Internal Transcribed Spacer rDNA Research Articles

Ocurrence of Clubroot Caused by Plasmodiophora brassicae on Kohlrabi in Korea

From 2016 to 2018, approximately 15% of kohlrabi were observed displaying significant clubroot symptoms in farmer’s fields in Jeju, Korea. The initial infection appeared as hypertrophy of root hairs, and as the disease progressed, galls formation occurred on the main roots, finally disease progress resulted in yellowing and wilting of leaves. Pathogenicity was proven by artificial inoculation of plants with resting spore suspension, fulfilling Koch’s postulates. The resting spore is one-celled, spherical and subspherical, colorless, and 3-5 mm in diameter. On the basis of the morphological characteristics and phylogenetic analyses of internal transcribed spacer rDNA, the causal agent was identified as Plasmodiophora brassicae. To our knowledge, this is the first report on the occurrence of P. brassicae on kohlrabi in Korea.

Development of a multiplex polymerase chain reaction assay for the parallel detection of harmful algal bloom-forming species distributed along the Chinese coast

Harmful algal blooms (HABs) have adverse effects on the marine ecological environment, public health, and marine economy. Thus, methods for the accurate and rapid identification of harmful algal species are urgently needed for the effective monitoring of the occurrence of HABS. A method for the parallel detection of harmful algal species must be established because various HAB-forming algal species coexist in the marine environment. This work developed a multiplex PCR (mPCR) method that can simultaneously detect six common HAB-forming microalgal species distributed along the coast of China: Karlodinium veneficum (Kv), Chattonella marina (Cm), Skeletonema spp., Scrippsiella trochoidea (St), Karenia mikimotoi (Km), and Prorocentrum donghaiense (Pd). Specific mPCR primers were designed from the internal transcribed spacer rDNA or large subunit rDNA gene of the target algal species. The mPCR conditions were optimized. Each mPCR primer was subjected to a cross-reactivity test with other microalgae to confirm the specificity of the developed mPCR system. The results of the system stability test indicated that the background concentration of DNA tested did not affect the performance of the established mPCR system. The results of the sensitivity test showed that the detection limit of the proposed mPCR system for Kv, Cm, Km, and Pd was 0.6 ng μL−1 and that for Skeletonema spp. and St was 0.06 ng μL−1. Additional mPCR analysis with spiked field samples revealed that the detection limit of the mPCR system for Km, Pd, and Kv was 60 cells, whereas that for Cm, Skeletonema spp., and St was 6 cells. The convenience and accuracy of the established mPCR assay were further validated through tests with field samples. The proposed mPCR assay is characterized by parallel analysis, strong specificity, and stability and can be used to supplement morphology-based detection methods for algal species.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Ixeridium dentatum in Korea

Ixeridium dentatum (Thunb. ex Mori) Tzvelev, known as toothed ixeridium, belonging to the family Asteraceae, is native to Korea and East Asia. In Korea, it has been used as a traditional herbal medicine for the treatment of hepatitis, indigestion, pneumonia, tumors, and hyperlipidemia. I. dentatum is commercially cultivated in some areas (over 100 ha) of Korea. In October 2017, symptoms of powdery mildew were observed on over 60% of the leaves and stems of I. dentatum plants in a garden at Chonnam National University located in Gwangju, Korea. Disease symptoms included white powdery colonies on the upper leaf surfaces and stems with or without necrosis. Appressoria of the fungus were indistinct to slightly nipple shaped. Conidiophores were cylindrical, 90.3 to 190.5 × 10.5 to 12.5 μm. Foot cells of conidiophores were 45.2 to 65.1 × 10.4 to 12.5 μm, cylindrical, or slightly constricted at the base, followed by zero to three shorter cells, forming conidia in chains. Conidia were ellipsoid to oval, 28.8 to 38.4 × 16.5 to 21.3 μm. Chasmothecia were not observed. Morphologically, the isolate was consistent with the description of Podosphaera xanthii (Castagne) U. Braun & N. Shishkoff (Braun and Cook 2012; Lee 2013; Meeboon and Takamatsu 2015). Genomic DNA was extracted directly from the mycelia and conidia using a gDNA prep kit (Solgent, Daejeon, Korea). The internal transcribed spacer (ITS) region of the rDNA of two isolates was amplified using the primers ITS1 and ITS4 as described by Lee (2015). A BLASTn search of the ITS sequences against the NCBI database indicated that the isolates EML-IDPW1726 and EML-IDPW1727 (GenBank accession nos. MG719995 and MG719996, respectively) matched P. xanthii (GenBank accession no. KY388505), with 100% (444/444 bp) sequence homology. Phylogenetic analyses based on the ITS rDNA indicated that EML-IDPW1726 and EML-IDPW1727 isolates were identical to P. xanthii. Therefore, the causal pathogen was identified as P. xanthii based on the morphology of its imperfect stage and molecular analyses. The pathogenicity of the biotrophic fungus was demonstrated by gently pressing the infected leaves onto healthy leaves of I. dentatum (Lee 2013). Plants were maintained in a greenhouse at 23 ± 2°C. Seven days after inoculation, symptoms similar to those observed under natural conditions developed on the inoculated leaves of I. dentatum plants. The fungus on inoculated leaves was morphologically identical to that first observed. Powdery mildew of I. dentatum caused by Golovinomyces cichoracearum and P. fuliginea has also been reported previously from Japan (Farr and Rossman 2017). P. xanthii (synonyms: P. fusca, Sphaerotheca fuliginea, and S. fusca) has been reported to cause powdery mildew on Ixeridium spp. (synonyms: Ixeris, Paraixeris, Youngia, Chondrilla, Prenanthes, and Lactuca [The Plant List 2013]) under the name of Sphaerotheca humuli var. fuliginea. To our knowledge, this is the first record of powdery mildew caused by P. xanthii on I. dentatum in Korea. The occurrence of powdery mildew on I. dentatum could pose a serious threat to the health of this plant, resulting in death and severe yield reduction.

Genetic uniformity characterizes the invasive spread of Neofusicoccum parvum and Diplodia sapinea in the Western Balkans

AbstractIn the past decade, trees and shrubs in the Western Balkans region have been damaged by canker and die‐back disease caused by Botryosphaeriaceae species. These pathogens include Neofusicoccum parvum and Diplodia sapinea. In this study, we determine genetic diversity and structure between populations of N. parvum and D. sapinea from Serbia and Montenegro (Western Balkans) using DNA sequence data of the internal transcribed spacer rDNA, translation elongation factor 1‐alpha, β‐tubulin‐2 and microsatellite markers. The relationship of both pathogens was compared for populations from the Continental (CR) and Mediterranean (MR) regions and for isolates of D. sapinea from Cedrus spp. and Pinus spp. Neofusicoccum parvum and D. sapinea were shown to have a low gene and genotypic diversity across the regions and hosts. All genotypes of D. sapinea found on Pinus spp. were also present on Cedrus spp. The CR and MR populations of both species were found to be only slightly separated from one another by a geographical barrier. Low genetic diversity and dominance of N. parvum and D. sapinea on non‐native trees suggests that these species have most likely been introduced into Western Balkans, possibly through the movement of infected plants.

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.

Trichoderma cyanodichotomus sp. nov., a new soil-inhabiting species with a potential for biological control.

During a biodiversity survey of Trichoderma (Ascomycota, Hypocreales, Hypocreaceae) in coastal and lake wetlands of China, a new species, Trichoderma cyanodichotomus, was isolated from Dongting Lake wetland of Hunan province. The strain TW21990-1 was characterized as having two types of conidia and producing a distinct blue-green pigment on potato dextrose agar and cornmeal dextrose agar. The taxonomic position was analyzed using three molecular markers, internal transcribed spacer rDNA, translation elongation factor 1-alpha, and RNA polymerase II subunit B, revealing less than 95.0% homology with all known Trichoderma species. The combined phylogenetic tree further identified T. cyanodichotomus as an independent subgroup belonging to Section Pachybasium, with no close relatives. In vitro antagonistic activity by dual-culture assay exhibited broad inhibition against various plant pathogens, including Botryosphaeria dothidea, Pythium aphanidermatum, Rhizoctonia solani, and Verticillium dahliae. In addition, TW21990-1 demonstrated moderate hydrolase activity of cellulase, chitinase, β-1,3-glucanase, and protease, which might be involved in mycoparasitism. Greenhouse experiments showed strong biocontrol effects against tomato damping-off incited by P. aphanidermatum, together with increased seedling height and weight gain. The identification of T. cyanodichotomus will provide useful information for sufficient utilization of fungal resources.

Phacidiaceae endophytes ofPicea rubensin Eastern Canada

More than 100 fungal endophyte strains belonging to the family Phacidiaceae were isolated from surface-sterilized Picea rubens Sarg. needles collected from the Acadian Forest Region in New Brunswick, Canada. The strains were characterized morphologically by their asexual states, and phylogenetic analyses were conducted using the nuclear internal transcribed spacer rDNA (ITS) marker and the second largest subunit of ribosomal polymerase II (RPB2). Morphological and phylogenetic data revealed seven species: Darkera cf. parca; Strasseria geniculata; two novel Phacidium species: Phacidium dicosmoanum and Phacidium faciforme; and three novel monotypic genera described to accommodate distinct species: Calvophomopsis rubenticola, Cornibusella ungulata, and Gloeopycnis protuberans. Further analyses of Darkera spp. were performed with ITS and partial translation elongation factor 1-α (TEF1α), and the results suggest that D. parca is a species complex. Phacidiaceae includes hundreds of known species that are unrepresented by sequence data; therefore, ITS sequences were generated from herbarium material including type specimens of Darkera parca, Phacidium lunatum, and specimens of Allantophomopsiella, Allantophomopsis, Bulgaria, Phacidium, and Pseudophacidium species. The description of novel species combined with morphological observations and reference sequences will facilitate the identification of conifer endophytes, both from specimens or cultures and in environmental sequence data, and improve our understanding of this large and mostly neglected family.

Molecular Characterization and Identification of Stubby Root Nematode Species From Multiple States in the United States.

Stubby root nematodes (SRN) are important plant parasites infecting many crops and widely distributed in many regions of the United States. SRN transmit Tobacco rattle virus, which causes potato corky ringspot disease, thereby having a significant economic impact on the potato industry. In 2015 to 2017, 184 soil samples and 16 nematode suspensions from North Dakota, Minnesota, Idaho, Oregon, Washington, South Carolina, North Carolina, and Florida were assayed for the presence of SRN. SRN were found in 106 soil samples with population densities of 10 to 320 SRN per 200 g of soil and in eight of the nematode suspensions. Sequencing of ribosomal DNA (rDNA) or species-specific polymerase chain reaction assays revealed the presence of four SRN species, including Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus. Accordingly, their rDNA sequences were characterized by analyzing D2-D3 of 28S rDNA, 18S rDNA, and internal transcribed spacer (ITS) rDNA obtained in this study and retrieved from GenBank. Both intra- and interspecies variations were higher in ITS rDNA than 18S rDNA and D2-D3 of 28S rDNA. Based on phylogenetic analysis, the four SRN species formed a monophyletic group, with P. allius more closely related to P. porosus than P. minor and T. obtusus. Indel variation of ITS2 rDNA was present in P. allius populations from the same geographic regions. This study documented the occurrence of SRN species across multiple states. The intra- and interspecies genetic diversity of rDNA in this study will provide more information for understanding the evolutionary relationships of SRN and will be valuable for future studies of SRN species identification and management.

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.

Pileospora piceae gen. et sp. nov. (Septorioideaceae, Botryosphaeriales) from Picea rubens

During a survey of conifer-associated fungi from the Acadian forest in Eastern Canada, an unidentified endophyte species was isolated from surface-sterilized red spruce (Picea rubens) needles. Conidiomata developed in vitro and produced dimorphic conidia, which provided morphological evidence of its position within Botryosphaeriales. Corresponding conidiomata were also observed on a dead red spruce twig. Phylogenetic analyses using nuc internal transcribed spacer rDNA (ITS) and partial 28S nuc rDNA (LSU) sequences confirmed its relationship within Botryosphaeriales, in a polytomy containing Saccharataceae and Septorioideaceae. Based on morphological and phylogenetic evidence, we describe a novel monotypic genus and species as Pileospora piceae, tentatively placed within Septorioideaceae. ITS similarity shows P. piceae was previously detected in an unrelated study as an unidentified fungal endophyte of black spruce (Picea mariana) from Eastern Quebec.

Morosphaeria muthupetensis sp. nov. (Morosphaeriaceae) from India: morphological characterization and multigene phylogenetic inference

Abstract A novel species of Morosphaeria, Morosphaeria muthupetensis, saprobic on intertidal mangrove wood of Rhizophora mucronata from Muthupet mangroves on Kaveri River Delta, Tamil Nadu along the East coast of India, is described in this paper. Morosphaeria muthupetensis is characterized by large sub-globose to lenticular ascomata; long cylindrical to clavate, short pedicellate asci; small, fusiform to ellipsoidal ascospores with acute ends when compared to other Morosphaeria species. A multigene phylogeny obtained from a combined nuclear large subunit, small subunit, internal transcribed spacers rDNA and translation elongation factor 1-alpha sequence analysis revealed that M. muthupetensis is phylogenetically distinct and nested in between Morosphaeria velatispora and Morosphaeria ramunculicola and all these species constitute a strongly supported monophyletic clade sister to the genus Helicascus. Detailed descriptions and illustrations of M. muthupetensis are presented and compared with other Morosphaeria species.

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.

Identification and characterization of Neopestalotiopsis clavispora associated with leaf blight of small cardamom (Elettaria cardamomum Maton)

AbstractLeaf blight is a major foliar disease prevalent in all cardamom‐cultivating tracts, manifesting in diverse forms of symptoms. In this study, six symptomatological variants were delineated based on the expression of foliar symptoms in cardamom genotypes (Malabar, Mysore and Vazhukka) and designated as SV 1 to SV 6. Among the symptomatological variants, SV 1, SV 2, SV 3 and SV 6 were more pronounced in Vazhukka, while SV 4 and SV 5 were prominent in Malabar type. Subsequent isolation from the variants yielded whitish colonies, which were correspondingly coded as SV 1 to SV 6. The conidia were fusiform, five‐celled, with three median versicoloured cells, two terminal hyaline cells and measured 23.1–27.25 × 3.84–4.43 μm. The apical cells had two to three tubular, flexuous, unbranched appendages, whereas the basal appendage was single, tubular and unbranched. Based on conidial characteristics and molecular characterization with internal transcribed spacer rDNA region, partial β‐tubulin, translation elongation factor 1 alpha and large subunit (28S) of the nrRNA genes revealed identity of the pathogens as Neopestalotiopsis clavispora. The pathogenicity test was performed on Malabar, Mysore and Vazhukka genotypes, and Koch’s postulates were proved. In‐vitro interaction at three temperature regimes indicated that N. clavispora was inhibitory to Colletotrichum gloeosporioides at 10 and 30°C. Among the fungicides, carbendazim, propiconazole and carbendazim‐mancozeb completely arrested hyphal growth of N. clavispora under in‐vitro conditions. This study constitutes first report on the association of Neopestalotiopsis clavispora with leaf blight disease of small cardamom.

Molecular detection of fungal pathogens in clinical specimens by 18S rDNA high-throughput screening in comparison to ITS PCR and culture

The rising incidence of invasive fungal infections and the expanding spectrum of fungal pathogens makes early and accurate identification of the causative pathogen a daunting task. Diagnostics using molecular markers enable rapid identification of fungi, offer new insights into infectious disease dynamics, and open new possibilities for infectious disease control and prevention. We performed a retrospective study using clinical specimens (N = 233) from patients with suspected fungal infection previously subjected to culture and/or internal transcribed spacer (ITS) PCR. We used these specimens to evaluate a high-throughput screening method for fungal detection using automated DNA extraction (QIASymphony), fungal ribosomal small subunit (18S) rDNA RT-PCR and amplicon sequencing. Fungal sequences were compared with sequences from the curated, commercially available SmartGene IDNS database for pathogen identification. Concordance between 18S rDNA RT-PCR and culture results was 91%, and congruence between 18S rDNA RT-PCR and ITS PCR results was 94%. In addition, 18S rDNA RT-PCR and Sanger sequencing detected fungal pathogens in culture negative (N = 13) and ITS PCR negative specimens (N = 12) from patients with a clinically confirmed fungal infection. Our results support the use of the 18S rDNA RT-PCR diagnostic workflow for rapid and accurate identification of fungal pathogens in clinical specimens.

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.

First Report of Two Colletotrichum Species Associated with Bitter Rot on Apple Fruit in Korea – C. fructicola and C. siamense

Bitter rot caused by the fungal genus Colletotrichum is a well-known, common disease of apple and causes significant yield loss. In 2013, six fungal strains were isolated from Fuji apple fruits exhibiting symptoms of bitter rot from Andong, Korea. These strains were identified as Colletotrichum fructicola and C. siamense based on morphological characteristics and multilocus sequence analysis of the internal transcribed spacer rDNA, actin, calmodulin, chitin synthase, and glyceraldehyde-3-phosphate dehydrogenase Pathogenicity tests confirmed the involvement of C. fructicola and C. siamense in the development of disease symptoms on apple fruits. This is the first report of C. fructicola and C. siamense causing bitter rot on apple fruit in Korea.

Use of ITS DNA barcode for identification of Jewels of Opar (Talinum paniculatum) collected in Thanh Hoa, Vietnam

Jewels of Opar (Talinum paniculatum) is a herbaceous plant known for its highly medicinal value. This medicinal plant is listed in the Red List of Vietnam’s medicinal plants and in the handbook of Vietnam’s medicinal plants, both documents warranting its necessity for protection. Jewels of Opar contains bioactive substances such as phytosterols, saponins, flavonoids, tannins, steroids, and many other inorganic substances. Jewels of Opar plants collected in Thanh Hoa, Vietnam, are tree-like herbs, their green trunks grows straight up and then branch out. The shapes of the leaves vary; they are generally oval or ovate-oblong in shape, with wavy veins. The flowers of this tree have five reddish purple wings, two sepals, more than ten stamens, and a spherical ovary. Both the fruits and their seeds are small and black. Tuberous roots are cylindrical with many small roots, and the tubers themselves are sweet and spicy. Internal transcribed spacer (ITS) region isolated from T. paniculatum plants in Thanh Hoa, Vietnam is 643 bp in length. Based on the nucleotide sequences of the ITS region and using the basic local alignment search tool (BLAST) in theNational Center for Biotechnology Information (NCBI), the Jewels of Opar samples collected in Thanh Hoa, Vietnam were determined to belong to T. paniculatum species, Talinumgenus, Portulacaceae.

Phomopsis Black Root Rot of Cucumber in Washington State Caused by Diaporthe sclerotioides

Phomopsis black root rot of cucumber (Cucumis sativus L.), caused by Diaporthe sclerotioides (syn. Phomopsis sclerotioides), has been reported in Austria, Britain, Canada, Denmark, France, Germany, Italy, Japan, Malaysia, the Netherlands, and Norway (Shishido et al. 2016). In August 2017, wilted plants were observed in patches comprising 10% of a 5-ha organic cucumber crop in Skagit Co., WA, after fruit had formed. Crowns, tap roots, and secondary roots had pink to brown, dry, corky lesions, black pseudostromata, and rectangular pseudosclerotia within cortical cells. Sections of symptomatic roots and crowns (≤10 mm long) of eight plants selected randomly were surface-sterilized in 0.6% NaOCl for 3 to 5 min, triple-rinsed in sterilized water, dried on sterilized blotters, plated on water agar, and incubated at 24 ± 2°C with a natural day/night cycle. Hyphae were transferred to 1/2-strength PDA amended with chloramphenicol (100 ppm). After 2 weeks, each isolate covered the plate with gray-brown, dense mycelium. Flat, brown-black, irregular pseudostromata formed in the medium. Five isolates were grown in 1/2-strength potato dextrose broth on a shaker for 7 days, mycelia harvested, DNA extracted, and the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of ribosomal DNA amplified using universal eukaryotic primers UN-UP18S42 and UN-LO28S576B (Schroeder et al. 2006). The sequences of isolates Dsl004, Dsl005, Dsl006, and Dsl008 (GenBank accession nos. MG434673.1, MG434674.1, MG434675.1, and MG434676.1, respectively) had 100% identity to that of a D. sclerotioides isolate (NR_111069.1); the sequence of Dsl002 (MG434672.1) had 99% identity because of a 7-nt insertion in the ITS1 region. All five isolates and DNA of D. sclerotioides ATCC18585 (ex-type culture) produced the expected 392-bp band of amplified DNA from the ITS rDNA using the highly specific primers CPs-1 and CPs-2 (Shishido et al. 2010). In addition, a 566-nt consensus sequence of the TEF 1-α gene of Dsl002, Dsl004 to Dsl006, and Dsl008 (MG868937) generated by PCR assay with primers EF-2F and EF-5AR (Taskin et al. 2010) was 100% identical to that of D. sclerotioides ATCC18585 (MG868936). Pathogenicity of the five isolates on cucumber was verified in a greenhouse using two methods, direct-seeding and transplanting. Two-week-old colonized PDA plates of each isolate were cut into 3 mm³ pieces and mixed in Sunshine Potting Mix #1 (Sungro Horticulture, Canada) (half a plate/1.0 liter potting mix/15-cm-diameter pot). A nontreated seed (cv. Turbo) was then planted in the medium in each of five replicate pots/isolate, and a 10-day-old Turbo seedling was transplanted into each of five replicate pots/isolate. For the control treatment, potting medium was amended with noncolonized PDA (five direct-seeded and five transplanted replicates). Plants were maintained at 18 to 26°C with 10 h light/day. Dsl004, Dsl005, Dsl006, and Dsl008 caused wilting and death of plants in 3 weeks with the direct-seeding method, and in 4 to 5 weeks for the transplanting method. Dsl002 caused stunting and minor wilting. Control plants remained healthy. The roots of inoculated plants had corky lesions with pseudostromata and pseudosclerotia. D. sclerotioides was reisolated from roots of inoculated plants but not control plants. The species was confirmed morphologically and by ITS rDNA and TEF 1-α sequencing. This is the first report of Phomopsis black root rot of cucumber caused by D. sclerotioides in Washington State or anywhere in the U.S.A. The disease was reported in greenhouse cucumber in neighboring British Columbia, Canada, in 1972 (Ormrod and Christie 1972). Research is needed to assess prevalence of the pathogen and management strategies to limit spread of the disease.