenous cells, conidiogenous cells with a distinct sympodial rachis, and single-cell conidia that was globose to subglobose, obovoid, smooth, and hyaline. The morphological features of species accepted in Tritirachium are included in this study.

<p><strong><em>Urocystis piptatheri</em></strong><strong> sp. nov. (Urocystaceae), a new species of smut fungus on <em>Piptatherum laterale</em> (Poaceae) from Pakistan</strong></p>

A new species of smut fungi, Urocystis piptatheri is described from Kumrat Valley, Pakistan based on morphological characters and molecular phylogenetic analysis of nuclear ribosomal DNA comprising the internal transcribed spacer (ITS) regions along with large subunit (LSU). The new species is characterized by the large size of its spore balls, size and number of central spores, spore wall thickness, sori in leaves and by the host plant species (Piptatherum laterale). Phylogenetic analysis of nuclear ribosomal ITS and LSU nucleotide sequences confirmed the novelty of Urocystis piptatheri. A key for the identification of Urocystis species on Pipthatherum spp. is provided.

Species-specific Identification of Vibrio sp. based on 16S-23S rRNA gene internal transcribed spacer.

To explore a prokaryotic species-specific DNA marker, 16S-23S rRNA gene internal transcribed spacer (ITS) sequence for identification and classification of Vibrio. Five hundred and seventy four ITS sequences from 60 Vibrio strains were collected, then the primary and secondary structures of ITS sequence were analysed. The ITS was divided into several subunits, and the species-specificity of these subunits were evaluated by blast. The variable subunit of ITS showed high species-specificity. A protocol to identify a Vibrio species based on ITS analysis was developed and verified. Both the specificity and sensitivity were 100%. The phylogeny analysis of Vibrio based on ITS showed that ITS devised a better classification than 16S rDNA. Finally, an identification method of Vibrio based on ITS sequencing in food samples was developed and evaluated. The results of ITS sequencing were (100%) consistent with the results identified by ISO standard. Vibrio could be accurately identified at the species level by using the ITS sequences. The present study suggests that the ITS can be considered as a significant DNA marker for identification and classification of Vibrio species, and it posed a new path to screen the Vibrio in food sample.

Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens.

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.

Genetic variation and survival of Erysiphe necator in tropical India

The heterothallic, biotrophic fungus Erysiphe necator causes powdery mildew on grapes which impacts the yield and quality of fruits and their processed products. Two genetic groups, A and B, have been described which overwinter either as flag shoots or chasmothecia. The genetic structure of the Indian isolates and their oversummering mode in tropical regions is not known. In this study, population genetic analysis of 26 and 134 E. necator isolates from temperate and tropical regions of India, respectively, was performed. PCR analysis showed presence of both genetic groups A and B in temperate regions and only genetic group B in tropical regions. Flag shoots were not observed in tropical region. Weather data analysis and temperature sensitivity of E. necator conidia indicate the possibility of the pathogen oversummering as conidia on bark. Sequence analysis of ITS (Internal Transcribed Spacer), IGS (Intergenic Spacer), TUB2 (Beta-Tubulin) and EF1-α (Translation Elongation Factor 1-α) genes covering 1762 nucleotides from 22 isolates showed the presence of four haplotypes, A-IN1, B-IN1, B-IN2 and B-IN3 in temperate regions and only haplotype B-IN3 in tropical regions. Haplotypes A-IN1 and B-IN1 were similar to haplotypes of group A and B, respectively, reported from USA, Europe and Australia. The other two haplotypes, B-IN2 and B-IN3 grouped with haplotypes from USA and are new reports from India. The diversity and haplotype structure suggest that powdery mildew was introduced in north India and then moved to tropical regions corroborating historical reports of the introduction of grapevines in India. Occurrence of a genetic group with lower fungicide sensitivity and greater aggressiveness might explain the difficulties to manage this disease in Maharashtra, India.

Characterization of Bipolaris sorokiniana and Alternaria sesami isolates obtained from sesame (Sesamum indicum L.) in Egypt

Keeping in view the losses caused by the leaf spot disease of sesame, the present study was carried out to characterize fungal pathogenic causes leaf spot disease of sesame in Egypt. During the current study, Bipolaris sorokiniana and Alternaria sesami isolates were characterized by microscopy, colony characteristic and its pathogenicity. The identities of the fungi were confirmed by analysis of rDNA ITS (internal transcribed spacer). Fourteen RAPD primers were screened with the DNA of the fungal isolates to detect the polymorphism among these isolates. The estimated genetic similarities ranged from 74% to 82% with Bipolaris isolates, and from 85% to 96%with Alternaria isolates revealing very high levels of genetic similarity among the studied accessions. Significant differences in aggressiveness among the isolates of both pathogens were observed after artificial inoculation of sesame plants using spray inoculation methods. The disease reaction of three varieties of sesame to both fungi reveled that, the variety Shandawil-3 showed the highest degree of susceptibility to both pathogens. The host range of the isolates of both pathogens were tested on 11 plant species belonged to three families. All isolates of Bipolaris sorokiniana were pathogenic on tomato, maize, broad bean, bean, sorghum and wheat. On the other hand, the isolates of Alternaria sesami markedly varied in its pathogenicity on tomato, broad bean, bean and sorghum and were nonpathogenic to other tested plant species.In conclusions from our results, the causal pathogen Bipolaris sorokiniana is considered the most prefiling pathogen among the sesame leaf spot causal pathogen in Egypt.

Molecular Identification and Genetic Diversity of Thalassia hemprichii Through DNA Barcoding Using Internal Transcribed Spacer gene (ITS) from Awur Bay Jepara, Indonesia

Thalassia hemprichii is a type of seagrass that dominates in Awur Bay of Jepara. Conventional identification has many obstacles and the environmental pressure causes seagrass stand damage and incomplete. This leads to difficulty in morphological identification. Therefore, it is necessary to do molecular identification. DNA barcode using ITS gene is one method of molecular identification using short sequences that are efficient and have a high level of accuracy. The purposes of this research were to identify Thalassia hemprichii using DNA barcode of the Internal Transcribed Spacer (ITS). The research methods includeds DNA extraction using CTAB, PCR amplification of ITS fragment, electrophoresis, DNA sequencing, and analysis with MEGA 6 and BLAST. The result of this research showed that all of the samples studied were Thalassia hemprichii. DNA barcode of ITS is a gene marker which capable of identifying seagrass species of Thalassia hemprichii.

Diaporthe nebulae sp. nov. and First Report of D. cynaroidis, D. novem, and D. serafiniae on Grapevines in South Africa.

Diaporthe species cause Phomopsis cane and leaf spot as well as Phomopsis dieback on grapevines. Symptoms of Phomopsis dieback have increasingly been observed over the past few years. In order to assess the current status of Diaporthe on grapevines in the Western Cape Province of South Africa, isolations were made from dormant grafted nursery vines, dormant rootstock canes, and dying or dead spurs of field vines. Cultures identified as Diaporthe based on morphological features were further identified to species level by sequencing the internal transcribed spacers (ITS) 1 and 2 and 5.8S rRNA and, for a representative subsample of isolates, the partial beta-tubulin (tub2) and translation elongation factor 1-alpha (EF1-α) genes. Phylogenetic analysis of the combined ITS, tub2, and EF1-α data revealed nine Diaporthe species associated with grapevines during this survey. One of these represents a new species, D. nebulae sp. nov., and three other species, namely D. novem, D. cynaroidis, and D. serafiniae, are reported on grapevines in South Africa for the first time. Species-specific primers were designed for PCR identification of D. ampelina, D. ambigua, and D. foeniculina. Pathogenicity studies conducted on detached grapevine shoots indicated D. ampelina, D. novem, and D. nebulae sp. nov. as the most virulent species.

Genetic Diversity of the Pear Scab Fungus Venturia nashicola in Korea

Scab disease caused by Venturia nashicola is of agroeconomic importance in cultivation of Asian pear. However, little is known about the degree of genetic diversity in the populations of this pathogen. In this study, we collected 55 isolates from pear scab lesions in 13 major cultivation areas in Korea and examined the diversity using sequences of internal transcribed spacer (ITS) region, β-tubulin (TUB2), and translation elongation factor-1α (TEF-1α) genes as molecular markers. Despite a low level of overall sequence variation, we found three distinctive subgroups from phylogenetic analysis of combined ITS, TUB2, and TEF-1α sequences. Among the three subgroups, subgroup 1 (60% of isolates collected) was predominant compared to subgroup 2 (23.6%) or subgroup 3 (16.4%) and was distributed throughout Korea. To understand the genetic diversity among the subgroups, RAPD analysis was performed. The isolates yielded highly diverse amplicon patterns and none of the defined subgroups within the dendrogram were supported by bootstrap values greater than 30%. Moreover, there is no significant correlation between the geographical distribution and the subgroups defined by molecular phylogeny. Our data suggest a low level of genetic diversification among the populations of V. nashicola in Korea.

Prevalence and genetic diversity of Enterocytozoon bieneusi in sheep in China

BackgroundEnterocytozoon bieneusi is a common species of microsporidia that not only influences human health but also threatens animal productive performance and value. However, there have been no systematic studies of the prevalence of E. bieneusi in sheep in China.ResultsA total of 953 fecal specimens were collected from sheep from 11 provinces across five regions of China and analyzed for E. bieneusi by nested PCR targeting the ribosomal internal transcribed spacer (ITS). Enterocytozoon bieneusi infections were detected in four regions, with an overall infection rate of 20.4% (194/953). The highest infection rate was detected in pre-weaned lambs (25.0%), followed by post-weaned lambs (22.2%) and adult sheep (14.6%). Enterocytozoon bieneusi was found in nine of the 11 tested provinces, with infection rates between 2.9–51.7%. Eleven genotypes were identified based on ITS analysis, including seven known genotypes (BEB6, CHG1, CHG3, CHS7, CHS8, COS-I and NESH5) and four novel genotypes (CHHLJS1, CHHLJS2, CHNXS1 and CHXJS1). All 11 genotypes were clustered into group 2, and the zoonotic genotype BEB6 was the dominant genotype (n = 129, 66.5%) in sheep.ConclusionThe prevalence of E. bieneusi was studied in five regions representing most areas where sheep are bred in China. This is the first report of E. bieneusi infection in sheep for seven Chinese provinces. Geographical differences were detected in the distribution of E. bieneusi genotypes, but no differences were found among sheep in different age groups. The zoonotic genotype BEB6 was the dominant genotype, indicating that sheep are a potential source of zoonotic microsporidiosis in China. These results improve our knowledge of the epidemiology of E. bieneusi in sheep in China.

First Report of a Leaf Spot Caused by Boeremia exigua var. exigua on Hydrangea paniculata in Italy

Panicle hydrangea (Hydrangea paniculata) is a perennial ornamental plant belonging to the Saxifragaceae commonly used in gardens, characterized by massive upright flowers from midsummer to autumn. Starting from the end of August 2017, a previously unknown leaf spot was observed on 2- to 3-year-old plants of H. paniculata grown in borders in a garden located in Valle Cervo near Biella, northern Italy (45°36′00″N, 8°03′00″E). Ten out of 20 plants grown in this garden were affected, with 30 to 40% of leaves showing symptoms, consisting initially of circular necrotic spots, with diameter ranging between 20 and 80 mm, well defined by a brown margin. Severely infected leaves became chlorotic and abscised. The disease started from basal leaves on plants grown in shadow, at high relative humidity. In several isolations carried out from infected tissues on potato dextrose agar amended with 25 mg/liter of streptomycin sulfate a fungus was consistently isolated. After 15 days of incubation, black pycnidia, 127 to 255 μm diameter, developed, releasing hyaline, elliptical, nonseptate conidia, measuring 4.3 to 10.3 μm (average 6.9 μm) × 1.9 to 4.1 μm (average 2.7 μm). On the basis of its morphological characteristics, the fungus was identified as Phoma sp. (Boerema et al. 1976). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis of the 517 bp obtained showed 100% homology with Boeremia exigua (formerly Phoma exigua) (GenBank accession nos. MF039478, MF599109, and MF599108). The nucleotide sequence has been assigned the GenBank accession number MG766873. Because ITS analysis was not able to identify the varietas of this species, additional genes (elongation factor 1 alpha gene [EF1α] and β-tubulin [BTUB]) were amplified with primers EF1-728F/EF2 and Bt2a/Bt2b, respectively, and sequenced. BLAST analysis of the EF1α 449-bp sequence (GenBank accession no. MG970366) and the BTUB 372-bp sequence (GenBank accession no. MG970365) showed 100 and 99% similarity with the sequences of B. exigua var. exigua (GenBank accession nos. KY550228 and KR010463, respectively). Pathogenicity tests were performed by spraying leaves of healthy 24-month-old H. paniculata plants grown in pots at temperatures between 16 and 22°C with a spore and mycelial suspension (10⁶ CFU/ml). After artificial inoculation, leaves were covered with transparent plastic bags for 5 days. Plants sprayed only with sterile water served as a control. Three plants were used for each treatment. Lesions similar to those seen in naturally infected leaves developed on leaves 7 to 10 days after inoculation in all three inoculated plants, whereas control plants remained healthy. The same fungus was consistently reisolated from the lesions. The pathogenicity test was carried out twice. To our knowledge, this is the first report of B. exigua var. exigua on H. paniculata in Italy. The pathogen has been previously reported on H. macrophylla in Italy and the United States (Farr and Rossman 2017; Garibaldi et al. 2006). The disease is at present limited to a few gardens in the mountain region where it was observed.

Caracterisation D’isolats Locaux De Metarhizium Spp, Champignon Entomopathogene De Cosmopolites Sordidus Germar, Isoles Des Bananeraies En Cote d’Ivoire

Chemical insecticides have been commonly used against the black weevil, Cosmopolites sordidus, in banana plantations which caused huge economic and agricultural losses. Despite their benefits, pesticides can be hazardous to both humans and the environment. Intensive research in substituting chemical insecticides with biological control agents have been driven. The biological and molecular characteristics of ten strains of Metarhizium spp, isolated from dead weevils, collected in banana plantation in Côte d'Ivoire were studied. The study aims to identify the species of Metarhizium sp. The biological and molecular characteristics namely: length and width of the spores, colour of aerial mycelium as well as its variation over time, colour on the setback of the box, production of the diffusible pigment and the analysis of the region ITS (Internal Transcribed Spacer) were evaluated. The colonies presented at first a flat white mycelium cottony or fluffy appearance on which the spores formed small islets. After 21 days of incubation at 32°C. in the dark, the mycelium takes on the periphery a greenish coloration with a powdery appearance. The relief is fairly flat. The spores are ellipsoid in shape of 3 μm in length and 1 μm. The amplification by ITS-DNA PCR generated a specific fragment of 550 bp. The analysis of the ITS region of the sequences of these ten (10) isolates showed maximal correspondence with different strains of M. anisopliae and Metarhizium pinghaense.

Novel Collophorina and Coniochaeta species from Euphorbia polycaulis, an endemic plant in Iran

During a study on the biodiversity of yeasts and yeast-like ascomycetes from wild plants in Iran, four strains of yeast-like filamentous fungi were isolated from a healthy plant of Euphorbia polycaulis in the Qom Province, Iran (IR. of). All four strains formed small hyaline one-celled conidia from integrated conidiogenous cells directly on hyphae and sometimes on discrete phialides, as well as by microcyclic conidiation. Two strains additionally produced conidia in conidiomata that open by rupture. The internal transcribed spacer (ITS) sequences suggested the placement of these strains in the genera Collophorina (Leotiomycetes) and Coniochaeta (Sordariomycetes), respectively. Blast search results on NCBI GenBank and phylogenetic analyses of ITS, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the translation elongation factor 1α (EF-1α) sequences, and the nuclear large subunit ribosomal gene (LSU), partial actin (ACT), and β-tubulin (TUB) sequences, respectively, revealed the isolates to belong to three new species, that are described here as Collophorina euphorbiae, Coniochaeta iranica, and C. euphorbiae. All three species are characterised by morphological, physiological, and molecular data.

Genetic diversity within dominant Enterocytozoon bieneusi genotypes in pre-weaned calves

BackgroundCattle are commonly infected with the microsporidian parasite Enterocytozoon bieneusi. Sequence characterization of E. bieneusi in these animals at the ribosomal internal transcribed spacer (ITS) locus had identified I, J and BEB4 as the dominant genotypes. However, current studies on E. bieneusi in dairy cattle are mostly on infection rates and genotype distribution. This study aims to examine the intragenotypic diversity within dominant E. bieneusi genotypes in pre-weaned dairy calves in Shanghai, China.MethodsEnterocytozoon bieneusi genotypes and subtypes were identified by PCR sequence analysis of ITS and multilocus sequence typing (MLST), based on material from farms. Chi-square test was used to examine differences in E. bieneusi infection rates between farms or age groups.ResultsThe overall infection rate of E. bieneusi was 26.5% (214/809), ranging from 12.6% (Farm 5) to 38.5% (Farm 4). Infection rates increased with age during early life, with the peak infection rate (43.0%; 43/100) occurring at six weeks. Four genotypes were present, including J (n = 145, 67.8%), BEB4 (n = 59, 27.6%), CHN4 (n = 4, 1.9%) and CHN15 (n = 1, 0.5%), with the former two belonging to Group 2 and the latter two belonging to Group 1. Differences were detected in the distribution of the dominant genotypes J and BEB4 among five study farms. Altogether, 10 multilocus genotypes (MLGs) were identified in the two dominant ITS genotypes, including MLG-J1 to MLG-J8 of genotype J and MLG-B1 to MLG-B2 of genotype BEB4. MLG-B1 and MLG-B2 were recovered in Farms 1, 2 and 5, whereas MLG-J1 to MLG-J5 and MLG-J6 to MLG-J8 were found in Farms 3 and 4, respectively.ConclusionsThere is extensive genetic heterogeneity within the dominant E. bieneusi genotypes J and BEB4 in dairy calves in Shanghai, China, and MLST should be used in molecular epidemiological studies of E. bieneusi in cattle.

Identification and Validation of Amaranthus Species‐Specific SNPs within the ITS Region: Applications in Quantitative Species Identification

ABSTRACTThe Amaranthus genus consists of as many as 70 species, many with similar morphology. The development and validation of a DNA barcode specific to key amaranths would aid in plant identification. These barcodes can be used to develop assays for single‐species identification, critical for species surveillance and contaminant screening. A reference panel of 75 internal transcribed spacer (ITS) sequences from the National Center for Biotechnology Information (NCBI) across 11 Amaranthus spp. was analyzed for species‐specific single‐nucleotide polymorphisms (SNPs). Identified SNPs were validated using 92 accessions of an Amaranthus spp. diversity panel. Of the 75 investigated ITS sequences from NCBI, 13 were identified as potentially mislabeled. Phylogenetic analysis of ITS from the reference panel distinguished 9 of the 11 investigated species. Nine SNPs were validated as species specific. Five species were distinguished with single SNPs. To illustrate the utility of the ITS SNP analysis, a quantitative assay for Amaranthus tuberculatus (Moq.) Sauer identification was constructed targeting SNP 73G and validated using simulated population samples. Results from this research will aid in population screening for A. tuberculatus, and in the development of other quantitative detection assays for Amaranthus.

On the Monophyly ofMacrolobiumSchreb., an Ecologically Diverse Neotropical Tree Genus (Fabaceae-Detarioideae)

Premise of research. The Neotropical endemic Macrolobium is one of the most species-rich genera (ca. 75 species) within subfamily Detarioideae (Fabaceae, alternatively Leguminosae). Two sections distinguished by floral morphology have been recognized in the past. Although morphologically diverse, species within the genus share several characters, including a single well-developed petal in adaxial position. However, previous analyses based on plastid markers have suggested that the genus is not monophyletic. We produce the most densely sampled molecular phylogeny of Macrolobium and test the monophyly of the genus and the two sections.Methodology. We analyzed nucleotide sequence data from the nuclear ribosomal internal transcribed spacer (ITS) and plastid (matK, trnG) genomes using Bayesian and maximum likelihood analyses.Pivotal results. The combined analysis retains Macrolobium as a monophyletic genus, with two well-supported subclades corresponding to the two recognized sections. Macrolobium pendulum is the only species placed in a section different from its taxonomic treatment. The relationships recovered with the plastid markers differ slightly from the combined and ITS analyses, but without significant support.Conclusions. Macrolobium is shown to be a monophyletic genus and to contain two well-supported and morphologically defined sections with differing Amazonian and Andean/Central American distributions corresponding to the Gentry pattern. Species are also found to group partly according to habitat preferences and leaf morphology. Both sections contain groups of multijugate and unijugate species, and there appear to have been multiple shifts of this character.

Morphological and molecular characterisation of three species of Paratylenchus Micoletzky, 1922 (Tylenchida: Paratylenchidae) from China, with a first description of the male P. rostrocaudatus

Summary In a recent survey of plant-parasitic nematodes associated with forest in China, three species of Paratylenchus, including P. aculentus, P. leptos and P. rostrocaudatus, were found. Paratylenchus leptos and P. rostrocaudatus are new records for China and the male of P. rostrocaudatus is described for the first time. Morphological descriptions, morphometrics and light microscopic photos are provided for these three species. Molecular characterisation of these species using ribosomal RNA (rRNA) D2-D3 expansion domains of large subunit (LSU D2-D3), internal transcribed spacer (ITS) and small subunit (SSU) sequences are also given. Except for the ITS sequence of P. aculentus, other sequences were amplified for the first time. Phylogenetic relationships within the genus Paratylenchus are discussed as inferred from the analyses of LSU D2-D3, ITS and SSU.

Amanita glarea, a new species in section Vaginatae from Pakistan

A new species, Amanita glarea, is described and illustrated from Pakistan. Molecular phylogenetic data derived from internal transcribed spacer (ITS) and nuclear ribosomal large subunit (LSU) regions along with morphological characters make it distinct from other currently known Amanita species. Amanita glarea is characterized by a grayish brown, slightly umbonate, pileus having universal veil remnants and striate margins, a long ringless stipe with a saccate volva bearing yellowish brown patches on the verrucose surface, inamyloid globose to subglobose basidiospores, and a filamentous pileipellis with some inflated round to elongated cells. Phylogenetic analysis of nuclear ribosomal ITS and LSU nucleotide sequences resolve A. glarea with other taxa in Amanita section Vaginatae.