Identical Internal Transcribed Spacer Sequences Research Articles

First record of Diplodia gallae causing branch cankers on declining scarlet oak (Quercus coccinea) in Virginia.

Members of Botryosphaeria s.l. have an extensive history as cankering pathogens of stressed and declining oak trees in the eastern United States (Ferreira et al. 2021). The host range, distribution, and virulence among two closely related species, Diplodia corticola and D. gallae, remains unclear (Brazee et al. 2023). On 15 August 2023, a survey was conducted at a declining natural hardwood site in Shenandoah County, Virginia (GPS coordinates 38.922089, -78.606125). One mature Quercus coccinea tree that displayed scorched leaf margins and branch dieback was felled and a cankered branch from the crown was sampled (Fig. 1A and B). A 4-mm piece of necrotic tissue was selected from the margin of the canker, disinfected with 2.5% NaOCl, again with 70% ethanol, and air-dried before being placed on half-strength acidified PDA medium (pH 4.8) and incubated in the dark at 22 ± 2°C. After 5 days, four colonies were transferred to full-strength PDA medium and incubated in the dark at 22 ± 2°C. After 10 days, all four colonies displayed thick, gray, floccose mycelium and pigmented hyphae (Fig. 1C). Mycelia was harvested from 10-day-old colonies with a sterile pin and DNA was extracted using a Qiagen DNeasy Plant Pro Kit (Germantown, MD) according to the manufacturer's instructions. A fragment of the internal transcribed spacer (ITS) and translation elongation factor 1-α (tef1) loci were amplified using ITS4/ITS5 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) primer sets, respectively. The PCR amplicons were purified with ExoSap-IT (Affymetrix, Santa Clara, CA) and sequenced at Eurofins (Louisville, KY).&xa0; The raw nucleotide sequences were analyzed using Geneious 11.1.5 software (Biomatters, Auckland, NZ). All four colonies had identical ITS sequences. A 523 and 276-bp fragment of the ITS and tef1 loci, respectively, from isolate R1.2 was deposited into the GenBank database (accessions OR934498 and OR961039). A dataset of 43 strains consisting of 38,658 characters was aligned using MAFFT v7.49 (Katoh et al. 2013), and a concatenated ITS + tef1 maximum likelihood phylogenetic tree (1000 bootstraps) was built with PhyML 3.0 (Guindon et al. 2010) using the GTR substitution model. Isolate R1.2 was grouped with isolates of D. gallae although the species failed to form a well-supported clade (BS = 67) due to intraspecific variation (Fig. 1D). Koch's postulates were fulfilled by inoculating five healthy, containerized Q. coccinea trees (average stem caliper 5.3 cm) with isolate R1.2, with five plants as controls. After disinfecting the bark with 70% ethanol, a 0.5 mm section of the bark was removed 13 cm above the soil line with a sterile scalpel, and a 0.5 mm agar plug taken from the edge of a 10-day-old PDA culture was placed in the wound with the mycelium facing the cambial tissue, sealed with Parafilm, and maintained at 22 ± 4°C. The same procedure was performed on the control plants using sterile PDA plugs. After five weeks the bark was removed, and all five stems treated with R1.2 had necrotic lesions with a mean linear growth ([length+width]/2) of 9.2 ± 2.72 mm from the edge of the wound, which was significantly larger (P = 0.003) than the controls (1 ± 0.66 mm; Fig. 1E - L). Necrotic stem tissue was sampled as previously described, and the isolate recovered was confirmed as D. gallae based on morphology and 100% ITS sequence homology to isolate R1.2. D. gallae was not recovered from the control plants. In the United States, D. gallae has been isolated from Q. rubra and Q. velutina twig cankers in Maine, Massachusetts, New Hampshire, New York, and Vermont (Brazee et al. 2023). This is the first report of the species in Virginia causing branch cankers on Q. coccinea.

First Report of Kiwifruit Root Rot Caused by Globisporangium spinosum in China.

Kiwifruit is widely cultivated for its high vitamin C content and nutritional value. In January 2022, root rot symptoms were found in about 30% of Actinidia chinensis cv. Jinyan plants grafted on A. deliciosa rootstocks in an orchard located in Sanming (26.32°N, 117.23°E), Fujian Province of China. The affected plants appeared stunted, with brown and decaying roots, some of which were covered with white hyphae. To isolate the pathogen, the surfaces of typical symptomatic roots were sterilized for 30 s using 75% ethanol, followed by four rinses in sterile water, placing on potato dextrose agar (PDA), and incubating away from light at 25°C for 7 days. 16 Globisporangium-like isolates were obtained through hyphal tip isolation, displaying a milky-white appearance with irregular protuberances on the surface, and yellow-white backs with radial fold lines. The isolates were then cultured on corn meal agar for 5 days at 25°C in dark for morphological characteristics. Under microscope, the hyphae appeared as long strips without septa and 4.1 to 8.2 µm wide (average 6.7 µm), containing irregularly sized spherical droplets. Both terminal and intercalary hyphae swellings were observed; these appeared either spherical or subspherical, with some having projections. Their dimensions were 12.3 to 27.6 µm (average 17.3 µm). The oospores were mostly spherical, either plerotic or aplerotic, 11.8 to 22.3 µm wide (average 18.9 µm), with occasional projections. The antheridia were rod-shaped and curved, with one end attached to the oogonia. Amplification of the sequences of internal transcribed spacer (ITS) regions and cytochrome c oxidase subunit I (COI) were conducted using the primers ITS1/ITS4 (White et al. 1990) and OomCoxI-Levlo/OomCoxI-Levup (Robideau et al. 2011), respectively. The sequencing results revealed identical ITS and COI sequences in all 16 isolates. BLASTn analysis of the 969-bp ITS sequence ON202808 showed 99.38-99.59% similarity (965/971bp, 967/971bp) with the KJ162353 and AY598701 sequences from Globisporangium spinosum isolates, while the 700-bp COI sequence ON075783 showed 100% and 99.41% identity (680/680bp, 676/680bp) with the GenBank sequences HQ708835 and HQ708832, respectively, from G. spinosum. Phylogenetic analysis also showed that the obtained isolate (termed MA16) clustered with isolates from G. spinosum on the same evolutionary branch. For pathogenicity testing, four-month-old healthy Jinyan (A. chinensis) plants grown in sterilized media were transferred to sterile petri dishes covered with wet filter paper, and their roots were inoculated with a 5-mm-wide disk of MA16 when cultivated on PDA medium for 5 days. Miliang-1 (A. deliciosa) and Hongyang (A. chinensis) plants were treated similarly. The control groups each included three plants that were inoculated with non-colonized PDA. The plants were kept at 25 °C with a 12-/12-h light/dark cycle for 10 days when the inoculated plants exhibited root rot symptoms similar to those seen in the field, together with rotting and browning of the leaves. The control plants appeared healthy with no symptoms. After re-isolated from infected tissues, the pathogen was verified to be G. spinosum according to its ITS sequence, thus fulfilling the Koch's postulates. Recently, Pythium spinosum has been classified as G. spinosum according to whole-genome sequencing and phylogenomic analysis (Nguyen et al. 2022). Based on the morphological features and pathogenicity results, MA16 was identified as G. spinosum (van der Plaats-Niterink 1981; Huo et al. 2023). This report appears to be the first description of kiwifruit root rots caused by G. spinosum in China, and its identification will assist the development of strategies to counteract the disease.

Diddensiella parasantjacobensis f.a., sp. nov., a yeast species from forest habitats.

Six conspecific yeast strains, representing an undescribed species, were isolated from rotten wood collected in different locations in Hungary and Germany and an additional one from fungal fruiting body in Taiwan. The seven strains share identical nucleotide sequences in the D1/D2 domain of the nuclear large subunit (LSU) rRNA gene. The Hungarian and Taiwanese isolates share identical internal transcribed spacer (ITS) sequences as well, while the two German isolates differ from them merely by three substitutions and four indels in this region. The investigated strains are very closely related to Diddensiella santjacobensis. Along their LSU D1/D2 domain they differ only by one substitution from the type strain of D. santjacobensis. However, in the ITS region of Hungarian and Taiwanese strains we detected 3.5 % divergence (nine substitutions and nine indels) between the undescribed species and D. santjacobensis, while the German strains differed by 13 substitutions and nine indels from D. santjacobensis. This ITS sequence divergence has raised the possibility that the strains investigated in this study may represent a different species from D. santjacobensis. This hypothesis was supported by comparisons of partial translation elongation factor 1-α (EF-1α) and cytochrome oxidase II (COX II) gene sequences. While no difference and 1-2 substitutions among the partial EF-1α and COX II gene sequences of the strains of the undescribed species, respectively, were detected; the undescribed species differ by about 4 % (36 substitutions) and 10 % (50-51 substitutions) from D. santjacobensis in these regions. Parsimony network analysis of the partial COX II gene sequences also separated the investigated strains from the type strain of D. santjacobensis. In this paper we propose Diddensiella parasantjacobensis f.a., sp. nov. (holotype: NCAIM Y.02121; isotypes: CBS 17819, DSM 114156) to accommodate the above-noted strains.

Morphotypes of Ciborinia camelliae Kohn infecting camellias in Italy

Ciborinia camelliae Kohn is the causal agent of camellia flower blight. The pathogen infects only flowers of camellias, causing serious damage to the plant. Seventy-one strains were collected from six Italian regions and were characterized at the phenotypic and genetic level. Morphotypes were identified based on their phenotypic differences in culture media. The Italian population consisted of eleven morphotypes. Internal transcribed spacer (ITS) regions of twenty-two isolates belonging to the different morphotypes were sequenced. All morphotypes have identical ITS sequences. The closest match of the ITS with the strain ICMP 19812 of Ciborinia camelliae from New Zealand (Massey University Arboretum, Palmerston North, 100% sequence identity), and the morphological features confirmed the presence of a variable population of C. camelliae in six different Italian regions.

Vegetative Growth and Molecular Identification of <i>Fusarium equiseti</i> Isolated from Wilt Disease of <i>Centella asiatica</i> L. in Bangladesh

Centella asiatica (L.) is commonly known as Thankuni plant and has ethnobotanical importance in Bangladesh. Present experiment was conceded to investigate the wilt disease of C. asiatica, vegetative growth and molecular characterization of pathogenic fungi. Pathogenic fungus, Fusarium equiseti was identified as a causal agent of wilt disease in C. asiatica. The effect of culture media on the mycelial growth of F. equiseti showed the highest (89.25 mm) on potato dextrose agar (PDA) medium followed by carrot agar (CA) medium and the lowest growth (40.25 mm) was measured in HA medium. The optimal temperature and pH for mycelial growth of F. equiseti were 30°C and 7, respectively. The genetic variation of the selected species of fungi, the internal transcribed spacer (ITS) region was amplified using ITS4 and ITS5 primers and sequenced. The PCR product of the ITS region of F. equiseti was 535 bp. Phylogenetic tree of thirty-seven strains of Fusarium sp. based on the nucleotide sequences of the ITS region using the neighbor-joining method with 1000 bootstrapping indicated that 98% - 100% identity with MN886590.1 JUF0046 (F. equiseti). ITS sequences are generally constant, or show little variation within species, but vary between species in a genus. The ITS region is relatively short and can be easily amplified by PCR using universal single primer pairs. Genetic distance exhibited high level of similarity with identical ITS sequences. To date, no published research articles are found on the molecular identification of F. equiseti, the causal agent of fusarium wilt disease of C. asiatica in Bangladesh.

Use of Internal Transcribed Spacer Sequence Polymorphisms as a Method for Trichomonas vaginalis Genotyping.

Trichomonas vaginalis is the most common non-viral, sexually transmitted pathogen with a worldwide distribution. The aim of the present study was to design a new genotyping tool for T. vaginalis isolates using internal transcribed spacer (ITS) sequences. First, a total of 20 cryopreserved T. vaginalis isolates were thawed and genomic DNA was isolated from fresh cultures. A polymerase chain reaction (PCR) was performed to amplify the ITS regions and the amplicons were sequenced. These sequences were aligned with others from Genbank and polymorphisms were detected. At last, each ITS sequence was given a different sequence type. More than 99% homology was observed among sequences. Of 20 isolates, five had identical ITS sequence to reference (L29561) defined as ITST1. Moreover, 13 had A58 deletion (ITST10), one had C203T mutation (ITST2), and one had both A58 deletion and C203T mutation (ITST11). ITS typing of T. vaginalis sequences on Genbank revealed a total of 11 ITS types with the predominance of ITST1 (44.4%) globally. ITS typing seems to be an applicable and useful tool for a better understanding of molecular epidemiology as well as for the dissemination of T. vaginalis clones.

Cyst-theca relationship and phylogenetic positions of Scrippsiella plana sp. nov. and S. spinifera (Peridiniales, Dinophyceae)

Species belonging to the dinophyte genus Scrippsiella are frequently reported in marine waters, but information on their distribution in brackish environments is limited. Here we describe a new species, S. plana, through incubation of non-calcified cysts from sediments collected in the South China Sea and Caspian Sea. The vegetative cells consist of a conical epitheca and a rounded hypotheca with the plate formula of Po, X, 4′, 3a, 7′′, 5C+t, 5S, 5′′′, 2′′′′. It differs from other Scrippsiella species by its flattened body in dorsoventral view and a small first anterior intercalary (1a) plate (half the size of plate 3a). Scrippsiella plana strains from the South China Sea and Caspian Sea share identical internal transcribed spacer (ITS) sequences, and show phenotypic plasticity and local adaptation in growth rate at various salinities, consistent with the environments in which they originated. In addition, two strains of S. spinifera were obtained by incubating ellipsoid cysts with calcareous spines from sediments collected along the Turkish and Hawaiian coast. They also share identical ITS sequences and differ from Duboscquodinium collinii (a parasite of tintinnids) only at two base pair positions (in the ITS2 region). Molecular phylogeny based on ITS and large subunit ribosomal DNA (LSU rDNA) sequences revealed that S. plana was nested within the Calciodinellum (CAL) clade and S. spinifera within the S. trochoidea (STR) clade. The phylogenetic position of ‘Peridinium’ wisconsinense is reported for the first time, which supports multiple transitions of the Peridiniales to freshwater.

The infrageneric taxonomy ofChaerophyllum(Apiaceae) revisited: new evidence from nuclear ribosomal DNA ITS sequences and fruit anatomy

Evolutionary relationships among 65 Chaerophyllum spp. were inferred from nuclear ribosomal DNA internal transcribed spacer (ITS) sequence variation. Thirty-one species, represented by 158 mericarp samples, were analysed for fruit anatomical character variation, employing phylogenetic and phenetic methods to explore their congruence with infrageneric divisions based on molecular data. Phylogenetic trees inferred from molecular data using maximum likelihood (ML) and Bayesian inference (BI) methods corroborated the division of the genus into four sections: Chaerophyllum, Dasypetalon, Physocaulis and Chrysocarpum. From among the newly sequenced species, the Greek endemic C. heldreichii was grouped with section Chaerophyllum, whereas the highly variable Asian C. reflexum–C. villosum complex formed an early-branching paraphyletic assemblage in section Chrysocarpum. The recently described C. karsianum has an identical ITS sequence to C. bulbosum, whereas C. aksekiense was clearly separated from the morphologically similar C. macrospermum. Our study confirmed the postulated synonymy of several species on the basis of morphology, but also demonstrated distant relationships between some morphologically similar species. With the exception of the monotypic section Physocaulis, we were unable to find carpological traits matching sectional divisions. We hypothesize that fruit characters evolved rapidly as a result of diversification of members of the genus in different habitats. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178, 298–313.

First Report of Foliar Blight Caused by Phytophthora capsici on Citrus reticulata Blanco cv. Nian Ju in Guangdong, China.

Citrus reticulata Blanco cv. Nian Ju, an important ornamental plant, is traditionally displayed during the Chinese Spring Festival because its golden fruits are a symbol of auspiciousness. In the spring of 2012, foliar blight was observed on 10 to 30% of the Nian Ju plants at four nurseries in Yangjiang, Guangdong Province, China. Initial symptoms appeared as brown to black foliar lesions, followed by expansion of spots into blight. Some young branches also had necrosis. During frequent rainfall and prolonged wet periods at 22°C to 30°C, white and dense mycelia and sporangia were observed on the infected seedlings. To isolate the causal organism, leaves and stems were cut into sections. Each section included some partial lesion and adjacent asymptomatic tissues. They were surface-disinfested in 1% sodium hypochlorite for 60 s, rinsed three times with sterile water, and placed on V8 juice agar (V8A) at 25°C. After 3 days, 10 isolates were obtained and purified by single-zoospore method. These isolates were identified to species level by sequencing the rRNA internal transcribed spacer (ITS) region. Four representative isolates had an identical ITS sequence (GenBank Accession No. KF750568), which had 99% homology with Phytophthora capsici sequences in GenBank. In addition, all recovered isolates were identical in morphological characteristics. They produced caducous, papillate, and ovoid to ellipsoid sporangia (Length × width = 46.2 ± 7.7 × 23.6 ± 11.3 μm), often with a tapered base. The average length of pedicels was 33.3 ± 4.5 μm. All isolates are A2 mating type. They produced gametangia when paired with an A1 tester of P. capsici isolated from pepper on V8A. Plerotic oospores were 25.3 ± 2.1 μm in diameter. Amphigynous antheridia were 13.6 ± 2.8 μm long and 11.2 ± 0.9 μm wide. Oogonia were 27.4 ± 3.2 μm in diameter. To determine the pathogenicity, three 3-year-old potted C. reticulata cv. Nian Ju plants were sprayed with 20 ml of zoospore suspension from one representative isolate at 105 per ml. Two control plants were sprayed with 20 ml distilled water. All plants were then maintained at 90% relative humidity at 25°C with a 12-h photoperiod. Symptoms similar to those observed in the nurseries developed on all inoculated plants but not on any control plants after 10 days. The pathogenicity test was repeated once and similar results were obtained. P. capsici was recovered from all inoculated plants and resultant isolates had identical morphology to that of the isolates used for inoculation. P. capsici has a relatively broad host range including pumpkins, cucumbers, peppers, beans, squashes, and spinach (1,2). To our knowledge, this is the first report of foliar blight of C. reticulata cv. Nian Ju caused by P. capsici. This study indicates that P. capsici is potentially an important pathogen of C. reticulata cv. Nian Ju plants and further investigations into its epidemiology and development of site-specific integrated management programs for this new disease are warranted.

Morphology and phylogeny of Scrippsiella enormis sp. nov. and S. cf. spinifera (Peridiniales, Dinophyceae) from the China Sea

Gu H., Luo Z., Liu T. and Lan D. 2013. Morphology and phylogeny of Scrippsiella enormis sp. nov. and S. cf. spinifera (Peridiniales, Dinophyceae) from the China Sea. Phycologia 52: 182–190. DOI: 10.2216/12-036.1The genus Scrippsiella contains approximately 20 species that are widespread in coastal and oceanic areas. Classification of Scrippsiella traditionally relied on cyst morphology because the plate pattern was rather conserved. A new species, S. enormis sp. nov. was obtained by incubating a noncalcified cyst from sediments collected in the East China Sea. The vegetative cells consisted of a conical-convex epitheca and a round hypotheca with the plate formula of po, x, 4′, 3a, 7″, 6c (5c+t), 5s, 5‴, 2″″. It differed from other Scrippsiella species by possessing an asymmetrical 1′ and generating noncalcareous, spherical cysts with paratabulation. Phylogenetic analyses based on internal transcribed spacer (ITS) and 5.8S rDNA sequences revealed that S. enormis was nested within the Calciodinellum clade. In addition, two strains of S. cf. spinifera (strains SSFC02, SSFC03) were obtained by incubating calcareous cysts from sediments collected in the South China Sea. They shared identical ITS sequence and formed a sister clade of the S. trochoidea species complex, suggesting little phylogenetic significance of antapical spines in Scrippsiella.

First Report of Flower Anthracnose Caused by Colletotrichum karstii in White Phalaenopsis Orchids in the United States.

In October 2010, a Colletotrichum species was isolated from white Phalaenopsis flowers growing in a greenhouse in San Francisco, CA. This Phalaenopsis is a common commercial orchid hybrid generated mostly likely from Phalaenopsis amabilis and P. aphrodite. The white petals showed anthracnose-like lesions where necrotic tissue is surrounded by a ring of green tissue. The green halo tissues around the necrotic tissue contain functional chloroplasts. One-centimeter disks were cut around the necrotic sites and surface-sterilized with 95% ethanol and 0.6% sodium hypochlorite. The disks were placed on potato dextrose agar (PDA) medium to establish cultures. Pure cultures were obtained by subculturing hyphal tips onto fresh PDA plates. The generated colonies had white aerial mycelia and orange conidial mass. The color of the reverse colony varies between colorless and pale orange. Microscopic observations identified the conidia as cylindrical, straight, and rounded at both ends. In addition, the conidia were approximately 15.0 to 18.0 μm long and 5.0 to 6.5 μm in diameter. These observed morphological features suggested that these isolates possessed the same characteristics as previously described for Colletotrichum karstii, a species considered as part of the C. boninense species complex (1). Four putative independent Colletotrichum isolates were recovered (DED9596, DED9597, DED9598, and DED9599). To confirm the Colletotrichum isolates as the causative pathogen, healthy white Phalaenopsis flowers (five total) in a whole plant were sprayed with a conidial suspension (approximately 1.2 × 106 conidia/ml) of the isolates and incubated at 20°C and 100% relative humidity with cycles of 16 h light and 8 h of darkness. Approximately 1 ml of conidial suspension solution was used for each flower. The plants were watered regularly and flowers were sprayed with sterile double-distilled water daily. As negative controls, five flowers in a whole plant were sprayed with water. Fifteen to twenty days after inoculation, lesions started to form on the petals sprayed with the putative Colletotrichum isolates. All controls remained healthy. The Colletotrichum-inoculated flowers remained alive and did not die as a result of the infection. This same experiment was repeated and the same results were obtained. DNA was extracted from the necrotic regions of the petals infected by the pure cultures of the four isolates and used to sequence the 18S rRNA ITS (internal transcribed spacer) region. All four isolates gave identical ITS sequences. Analysis of the obtained representative sequences (GenBank Accession No. JQ277352) suggested that the isolated pathogen as C. karstii. Using the published ITS data for the C. boninense species complex (1), a phylogenetic tree was generated via the maximum likelihood method. This created tree places the isolates in the same group as C. karstii. This type of C. karstii infection in Phalaenopsis orchid petals was not documented in the U.S. before, although it has been reported in China and Thailand (2). To our knowledge, this is the first report of infection and green island formation caused by C. karstii on orchid flower in the United States.

Species pattern and phylogenetic relationships ofTrichodermastrains in rice fields of Southern Caspian Sea, Iran

As a first step of a project aimed at the identification of potential biocontrol agents of Rhizoctonia solani, the rice sheath blight fungus, we surveyed the biodiversity of the genus Trichoderma based on sequence of the internal transcribed spacer (ITS) 1 and 2 of the ribosomal RNA gene cluster in paddy fields in Mazandaran province, Northern Iran. Amongst the six obtained species of Trichoderma, T. harzianum and T. virens proved to be the most frequent species in this habitat. Sequence alignment and phylogenetic analysis revealed that the T. harzianum isolates can be divided into 14 different ITS genotypes clustering in four groups. Our results are in agreement with previous molecular studies, which also revealed that T. harzianum is a complex species comprising more or less different ITS genotypes. T. virens was not as diverse as T. harzianum and three different genotypes were distinguished which constituted only one cluster. All T. atroviride and T. hamatum strains had identical ITS sequences.

First Report of Oidium longipes as the Causal Agent of Petunia Powdery Mildew in the United Kingdom

In autumn 2009, during a survey of powdery mildews of solanaceous plants in the United Kingdom, petunia (Petunia × hybrida) plants showing typical symptoms of powdery mildew infections were repeatedly collected in East Malling, Rochester, and Sandringham, UK. Leaves, stems, and petals of the collected plants, grown as outdoor ornamentals, were covered by dense, sporulating, white mycelium. Conidia were ellipsoid-cylindrical, measured 20 to 30 × 10 to 15 μm, and were produced in chains. Germ tubes arose from the ends of conidia and terminated in simple, unlobed apices. Some of the conidiophores were extremely long, up to 250 μm, because the second or third cell, or sometimes the foot cell, was up to 105 to 170 μm long. Other conidiophores were shorter, with no exceptionally long cells, but all of them exhibited a few characteristics in common: their width increased from base to top, sometimes enlarging considerably at a particular point of the foot cell, and basal septa were usually located 7 to 30 μm from the point of branching. Hyphal appressoria were nipple shaped. The teleomorph stage was not found. On the basis of these characteristics, the fungus was identified as Oidium longipes, a recently described (4) and little known pathogen of petunia and other solanaceous plants (1,3). To support the identification of this fungus, DNA was extracted from conidia collected with sterile brushes from single leaves collected in Sandringham, East Malling, and Rochester with a Qiagen DNeasy Plant Kit (Qiagen, Hilden, Germany), and the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA was amplified and determined as described in Jankovics et al. (2). The three identical ITS sequences, deposited in GenBank under Accession Nos. HM156495, HM156496, and HM156497, were identical to several ITS sequences of O. longipes, such as AF250777, EU327324, and EU327325. This has also supported that the disease was caused by this species. Herbarium specimens were deposited under the Accession Nos. HAL 2373F, HAL 2374F, and HAL 2375F at the Herbarium of Martin Luther University, Halle, Germany. To our knowledge, this is the first report of O. longipes in the UK.

Identification of Pyrenophora teres f. maculata, Causal Agent of Spot Type Net Blotch of Barley in North Dakota.

Net blotch of barley (Hordeum vulgare L.) caused by the fungus Pyrenophora teres (anamorph Drechslera teres) is found in two forms, net form net blotch (NFNB) and spot form net blotch (SFNB). When inoculated on susceptible varieties, P. teres f. teres produces lesions with a characteristic net-like pattern surrounded by necrosis or chlorosis (NFNB), whereas P. teres f. maculata produces lesions consisting of spots surrounded by necrosis or chlorosis (SFNB). Recently, epidemics of SFNB have occurred throughout the world (4). Currently, net blotch is a significant foliar disease of barley in the North Dakota-Northwestern Minnesota agricultural region, a leading barley-production area. Diseased barley leaf tissue was collected annually from 2004 to 2008 in Fargo and Langdon, ND. Diseased leaves were incubated to promote sporulation. Ten single-spore isolates of P. teres collected from each location each year were tested for virulence by inoculation on 20 commonly used barley net blotch differential lines. Among the 100 isolates collected, one isolate collected in Fargo in 2006 (FGOH06Pt-8) and one isolate collected in Langdon in 2008 (LDNH08Pt-4) were identified as P. teres f. maculata due to their induction of spot-type lesions across the differential set. Conidial morphology of the two isolates was similar to P. teres f. teres isolates. A pathogenicity test of all isolates was performed on regional barley cvs. Tradition, Robust, and Lacey as well as barley lines Rika and Kombar (1) as previously described (3). The net form isolate 0-1 and spot form isolate DEN2.6 (obtained from B. Steffenson, University of Minnesota) were used as controls. The P. teres f. teres isolate 0-1 produced typical net type symptoms on all barley lines except the resistant line Rika, in which only small, dark spots were observed. DEN2.6 produced pin-point spot-like lesions with an extensive yellow halo on Robust, Lacey, Rika, and Kombar, but without chlorosis on Tradition. The two newly identified isolates induced elliptical spot-type lesions measuring 3 × 6 mm, larger than those produced by P. teres f. maculata isolate DEN 2.6, suggesting a higher level of virulence. We constructed a neighbor-joining phylogenetic tree using ClustalW2 ( http://www.ebi.ac.uk/ ) based on sequence identity of the internal transcribed spacer (ITS) region from 0-1 (GenBank No. GU014819), DEN2.6 (GenBank No. GU014820), FGOH06Pt-8 (GenBank No. GU014821), and LDNH08Pt-4 (GenBank No. GU014822) as well as P. teres f. maculata, P. teres f. teres, and P. tritici-repentis (causal agent of tan spot of wheat) accessions obtained from GenBank (2). All P. teres isolates clustered together and were clearly separated from the P. tritici-repentis cluster. Isolates FGOH06Pt-8 and LDNH08Pt-4 had identical ITS sequences and differed from DEN2.6 by only a single nucleotide. To our knowledge, this is the first report of P. teres f. maculata in North Dakota. Resistance to SFNB should now be considered in local barley breeding programs and cultivar releases. Reference: (1) M. Abu Qamar. Theor. Appl. Genet. 117:1261, 2008. (2) R. M. Andrie et al. Fungal Genet. Biol. 45:363, 2008. (3) Z. Lai et al. Fungal Genet. Biol. 44:323, 2007. (4) M. S. McLean et al. Crop Pasture Sci. 60:303, 2009.

A molecular phylogeny of the subtribe Pterostylidinae (Orchidaceae): resolving the taxonomic confusion

In the past decade, two major classification schemes of the subtribe Pterostylidinae have resulted in taxonomic confusion because a single well known genus was split into a large number of new genera and subgenera, many of which are difficult to discriminate accurately. These classifications have not been accepted widely among systematists because of poor phylogenetic support for several genera. Analyses of the internal transcribed spacer (ITS) region of nuclear rDNA in a large number of species and samples facilitate further clarification of the relationships within the Pterostylidinae. The phylogenetic trees were reconstructed using parsimony and Bayesian methods. These phylogenetic trees indicate that subtribe Pterostylidinae is monophyletic, and support the concept of a single genus, Pterostylis R.Br. sensu lato within the Pterostylidinae. Two clades representing subgenera correlate with the morphology of the lateral sepals, whereas several of the previously erected genera consistently have poor support. The proposed subgenera are divided further into 10 sections. Several closely related taxa with identical ITS sequences require further scrutiny by population-level molecular techniques to determine their taxonomic status.

First Report of Phytophthora sansomeana Causing Wilting and Stunting on Corn in Ohio.

During the spring of 2004, corn seedlings with symptoms of wilting and stunting were observed in corn fields with emergence problems in Madison and Brown counties, Ohio. Phytophthora isolates were recovered from sections of root tissue of diseased seedlings placed on dilute V8 media amended with pentachloronitrobenzene, iprodione, benlate, neomycin sulfate, and chloramphenicol. Colonies were rosaceous on potato dextrose agar, with a growth rate of 5 mm per day. Homothallic isolates with paragynous antheridia were observed on lima bean agar (LBA); oogonia were 35 to 50 μm in diameter. Sporangia were ovoid to obpyriform, nonpapillate, with an average size of 49 × 30 μm. Pathogenicity was tested on corn seeds using a petri dish assay with 3-day-old cultures on LBA and a sand-cornmeal cup test amended with inoculum from 7-day-old cultures on LBA (1). After 1 week in the petri dish assay, the seeds failed to germinate completely and were covered with white, fungal-like, aerial mycelia and the pathogen was recovered from brown discolored radicle roots. In the cup assay, 2-week-old seedlings developed the same symptoms observed in the field; the pathogen was also isolated from brown discolored roots. In both assays, no symptoms developed in the noninoculated controls. Both pathogenicity tests were repeated two times. Genomic DNA was extracted from mycelia of two isolates and the internal transcribed spacer (ITS) region was amplified and sequenced using ITS6/ITS4 primers (2). Both isolates had identical ITS sequences (GenBank Accession No. GQ853880). A BLAST search of the NCBI database showed 100% homology with the sequence of the haplotype isolate of Phytophthora sansomeana (Accession No. EU925375). P. sansomeana is a new species characterized principally by a large oogonial diameter (37 to 45 μm), rapid growth rate (7 to 10 mm/day), and an ITS sequence falling in Cooke's clade 8 (4). Pathogenicity tests, morphological characteristics, and the ITS sequence analysis indicate that P. samsomena is the causal agent of the symptoms observed on corn seedlings. P. sansomeana has been reported as a pathogen of soybean in Indiana, Douglas-fir in Oregon, and weeds in alfalfa fields in New York (4). To our knowledge, this is the first report of P. sansomeana infecting corn in Ohio, albeit other isolates have previously been recovered from soybean in the state. There are four previous reports of Phytophthora spp. affecting corn in the United States and Mexico (3). Crop rotation will have little effect in management of this pathogen since corn and soybean are produced in the same fields continuously throughout the state.

Morphological and molecular phylogenetic analysis of two Saprolegnia sp. ( Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp ( Carassius auratus gibelio Bloch) and BMY isolated from zebra fish ( Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates underwent repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. Our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (I) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species.

Molecular and Morphological Study of Species-Level Questions Within the Dragonfly Genus <I>Sympetrum</I> (Odonata: Libellulidae)

This study combines morphological and molecular data to address several questions of species validity within the dragonfly genus Sympetrum. We compared morphological characters (genitalia and other putatively diagnostic characters) and DNA sequences from mitochondrial cytochrome oxidase I (COI) and nuclear internal transcribed spacer (ITS) regions between these disputed taxa and their close relatives. Specimens of Sympetrum nigrescens Lucas shared COI haplotypes with Sympetrum striolatum (Charpentier), and no morphological characters consistently diagnosed S. nigrescens, which therefore becomes a junior synonym of S. striolatum. Similarly, Sympetrum occidentale Bartenev shared identical COI and ITS sequences with Sympetrum semicinctum (Say), and the supposed diagnostic morphological characters overlapped with the intraspecific variation within S. semicinctum. Sympetrum occidentale becomes a junior synonym of S. semicinctum. In a third case, the genetic distance between Sympetrum signiferum Cannings & Garrison and Sympetrum vicinum (Hagen) was lower than that found between most undisputed species. However, the morphological characters that distinguish S. signiferum from S. vicinum were distinct and consistent, and they supported the retention of S. signiferum as a valid species. In the fourth case, neither morphological nor genetic data were able to distinguish Sympetrum janeae Carle consistently from Sympetrum internum Montgomery, or Sympetrum rubicundulum (Say); in addition, genetic distances between individuals of S. internum and S. rubicundulum were small or nonexistent. Further studies are necessary to test the species status of S. janeae and its close relatives.

Comparative studies on morphology, ITS sequence and protein profile of Alexandrium tamarense and A. catenella isolated from the China Sea

The Alexandrium tamarense species complex is a closely related cosmopolitan toxigenic group of morphology-based species, including A. tamarense, A. catenella and A. fundyense. This study investigated the morphology, internal transcribed spacer (ITS) sequence and protein profile of A. tamarense and A. catenella grown in the same culture conditions using a combination of scanning electronic microscope (SEM), molecular and proteomic approaches. The results showed that all Alexandrium strains had the plate formula of Po, 4′, 6″, 6C, 8S, 5″′, 2″″. The ventral pore, a key conventional morphological feature to discriminate A. tamarense and A. catenella, was usually present in the first apical plate of ten A. tamarense strains, however, it was found to be absent in some cells of one Alexandrium strain, ATGX01. A. tamarense and A. catenella shared an identical ITS sequence with a minor variation at intraspecific level. Protein profiles of A. catenella DH01 and A. tamarense DH01, isolated from the same region of the East China Sea, showed no significant difference, the similarity of protein profiles of the two species reached 99% with a few proteins unique to one or the other. The present results suggest that the ventral pore is not a consistent morphological feature in the Alexandrium genus, and that A. tamarense and A. catenella are conspecific and should be redesignated to one species.

The Identification of Vahlkampfiid Amoebae by ITS Sequencing

We have determined the internal transcribed spacer (ITS) sequences (including the 5.8S ribosomal DNA) of 30 strains of 14 species belonging to eight vahlkampfiid genera. Each previously described species has a specific ITS sequence, except for Tetramitus aberdonicus, Tetramitus thorntoni, and Tetramitus jugosus, which have identical ITS sequences. The latter three may therefore constitute a single species despite their apparent phenotypic differences. The ITS sequence appears to be conserved within a species. The species Willaertia magna appears to be ubiquitous. The 5.8S rDNA sequences of Singhamoeba horticola and Learamoeba waccamwensis indicate that they do not represent different genera, but both belong to the genus Tetramitus. The ITS sequences of 16 undescribed vahlkampfiid isolates were determined. Based on these sequences, seven isolates were identified as belonging to described species, while nine probably represent seven new species. Five of these presumed new species belong to the genus Tetramitus, and one each to the genera Vahlkampfia and Paravahlkampfia.