Internal Transcribed Spacer rDNA Sequences Research Articles

First report of southern blight of mung bean caused by Sclerotium rolfsii in China

Mung bean (Vigna radiata) is an important legume crop in China. An emerging mung bean disease with symptoms resembling southern blight was detected in China. This study was conducted to confirm and characterize the responsible pathogen through cultural and morphological characterization, and molecular detection using multiple specific primers and multiple-clone sequence analysis of the rDNA internal transcribed spacer (ITS) region and the translation elongation factor 1α (EF1α) and RNA polymerase II subunit 2 (RPB2) genes. All isolates obtained from mung bean were identified as Sclerotium rolfsii. Interestingly, we found that rDNA ITS, EF1α, and RPB2 sequences of all isolates consisted of two distinct types, which likely originated from different nuclei. Our findings indicated that S. rolfsii isolates were heterokaryotic and contain a heterozygous genome. Two trials of pathogenicity and host range revealed that all S. rolfsii isolates were not only strongly virulent to mung bean but also to several other important crops, indicating their potential threat to future agricultural production. To our knowledge, this is the first report of S. rolfsii causing southern blight on mung bean in China.

<p><strong><em>Neowollea manoromense</em> gen. & sp. nov. (Nostocales, Cyanobacteria), a novel geosmin producer isolated from Thailand</strong></p>

In the present study, two new filamentous cyanobacterial strains (NUACC12 and NUACC15) were described based on a comprehensive study of morphological characteristics and molecular analyses. The novel strains were isolated from a shallow freshwater pond in the central region of Thailand. The morphological features of both strains are similar to those of Wollea species, W. ambigua, but are different in the dimensions of heterocyte and akinete cells as well as the absence of a sheath around the trichome. Phylogenetic analyses based on 16S rDNA and 16S–23S rDNA internal transcribed spacer sequences (ITS) revealed that new strains formed a robust supported clade which does not belong to the same clade with the type species of other cyanobacterial genera. Moreover, 16S rDNA sequence similarity between new strains and other putatively related taxa such as Wollea, Amphiheterocytum, Sphaerospermopsis, and Raphidiopsis raciborskii ranged from 96.1–97.4%, a level of similarity low enough for separating species differences. The unique patterns of the ITS secondary structure also clearly support two new strains representing a distinct cyanobacterial species. Considering all the results obtained in the present study, we here name the new genus Neowollea gen. nov., with the type species N. manoromense sp. nov. Based on morphology, ecology, 16S rDNA data and D1–D1′ helix structure, we also propose N. salina comb. nov. (basionym Wollea salina) as an additional species in the novel genus. Furthermore, the results of PCR amplification and GC/MS/MS analysis confirmed that two N. manoromense strains could produce an odorous compound, geosmin.

<p><strong>A morpho-molecular re-appraisal of <em>Polystigma fulvum</em> and <em>P. rubrum </em>(<em>Polystigma</em>, Polystigmataceae)</strong></p>

Collections of eleven Prunus specimens infected with Polystigma species from Belarus and Russia yielded two existing taxa: Polystigma fulvum (sexual morph) and Polystigma rubrum (asexual morph). DNA based phylogenies of large subunit nuclear rDNA (LSU) and nuclear ribosomal internal transcribed spacer (ITS) are provided for the first time for Polystigma fulvum and its placement is confirmed within Polystigmataceae. The concatenated LSU and ITS DNA sequence data for P. rubrum, analyzed to infer its potential relationship to other Polystigma species, also show that strains of P. rubrum are segregated into two subclades with sufficient genetic differences. No significant differences in morphology or morphometry among the strains of P. rubrum between the two subclades, especially vis-à-vis the conidiogenous cell and conidium sizes are observed (P>0.05). Subspecies concept of P. rubrum complex is discussed.

Impact of myco-induced postharvest deterioration on the proximate composition of roots of sweetpotato (<i>Ipomoea batatas</i> [L] Lam) grown in Ebonyi State

Fungal pathogens have been indicted as important agents responsible for postharvest losses of either quantitative or qualitative terms. This study was aimed at investigating the impact of postharvest rot induced by morphologically and Partial Internal Transcribed Spacer rDNA Sequence characterized fungi- A. flavus, Penicillium expansum, A. awamori, A. niger, Rhizopus oryzae, Fusarium solani and Botrydiplodiae theobromae isolated from rotted sweetpotato roots on the proximate composition of sweetpotato roots. Using a Completely Randomized Design with two replications, samples of healthy roots and fungi-infected roots of two sweetpotato cultivars- ‘Tupiaochi’ and ‘Oyorima’ were analyzed for moisture, crude fibre, crude protein, ash, fat, carbohydrate and dry matter and the data were subjected to Analysis of Variance using SPSS and significant treatment means separated using Duncan’s New Multiple Range Test. Results revealed the corresponding proximate values for the sweetpotato cultivars- Tupiaochi and Oyorima to be 75.25% and 70.50%Moisture, 2.92 and 1.99%Crude Fibre, 2.62 and 5.05% Protein, 2.24 and 1.48% Ash, 0.44 and 1.34% Fat and 16.33 and 19.64% Carbohydrates respectively. Findings from the fungi impact assessment revealed that fungal attack depletes important proximate components such as Carbohydrates, crude fibre and moisture while increasing crude protein, fat and ash of the roots, indicating that sweetpotato roots attacked by these test fungi beyond two weeks are unfit for consumption as they will be deficient of the major food nutrients they should have supplied to the body. Proper handling, storage and mitigation of fungal spoilage of sweetpotato roots will ensure the conservation of their nutritional components.Keywords: Impact, Myco-induced, Proximate Composition, Postharvest Deterioration, Sweetpotato

Internal Transcribed Spacer rDNA and TEF-1α Gene Sequencing of Pathogenic Dermatophyte Species and Differentiation of Closely Related Species Using PCR-RFLP of The Topoisomerase II.

Objective Precise identification of dermatophyte species significantly improves treatment and controls measures of dermatophytosis in human and animals. This study was designed to evaluate molecular tools effectiveness of the gene sequencing and DNA-based fragment polymorphism analysis for accurate identification and differentiation of closely- related dermatophyte species isolated from clinical cases of dermatophytosis and their antifungal susceptibility to the current antifungal agents. Materials and Methods In this experimental study, a total of 95 skin samples were inoculated into mycobiotic agar for two weeks at 28˚C. Morphological characteristics of the isolated dermatophytes were evaluated. DNA was extracted from the fungal culture for amplification of topoisomerase II gene fragments and polymerase chain reaction (PCR) products were digested by Hinf I enzyme. Internal transcribed spacer (ITS) rDNA and TEF-1α regions of the all isolates were amplified using the primers of ITS1/4 and EF-DermF/EF-DermR, respectively. Results Based on the morphological criteria, 24, 24, 24 and 23 isolates were identified as T. rubrum, T. interdigitale, T. tonsurans and E. floccosum, respectively. PCR-restriction fragment length polymorphism (RFLP) results provided identification pattern of the isolates for T. rubrum (19 isolates), T. tonsurans (28 isolates), T. interdigitale (26 isolates) and E. floccosum (22 isolates). Concatenated dataset results were similar in PCR-RFLP, except six T. interdigitale isolates belonging to T. mentagrophytes. ConclusionOur results clearly indicated that conventional morphology and PCR-RFLP were not able to precisely identify all dermatophyte species and differentiation of closely related species like T. interdigitale and T. mentagrophytes, while ITS rDNA and TEF-1α gene sequence analyses provided accurate identification of all isolates at the genus and species level.

Morphology and phylogeny of a new species of Trichomerium from North-Western Himalayas, India

The present paper describes Trichomerium bhatii, a new species in the family Trichomeriaceae (Chaetothyriales, Ascomycota). This taxon was isolated as epiphyte from dead bark of Prunus cerasoides collected from North-Western Himalayan region of India. The identity of isolate was based on asexual-morphs, cultural characteristics and phylogenetic analyses of partial nuclear ribosomal 28S large subunit and complete internal transcribed spacer rDNA sequence data with high statistical support value. The new taxon has turned out to be distinct from other known species in the genus showing 95.26% (624/655 bp) identity with closely related T. gloeosporum MFLUCC10-0087 on the basis of rDNA internal transcribed spacer region. A brief description of asexual-morph of T. bhatii with morphological and molecular data is provided. To the best of our knowledge this is the first report on this genus from India. The type specimen is deposited in the Ajrekar Mycological Herbarium (as voucher culture) and ex type cultures are deposited in Indian Type Culture Collection (ITCC-WDCM 430), New Delhi, and National Fungal Culture Collection of India (NFCCI-WDCM 932).

Endophytic fungi from the branches of Camellia taliensis (W. W. Smith) Melchior, a widely distributed wild tea plant

Camellia taliensis (W. W. Smith) Melchior is a wild tea plant endemic from the west and southwest of Yunnan province of China to the north of Myanmar and is used commonly to produce tea by the local people of its growing areas. Its chemical constituents are closely related to those of C. sinensis var. assamica, a widely cultivated tea plant. In this study, the α diversity and phylogeny of endophytic fungi in the branches of C. taliensis were explored for the first time. A total of 160 fungal strains were obtained and grouped into 42 species from 29 genera, which were identified based on rDNA internal transcribed spacer sequence analysis. Diversity analysis showed that the endophytic fungal community of the branches of C. taliensis had high species richness S (42), Margalef index D' (8.0785), Shannon-Wiener index H' (2.8494), Simpson diversity index DS (0.8891), PIE index (0.8947) and evenness Pielou index J (0.7623) but a low dominant index λ (0.1109). By contrast, that in the branches of C. taliensis had abundant species and high species evenness. Diaporthe tectonigena, Acrocalymma sp. and Colletotrichum magnisporum were the dominant endophytic fungi. The phylogenetic tree was established by maximum parsimony analysis, and the 11 orders observed for endophytic fungi belonging to Ascomycota and Basidiomycota were grouped into 4 classes.

Characterization of the complete mitochondrial genome of Uvitellina sp., representative of the family Cyclocoelidae and phylogenetic implications

Mitochondrial (mt) DNA has been useful in revealing the phylogenetic relationship of eukaryotic organisms including flatworms. Therefore, the use of mitogenomic data for the comparative and phylogenetic purposes is needed for those families of digenetic trematodes for which the mitogenomic data are still missing. Molecular data with sufficiently rich informative characters that can better resolve species identification, discrimination, and membership in different genera is also required for members of some morphologically difficult families of trematodes bearing few autapomorphic characters among its members. Here, the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) and the complete mt genome of the trematode Uvitellina sp. (Cyclocoelidae: Haematotrephinae) was determined and annotated. The mt genome of this avian trematode is 14,217bp in length, containing 36 genes plus a single non-coding region. The ITS rDNA sequences were used for the pairwise sequence comparison of Uvitellina sp. with European cyclocoelid species, and the mitochondrial 12 protein-coding genes (PCGs) and two ribosomal RNA genes were used to evaluate the position of the family within selected trematodes. The ITS rDNA analysis of Uvitellina sp. showed less nucleotide differences with Hyptiasmus oculeus (16.77%) than with other European cyclocoelids (18.63-23.58%). The Bayesian inference (BI) analysis using the 12 mt PCGs and two rRNA genes supported the placement of the family Cyclocoelidae within the superfamily Echinostomatoidea (Plagiorchiida: Echinostmata). The availability of the mt genome sequences of Uvitellina sp. provides a novel resource of molecular markers for phylogenetic studies of Cyclocoelidae and other trematodes.

Molecular characterization of a new Trypanosoma (Megatrypanum) theileri isolate supports the two main phylogenetic lineages of this species in Japanese cattle.

Trypanosoma (Megatrypanum) theileri is a cosmopolitan, usually non-pathogenic, trypanosome of cattle transmitted by blood-sucking arthropods, mainly tabanid flies. Several T. theileri strains isolated from domestic and wild ruminants via co-culturing with mammalian feeder cells or blood cells have been characterized morphologically and genetically. Here, we cultured a new trypanosome isolate from a Holstein cow in Hokkaido, Japan, and performed morphological and molecular characterization studies. The new isolate (Obihiro strain) was co-cultivated with Madin-Darby bovine kidney (MDBK) cells in GIT medium supplemented with 10% fetal bovine serum. Trypomastigotes and epimastigotes, but not intracellular parasites, were identified in the culture. Analysis of the V7-V8 region of 18S rRNA sequences showed that the Obihiro strain is positioned within the subgenus Megatrypanum. A dendrogram based on whole internal transcribed spacer rDNA sequence showed that the Obihiro strain clustered in the lineage TthII together with the Japanese isolates of T. theileri, Esashi 9, and Esashi 12, and isolates from Zambia and the USA. T. theileri of the KM strain and a T. theileri-like trypanosome isolated from deer (TSD1 strain) clustered in the lineage TthI, separate from the Obihiro strain. Based on a partial cathepsin L-like protein gene analysis, the Obihiro strain clustered with isolates of the TthIIF genotype, which includes T. theileri from Vietnam, Sri Lanka, and Brazil. Our analyses of the T. theileri Obihiro strain provide relevant insights into its genetic diversity in Japanese cattle and corroborate the host specificity of cattle and deer trypanosomes of the subgenus Megatrypanum.

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.

New Taxon of Fungal Endophytes from Phrynium capitatum Willd: A Promising Ethnomedicinal Plant in Northeast India and its Systematic and Phylogenetic Analysis

The findings of the current study deal with the first time report on the diversity of fungal endophyte from the ethnomedicinal plant- Phrynium capitatum Willd., (Marantaceae). The plant is growing wildly in the forest of Papum Pare, Arunachal Pradesh, India, and traditionally been used for anti-diabetic, antihyperglycemic or analgesic effects. During the investigation, samples of the plant parts, viz., leaves, stem, and roots were collected for isolation of endophytic spp. The endophytic spp were identified based on the morphological, cultural, and reproductive structures (hyaline, ellipsoidal, aseptate, pycnidia, beta conidia, perithecia, asci, and ascospores). Further, the phylogenetic analysis of the isolated species was made, using the sequences of 5.8S and 28S rDNA internal transcribed spacer sequence 1 and 4. The largest number of fungal endophytes (39%) were isolated from the leaves, followed by the roots (31%) and stems (30%). Overall 35 fungal species have been isolated, out of which, thirty-four belongs to the class Ascomycetes, and one from the class zygomycetes. The highest species richness and frequency of colonization were recorded in the leaf. The observations show that Pestalotiopsis longiseta was the most dominant endophytic species followed by Diplodina microsperma, Nodulisporium hinnuleum, Aspergillus flavus , Diaporthe sp., Nigrospora oryzae and Lasiodiplodia viticola. However, Pestalotiopsis neglecta, Sordaria fimicola, Diplodina microsperma, Fusarium incarnatum, Preussia sp., Diaporthe sp., Aureobasidium sp.were common fungal endophytes isolated from P. capitatum

A new record of Trichocladium griseum in Korea: morphological and molecular characterization

A unrecorded species of Trichocladium, Trichocladium griseum, was isolated in 2017 during a survey of fungal diversity in Ulsan province, South Korea. This species was identified based on morphological characteristics and phylogenetic analysis of the internal transcribed spacer (ITS) rDNA and β-tubulin gene sequences. T. griseum has not yet been reported in South Korea. Thus, we report for the first time a new record of Trichocladium griseum in Korea, and we include the descriptions and morphological illustrations of this fungus.

Tolypocladium dujiaolongae sp. nov. and its allies

A common cicada pathogen occurring in bamboo forests of southern China is described as a new species, Tolypocladium dujiaolongae. The previously unknown anamorphic state was obtained from discharged ascospores. Based on its morphological characteristics and a molecular analysis of the internal transcribed spacer rDNA sequence, we found that the asexual features of T. dujiaolongae are similar to those of Tolypocladium species. The typical characteristics of the fungus are described. The fruiting bodies formed on artificial culture media were identical to those of natural specimens, strongly supporting the idea that the isolated strain is the asexual stage of T. dujiaolongae. Our results indicate that there is no relationship between Tolypocladium dujiaolongae and the famous traditional Chinese medicine Isaria cicadae.

Morphology, ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales, Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum.

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W.sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G.dorsalisulcum and G.impudicum, e.g., a short sulcal intrusion in G.dorsalisulcum; nuclear chambers in G.impudicum and G.dorsalisulcum; and a chloroplast enveloped by two membranes in G.impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G.dorsalisulcum, G.impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.

Development of DNA markers for Slmlo1.1, a new mutant allele of the powdery mildew resistance gene SlMlo1 in tomato (Solanum lycopersicum).

Reductions in growth and quality due to powdery mildew (PM) disease cause significant economic losses in tomato production. Oidium neolycopersici was identified as the fungal species responsible for tomato PM disease in South Korea in the present study, based on morphological and internal transcribed spacer DNA sequence analyses of PM samples collected from two remote regions (Muju and Miryang). The genes involved in resistance to this pathogen in the tomato accession 'KNU-12' (Solanum lycopersicum var. cerasiforme) were evaluated, and the inheritance of PM resistance in 'KNU-12' was found to be conferred via simple Mendelian inheritance of a mutant allele of the PM susceptibility locus Ol-2 (SlMlo1). Full-length cDNA analysis of this newly identified mutant allele (Slmlo1.1) showed that a 1-bp deletion in its coding region led to a frameshift mutation possibly resulting in SlMlo1 loss-of-function. An alternatively spliced transcript of Slmlo1.1 was observed in the cDNA sequences of 'KNU-12', but its direct influence on PM resistance is unclear. A derived cleaved amplified polymorphic sequence (dCAPS) and a high-resolution melting (HRM) marker were developed based on the 1-bp deletion in Slmlo1.1, and could be used for efficient marker-assisted selection (MAS) using 'KNU-12' as the source for durable and broad-spectrum resistance to PM.

Molecular phylogeny of the genus Corchorus (Grewioideae, Malvaceae s.l.) based on nuclear rDNA ITS sequences

A molecular phylogenetic analysis of the genus Corchorus (Grewioideae, Malvaceae s.l.) is presented, based on sequences of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 144 accessions representing 47 species. Several other genera from the subfamily Grewioideae, namely Pseudocorchorus, Triumfetta, Sparrmannia, Entelea, and Grewia, were included as outgroups. The monophyly of the genus was well supported by all phylogenetic analyses (maximum likelihood, Bayesian approaches, and parsimony), and Corchorus was divided into four major clades. The majority of African species formed a statistically highly supported and distinct clade separated from the other pantropically distributed species. Several endemic species from Australia, New Caledonia, and tropical America were nested within this distinct clade, indicating dispersal from Africa to the rest of the pantropics. Based on the taxa included in this study, the two cultivated species (C. olitorius and C. capsularis) shared a common ancestry with wild species of C. africanus, C. brevicornatus, C. pseudocapsularis, C. pseudo-olitorius, C. urticifolius, C. pilosus, C. orinocensis, and C. cunninghamii. Pseudocorchorus, previously considered an accepted genus, was nested within the genus Corchorus and shared a common ancestry especially with C. depressus and C. siliquosus. Based on morphological and anatomical similarity as well as the results of the present molecular findings, inclusion of the six Pseudocorchorus species into Corchorus is proposed, with Pseudocorchorus as a synonym of Corchorus. Of the included outgroup taxa, Triumfetta is the closest sister to Corchorus, while the common ancestor of Corchorus/Pseudocorchorus, Triumfetta, Sparrmannia, and Entelea is Grewia. A further phylogenetic study with more taxa mainly from Australia, together with additional molecular markers and morphological investigation, would help to test the hypothesis on the biogeography and growth form evolution of the genus Corchorus.

Nucleotide Sequence Database Comparison for Routine Dermatophyte Identification by Internal Transcribed Spacer 2 Genetic Region DNA Barcoding.

Conventional dermatophyte identification is based on morphological features. However, recent studies have proposed to use the nucleotide sequences of the rRNA internal transcribed spacer (ITS) region as an identification barcode of all fungi, including dermatophytes. Several nucleotide databases are available to compare sequences and thus identify isolates; however, these databases often contain mislabeled sequences that impair sequence-based identification. We evaluated five of these databases on a clinical isolate panel. We selected 292 clinical dermatophyte strains that were prospectively subjected to an ITS2 nucleotide sequence analysis. Sequences were analyzed against the databases, and the results were compared to clusters obtained via DNA alignment of sequence segments. The DNA tree served as the identification standard throughout the study. According to the ITS2 sequence identification, the majority of strains (255/292) belonged to the genus Trichophyton, mainly T. rubrum complex (n = 184), T. interdigitale (n = 40), T. tonsurans (n = 26), and T. benhamiae (n = 5). Other genera included Microsporum (e.g., M. canis [n = 21], M. audouinii [n = 10], Nannizzia gypsea [n = 3], and Epidermophyton [n = 3]). Species-level identification of T. rubrum complex isolates was an issue. Overall, ITS DNA sequencing is a reliable tool to identify dermatophyte species given that a comprehensive and correctly labeled database is consulted. Since many inaccurate identification results exist in the DNA databases used for this study, reference databases must be verified frequently and amended in line with the current revisions of fungal taxonomy. Before describing a new species or adding a new DNA reference to the available databases, its position in the phylogenetic tree must be verified.

Taxonomic assessment of the Oxytropis species from South-East of Kazakhstan

The genus Oxytropis DC. is one of the largest genera in the Fabaceae family. The most plant species belonging to the Oxytropis genus have an important medicinal value. Currently the botanical taxonomy of the genus is complicated due to existence of many subgenera and sections that developed based on morphological traits. Also, in the literature there is luck of knowledge on phylogeny of Oxytropis species from Central Asian region. Therefore, the purpose of the present study was the clarification of taxonomic relationship of two Oxytropis species from SouthEast of Kazakhstan (O. almaatensis Bajt. and O. glabra DC.). The study was based on using phylogenetic analysis and haplotype network assessment based on sequences ITS (internal transcribed spacers), which is DNA marker of nuclear genome. Plant materials of O. almaatensis were collected from 2 populations in two neighboring Gorges in Trans Ili Alatau Mountains, O. glabra plant material was obtained from Herbarium of the Department of Biodiversity and Bioresources, al-Farabi Kazakh National University. Based on DNA sequences of ITS the phylogenetic and network relationships were investigated by using Neighbor Joining and Median Joining methods, respectively. The nucleotide sequences of ITS of O. almaatensis and O. glabra were aligned with sequences of 29 Oxytropis references found in the NCBI database. Out of the 601 aligned positions of ITS 33 (5.6 %) sites were found to be polymorphic nucleotides and used in evaluation of the genetic relationship of species. Constructed MJ haplotype network showed a very high congruence with the NJ phylogenetic tree. MJ network provided valuable additional hints in clarification of the taxonomic relationship among species involved in the analysis. In this study phylogenetic NJ tree and MJ network based on the variation of ITS sequences confirmed the monophyletic origin of the genus. The ITS haplotype network suggested that O. glabra is very diverse species and possibly played important role in the evolutionary processes of the genus in Central Asian region. The study is additional contribution in the molecular taxonomy of complex Oxytropis genus.

Phoma herbarum Causing Leaf Spot of Largehead Atractylodes in Central China

Largehead Atractylodes (LA) is a perennial herbaceous plant (family Asteraceae) native to China. The rhizome of LA is the most commonly used Chinese herbal medicine to prevent early pregnancy loss due to miscarriage (You et al. 2013). From 2014 to spring 2017, a foliar disease with symptoms of leaf was observed on LA in a Huazhong medicinal plant garden, Enshi, Hubei Province, China. At temperatures between 23 and 28°C, 85% of plants were infected. The first symptoms of the disease appeared as round or irregular dark brown spots 1 to 4.5 mm in diameter with a yellow halo at the edge, which appeared on the leaves at an early stage of infection. The spots were brown to gray in the center and dark brown; in the end, scattered black specks appeared in the center of the spot. Severely infected leaves became chlorotic and abscised. Stems and flowers were not affected by the disease. Five isolates were obtained from symptomatic leaf samples after they were surface sterilized with 0.1% mercuric chloride solution and plated onto potato dextrose agar at 22°C for 1 week. All the isolates were similar in cultural and morphological characteristics, and one isolate (BZYB-1) was selected for further characterization. The isolate produced green to gray colonies with sparse aerial hyphae, and the reverse appeared brown to brown red in color. Black pycnidia were globose to elongate, 55.3 to 66.7 × 125.5 to 137.4 μm (n = 50) in diameter. Conidia were hyaline, elliptical, and smooth, 4.3 to 7.2 × 1.5 to 3.6 μm (n = 50). These characteristics are consistent with Phoma herbarum (Boerema et al. 2004), which was further confirmed by sequencing analysis of the internal transcribed spacer (ITS) region of rDNA. Primers ITS1 and ITS4 were used for polymerase chain reaction (PCR) (White et al. 1990), and the ITS rDNA sequence was deposited in GenBank (accession no. KY780194). BLAST analysis of the PCR product showed 99.9% identity to that of P. herbarum (KY849801) (Zhang et al. 2017). The pathogen was further identified to the species level as P. herbarum using gene sequences from 18S ribosomal RNA (MG757513) (Faisal et al. 2007). Pathogenicity was tested by spraying the foliage of 30 1-year-old plants with a suspension of 10⁶ conidia/ml in sterile distilled water. Each plant received 30 ml of the inoculum. Ten healthy potted plants were inoculated with sterilized water as a control. All plants were covered with plastic bags for 5 days after inoculation to maintain high relative humidity and were placed in a growth chamber at 22°C. The first foliar lesions developed on leaves 7 days after inoculation and were similar to those observed in the field. No symptoms developed on the control plants. The pathogenicity test was completed twice. P. herbarum was successfully reisolated from all artificially inoculated plants and confirmed by morphological characters, which completed Koch’s postulates. The fungus was identified as P. herbarum based on both morphological and molecular characteristics. To our knowledge, this is the first report of P. herbarum causing leaf spots and lesions on LA in China. Given the recent tremendous expansion of cultivation area for this herb in China, the economic importance of this disease is likely to increase with the greater prevalence of this host species.

Identification of Vietnamese native Dendrobium species based on ribosomal DNA internal transcribed spacer sequence

Identification of Vietnamese native Dendrobium species based on ribosomal DNA internal transcribed spacer sequence