Internal Transcribed Spacer Sequence Data Research Articles

Utility of ITS region sequence and structure for molecular identification of Tilia species from Hyrcanian forests, Iran

Nucleotide sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA and 5.8S gene were used to infer the phylogeny of Tilia species (represented by 13 distinct populations) growing in different geographical areas of Hyrcanian forests in northern Iran. Four well-supported lineages were revealed, including that of a new species, T. hyrcana, with stellate trichomes on both sides of the leaves and petiole. T. hyrcana is a well-supported cladospecies, with the ITS sequence and secondary structure following the diagnosable phylogenetic species concept, and is also characterized by a distinct morphology. A controversial species is Tilia rubra subsp. caucasica, with three different forms—an assemblage of taxa characterized by a lack of stellate trichomes on leaves—while Tilia begonifolia is distinguished by stellate trichomes on the underside of both leaves and petiole. The fourth lineage group, T. dastyla, is characterized by the presence of trichomes on the style. A single taxon found in the west of the Hyrcanian forest region is similar to T. begonifolia, but due to the former being located in a distinct group, a reassessment of the diagnostic morphology is recommended. ITS sequence data also suggested a closer relationship between T. rubra and T. begonifolia. Compensatory base change analysis was not strong enough to separate individual species within the Tilia genus. In general, the study supports the utility of ITS sequence data and secondary structure as accessory taxonomic characteristics with which to help clarify the systematics of the Tilia genus.

First Report of Pilidiella granati Causing Dieback and Fruit Rot of Pomegranate (Punica granatum) in Iran.

Iran is the largest producer of pomegranate (Punica granatum) in the world, with more than 60,000 ha currently in production. In the spring of 2011, a decline and dieback of young pomegranate trees (7 to 10 years old) were observed in the Kheir area of Fars Province. Dieback and twig blight developed toward the lower part of the stem, resulting in death of aerial tree parts and growing suckers from roots. Surface-disinfected tissues of diseased plants were plated on potato dextrose agar (PDA) and malt extract agar media. Isolates were separated into two groups that had either pale green or white aerial mycelia and sporulated after 5 to 7 days at 25°C. Pycnidia were globose and black with thin, membranous, pseudoparenchymatic walls, 80 to 140 μm in diameter. Conidia were hyaline, one-celled, elongate to fusiform, straight, and 11 to 17 × 4 to 6 μm (average 14 × 4.7 μm). Cardinal minimum growth temperatures were 8 to 10°C, optimum at 27 to 30°C, and maximum at 35°C. Radial growth rate at 30°C was 8 to 9 mm per day. Representative isolates were deposited in the CBS-KNAW Fungal Biodiversity Centre, the Netherlands (CPC 19625 = CBS 130974 and CPC 19626 = CBS 130975; GenBank JN815312 and JN815313, respectively). Genomic DNA was extracted with the UltraClean Microbial DNA Isolation Kit (MoBio Laboratories, Inc., Solana Beach, CA) and the internal transcribed spacer (ITS) region of the nrDNA operon of two isolates were sequenced as described previously (1). On the basis of morphology (3), the causal organism was identified as Pilidiella granati Sacc. This identification was corroborated by the ITS sequence data, which was identical for both colony types to GenBank HQ166057 (identities = 614 of 614 [100%]). Pathogenicity tests were conducted using two representative isolates from each group on 5-month-old P. granatum trees with 10 replicates under greenhouse conditions; 5-mm mycelial plugs from the edge of 7-day-old colonies on PDA were placed under the bark of twig wounds. Uncolonized PDA plugs were used as noninoculated controls. Pathogenicity was also tested on nonwounded fruit by placing colonized 5-mm-diameter mycelial plugs on surface-disinfected pomegranate fruits; noncolonized PDA plugs were used as controls. All treated fruit were placed in plastic bags and maintained at 25°C for 10 days. Isolates were found to be pathogenic on twigs after 2 months, giving rise to brown lesions that were 2 to 5 cm long. No lesions were observed on the controls. Furthermore, the fungus was reisolated from all infected tissues, satisfying Koch's postulates. On pomegranate fruit, the fungus colonized the fruit after 5 to 8 days, followed by the appearance of fruit rot symptoms leading to the formation of abundant pycnidia covering the skin after 10 days. No decay was observed in control inoculations. Pilidiella granati has previously been reported as a pathogen of P. granatum fruit from Europe, Asia, and the United States (2). To our knowledge, this is the first report of this pathogen causing dieback and fruit rot of pomegranate in Iran.

First Report of Leaf Spot Disease on Schisandra chinensis Caused by Phoma glomerata in China.

Schisandra chinensis (Turcz.) Baill is a perennial liana belonging to the Schisandra genus of the family Magnoliaceae, which is cultivated in China as an important medicinal plant. In the summer of 2008, we observed a previously unknown foliar disease on the schisandras in Jingyu and Antu counties and the cities of Ji'an and Hunchun in Jilin Province. Symptoms appeared on the apex, margin, and center of leaves. The infection initially manifested as pale brown, small, necrotic spots on the leaves. Subsequently, these lesions became grayish brown in the center and dark brown with slight protuberances at the margins. Finally, these lesions developed concentric rings with a clear boundary separating them from the healthy tissue, were round to elliptical or irregular in shape, and had a diameter of 3 to 5 mm. In severely infected leaves, these spots eventually covered the entire leaf. Black spots (pycnidia) were produced on the infected leaf tissues in a humid environment. Fungus from infected leaf tissues was isolated on potato dextrose agar. The cultures were initially pale brown and turned dark green with age. Embedded pycnidia were generally formed after 5 days. The pycnidia were agglutinating, globose to subglobose, and measured 60.0 to 212.0 × 33.6 to 268.0 μm. Abundant conidia (4.06 to 7.2 × 1.65 to 3.53 μm) exhibiting zero to three oil droplets were produced by an 8-day-old colony; these conidia were ovoid or ellipsoidal, colorless, and aseptate; they were similar to conidia of Phoma glomerata. The internal transcribed spacer (ITS) sequence of rDNA of the isolated pathogenic strain (PG11; GenBank Accession No. GU724511) had 100% identity to P. glomerata (GenBank Accession No. HM769279). Therefore, the pathogen was identified as P. glomerata (Corda) Wollenw. & Hochapfel on the basis of morphology and ITS sequence data. To validate Koch's postulates, schisandra leaves were spray inoculated with a 2.5 × 105 conidia/ml suspension of the isolated pathogen. An equal number of healthy plants were inoculated with sterile water (control). After inoculation, 10 plants were covered with plastic bags for 3 days and maintained in a growth chamber at 25°C. After 8 days, all inoculated plants showed symptoms identical to those observed on the schisandra leaves infected in the field, whereas the controls did not show any symptoms. Reisolation of the fungi from lesions of inoculated leaves confirmed that the causal agent was P. glomerata. Diseases caused by P. glomerata have been reported on some plants (1,2). However, to our knowledge, this is the first report of leaf spot disease caused by P. glomerata on S. chinensis in China as well as in the world.

Significant population genetic structure detected for a new and highly restricted species of Atriplex (Chenopodiaceae) from Western Australia, and implications for conservation management

Atriplex sp. Yeelirrie Station (L. Trotter & A. Douglas LCH 25025) is a highly restricted, potentially new species of saltbush, known from only two sites ~30 km apart in central Western Australia. Knowledge of genetic structure within the species is required to inform conservation strategies as both populations occur within a palaeovalley that contains significant near-surface uranium mineralisation. We investigate the structure of genetic variation within populations and subpopulations of this taxon using nuclear microsatellites. Internal transcribed spacer sequence data places this new taxon within a clade of polyploid Atriplex species, and the maximum number of alleles per locus suggests it is hexaploid. The two populations possessed similar levels of genetic diversity, but exhibited a surprising level of genetic differentiation given their proximity. Significant isolation by distance over scales of less than 5 km suggests dispersal is highly restricted. In addition, the proportion of variation between the populations (12%) is similar to that among A. nummularia populations sampled at a continent-wide scale (several thousand kilometres), and only marginally less than that between distinct A. nummularia subspecies. Additional work is required to further clarify the exact taxonomic status of the two populations. We propose management recommendations for this potentially new species in light of its highly structured genetic variation.

Molecular Systematic Study of Chrysosplenium Series Pilosa (Saxifragaceae) in Korea

To address systematic issues pertaining to series Pilosa of Korean Chrysosplenium, the phylogenetic relationships of the 35 accessions of Korean Chrysosplenium taxa were examined using the DNA sequence data obtained from the nuclear ribosomal internal transcribed spacer (ITS) regions and the psbK-I gene of the plastid genome. The results from both ITS and psbK-I sequence data indicated that Chrysosplenium flaviflorum formed a well-supported clade that merits species status of the taxon. The ITS phylogenetic trees detected two distinct lineages in Chrysosplenium pilosum var. fulvum. Geographically, one lineage was widely distributed throughout the country whereas the other was confined to Gyeonggi and Gangwon provinces. The plants sampled from Jeju Island, which have been recognized as C. pilosum var. sphaerospermum [C. hallaisanense] exhibited no DNA sequence feature segregating them from C. pilosum var. fulvum [C. barbatum] distributed in the mainland area of Korea. The plastid DNA sequence data was not fully in concordance with the nuclear ribosomal ITS data and the current taxa delimitation. These results suggest incomplete lineage sorting or that plastid genome capture may have occurred during the evolution of some taxa examined in the present study.

Phylogenetic relationships in Ranunculus species (Ranunculaceae) based on nrDNA ITS and cpDNA trnL-F sequences

The genus Ranunculus L., with a worldwide distribution, is the largest member of the Ranunculaceae. Here, nuclear ribosomal internal transcribed spacer (ITS) sequence data and chloroplast trnLF sequence data were used to analyze phylogenetic relationships among members of the annual and perennial (Group Praemorsa, Group Rhizomatosa, Group Grumosa and Group non-Grumosa) species of Ranunculus in Iran. In the strict consensus tree of nrDNA ITS sequence analyses, seven sub-clades were described, based on morphological, karyological, palynological, and ecological features. Within each clade, there were species belonging to more than one Group, and species of a single Group may fall into different clades, revealing that a classification based on underground system characters does not show natural interspecific relationships and must be revised. This is also apparent from trnL-F sequence analyses. Based on our results, the Praemorsa and Rhizomatosa Groups can be merged. The ITS sequence data show interspecific relationships more clearly than trnL-F sequence data. © 2011 Progress in Biological Sciences, Vol. 1, No.1, 41-47.

First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua and Agrostis stolonifera in New Jersey.

Waitea circinata var. circinata was first reported as the causal agent of brown ring patch on annual bluegrass (Poa annua L.) in the United States in 2007 (2). In early April to mid-June of 2009, circular to irregularly shaped yellow rings resembling symptoms of this disease were observed on an annual bluegrass putting green at Rutgers University in North Brunswick, NJ. Severely infected foliage eventually turned brown as the disease progressed. During the same time period, similar disease symptoms were observed on creeping bentgrass (Agrostis stolonifera L.) from a golf course in Bedminster Township, NJ. The disease reappeared in both locations in April of 2010. Five additional samples with similar symptoms on creeping bentgrass and annual bluegrass were received at Rutgers Diagnostic Laboratory from Paramus, Madison, Allamuchy, and Farmingdale, NJ between late April and early May of 2010. Portions of diseased leaf and sheath tissue that displayed symptoms of the disease were disinfested for 1 min in 0.5% NaOCl, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA) amended with 50 mg/liter of streptomycin sulfate. At the first sign of fungal growth, single hyphal tips were transferred to PDA. After 1 week at 25°C, white-to-orange mycelial colonies formed in culture and eventually turned brown with age. Minute sclerotia (≤3 mm), which followed the same color development pattern, formed within 10 days. These features are consistent with those described of W. circinata var. circinata (2,3). The internal transcribed spacer (ITS) region of the ribosomal RNA gene was amplified using primer pair ITS1/ITS4 and sequenced with ITS4 (GenBank Accession Nos. HQ166065 to HQ166071). BLASTn analysis of the ITS sequences showed a 99 to 100% similarity to W. circinata var. circinata sequences deposited in GenBank (1,2). Pathogenicity tests were conducted in 2010 using 6-week-old creeping bentgrass seedlings cv. Declaration inoculated with colonized oat grain that had been autoclaved and then infested with the Bedminster Township isolate. Eight colonized oat grains were uniformly spread around the crowns of seedlings grown in 10-cm-diameter pots. Control plants were treated with autoclaved grain. Plants were incubated at 25°C and high humidity maintained by misting the plants three times per day. Within 3 days postinoculation, foliage near infested grain turned chlorotic. All foliage in pots became completely blighted and spherical orange-brown sclerotia were observed on leaf sheaths by the eighth day. W. circinata var. circinata was consistently reisolated from inoculated plants (as confirmed by isolate morphology and ITS sequencing) but not from control plants. The ITS sequence data, morphological characters of the isolates, and pathogenicity tests demonstrate that W. circinata var. circinata is present in New Jersey. To our knowledge, this is the first report of W. circinata var. circinata infecting turfgrass in New Jersey.

Taxonomy of Desmodesmus serratus (Chlorophyceae, Chlorophyta) and related taxa on the basis of morphological and DNA sequence data

Fawley M.W., Fawley K.P. and Hegewald E. 2011. Taxonomy of Desmodesmus serratus (Chlorophyceae, Chlorophyta) and related taxa on the basis of morphological and DNA sequence data. Phycologia 50: 23–56. DOI: 10.2216/10-16.1The green alga Desmodesmus serratus is a common small Desmodesmus species. The highly variable morphology among strains and natural populations suggested that this taxon was in need of revision using a combination of molecular and morphological characters. Thirty-six isolates that were assigned to D. serratus on the basis of morphology or that were known to be closely related to D. serratus on the basis of the results of DNA sequence analysis were examined. These isolates were evaluated by light microscopy, electron microscopy, and phylogenetic analysis of DNA sequences from the nuclear ribosomal internal transcribed spacer (ITS) regions, nuclear 5.8S ribosomal DNA, and the plastid rbcL gene. Results of the analyses of ITS and rbcL sequence data were congruent. On the basis of the results of these analyses, we propose the new species Desmodesmus itascaensis, Desmodesmus perdix, Desmodesmus serratoides, Desmodesmus pseudoserratus, and Desmodesmus santosii. Two of these species, D. perdix and D. pseudoserratus, possess morphologies identical to that of D. serratus. However, these species were all clearly separated on the basis of the results of DNA sequence analysis. The new species as well as D. serratus form a monophyletic lineage that is allied with the small, spineless species, Desmodesmus costato-granulatus, Desmodesmus lunatus, Desmodesmus elegans, Desmodesmus ultrasquamatus, and Desmodesmus regularis.

A new species ofMelampsorarust onSalix elbursensisfrom Iran

SummaryA rust fungus was found causing stem cankers on 1‐ to 5‐year‐old stems ofSalix elbursensisin the north west of Iran. The rust also forms uredinia on leaves and flowers of the host willow. Light and scanning electron microscopy revealed that the new rust is morphologically distinct from severalMelampsoraspecies occurring on the willows taxonomically close toS. elbursensis, but indistinguishable fromMelampsora larici‐epitea. Examination of the internal transcribed spacer (ITS) region of the ribosomal DNA suggested that the rust fungus is phylogenetically close toMelampsora allii‐populinaandMelampsora pruinosaeonPopulusspp. Based on both the morphological characteristics and the ITS sequence data, the rust is described as a new species –Melampsora iranicasp. nov.

First Report of Marasmiellus mesosporus Causing Marasmiellus Blight on Seashore Paspalum.

Seashore paspalum (Paspalum vaginatum Sw.) is a newly cultivated C4 turfgrass that has exceptional salinity tolerance and is highly suited for use on golf courses in coastal areas. In October 2008 and June 2009, circular patches of blighted seashore paspalum ranging from 30 cm to >3 m in diameter were observed in fairways, tees, and roughs established with 'Supreme' seashore paspalum at Roco Ki Golf Club in Macao, Dominican Republic. Affected patches were initially chlorotic followed by reddish brown necrosis of leaves and leaf sheaths. Reddish brown-to-gray lesions were also observed on leaf sheaths during the early stages of necrosis. During periods of wet or humid weather from June through October, basidiocarps were produced on necrotic plant tissue and identified as Marasmiellus mesosporus Singer (2). Three isolates were obtained by plating symptomatic leaf sheaths that were surface sterilized with a 0.5% NaOCl solution on potato dextrose agar amended with 50 ppm each of streptomycin, chloramphenicol, and tetracycline (PDA+++). Sequences of the internal transcribed spacer (ITS) region of rDNA, obtained from these three isolates and three stipes of basidiocarps, were identical to each other and 99% similar to a M. mesosporus sequence deposited in the NCBI database (Accession No. AB517375). To confirm pathogenicity, a M. mesosporus isolate obtained from symptomatic plant tissue was inoculated onto 6-week-old P. vaginatum ('Seaspray') planted (0.5 mg seed/cm2) in 10-cm-diameter pots containing a mixture of 80% sand and 20% reed sedge peat. Two weeks prior to inoculation, the isolate was grown on a sterilized mixture of 100 cm3 of rye grain, 4.9 ml of CaCO3, and 100 ml of water. Infested grains were placed 0.5 cm below the soil surface for inoculation. Pots were inoculated with five infested grains or five sterilized, uninfested grains with three replications of each treatment. After inoculation, pots were placed in a growth chamber with a 12-h photoperiod set to 30°C during the day and 26°C at night. Approximately 20% of plants in inoculated pots were necrotic 7 days postinoculation and this increased to 75% by 21 days postinoculation. Diseased plants in inoculated pots exhibited symptoms similar to those observed in the field. Leaves were initially chlorotic with brown lesions on lower leaf sheaths and eventually turned necrotic, reddish brown, and collapsed. Pots receiving uninfested grains were healthy and showed no symptoms on all rating dates. At 21 days postinoculation, basidiocarps were observed emerging from three colonized plants at the base of the oldest leaf sheath near the crown. Three reisolations were made on PDA+++ from stem lesions surface sterilized with a 0.5% NaOCl solution. All reisolations were confirmed as M. mesosporus by culture morphology and ITS sequence data. M. mesosporus was previously reported causing disease on American beachgrass (Ammophila breviligulata Fernald) in North Carolina (1) and recently in Japan (3). The pathogen was initially placed in the genus Marasmius and reported as the cause of the disease Marasmius blight (1). Subsequent morphological observation found that the pathogen belonged in the genus Marasmiellus (2). To our knowledge, this is the first report of M. mesosporus causing Marasmiellus blight on seashore paspalum, a high-amenity turfgrass.

Phylogenetics and biogeography of the parasitic genusThesiumL. (Santalaceae), with an emphasis on the Cape of South Africa

Thesium is a large genus of parasitic shrubs belonging to tribe Thesieae of Santalaceae. It has a principally Old World distribution, with the greatest diversity being found in southern Africa. Little is known about the relationships within Thesium or its relationships with its closest relatives. In this article, we present a first estimate of species-level phylogenetic relationships in Thesium based on internal transcribed spacer (ITS) and trnL–trnF sequence data, and use this to explore the biogeographical history of the group. One hundred and four samples representing 72 Thesium spp. were included in a phylogenetic analysis. Plastid and combined data resolve Thesium as paraphyletic relative to Thesidium and Austroamericium with high posterior probability and bootstrap support. ITS sequence data place Thesidium as sister to a large Thesium clade, but with weak support. Ancestral range reconstruction and dating analysis suggest a southern African origin for the group, with a crown age of 39.1 ± 11.9 Mya, followed by dispersal into Europe and South America. A large clade of Cape species split in the Miocene from a clade comprising tropical species (25.5 ± 7.3 Mya) with the diversification of extant species beginning at 16.7 ± 6.3 Mya. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162, 435–452. ADDITIONAL KEYWORDS: hemiparasite – parasite – southern Africa – trans-Atlantic dispersal.

First report of corm rot disease caused bySclerotium rolfsiiin banana

A corm rot disease was observed for the first time in banana. The disease was found to occur in the majority of commercial cultivars grown in different banana-growing states of India. The incidence of the disease was up to 50% and found to occur at an altitude up to 3000 m asl. The pathogen was identified as Sclerotium rolfsii based on morphological characters and rDNA internal transcribed spacer sequence data. Bananas (Musa spp.) are the world's fourth most important global food crop grown in more than 120 countries (Molina and Valmayor 1999) and are the staple food for more than 400 million people (Swennen et al. 1995). India is the world's largest banana producer and 26 million tonnes are produced annually from 709 000 ha. However, various pests and disease problems, particularly Fusarium wilt, cause significant losses (Ploetz 2005). During a disease survey in different banana-growing regions of India in March 2005, a new wilt-like disease was observed in cv. Rasthali (Silk-AAB) in the Tirukattupalli area of Thanjavur district in Tamil Nadu, India. The important symptoms observed were yellowing of leaves from base to apex, extensive pseudostem sheath rot with profuse mycelial growth on the pseudostem (Fig. 1A, B). The colour of the rotted portion was yellowish-red to reddish-brown (Fig. 1B, C), with numerous brown sclerotia present. Splitting of the basal portion or the pseudostem sheath was observed, and the bottom leaves had desiccated. The infected plant emanated a mushroom odour. The inner tissues of the corm were spongy and colonised extensively with white mycelium (Fig. 2A). At higher altitudes of 2000-3000 m asl, the cortical portion of the corm was completely converted into a mass of fibre (like coconut fibre) with profuse growth of fungal mycelium (Fig. 2B). In some plants, the infection extended up to half of the medulla region. The emerging peeper was necrotic and colonised extensively with white mycelium. In some cases the mycelial growth was seen on the growing tip of the young bud and resulted in reddish brown necrosis. The symptoms were observed in all stages of plant growth. Samples of the diseased plant parts were collected and brought to the laboratory and cultured on potato dextrose agar (PDA). White mycelial growth was observed within a week. Subsequently, small round dark-brown sclerotia developed in

First Report of Pyrenophora seminiperda in Turkey and Greece.

Pyrenophora seminiperda (Brittleb. & D.B. Adam) Shoemaker (anamorph Drechslera campanulata (Lév.) B. Sutton) is a generalist seed pathogen that can cause high mortality in the seed banks of annual and perennial grasses and a minor leaf spot disease. Its current reported distribution is mainly temperate grasslands, deserts, and winter cereal-growing regions in Argentina, Australia, Canada, Egypt, New Zealand, South Africa, and the United States (2). P. seminiperda was originally described in France in the mid-1800s, but there are no recent reports from eastern Europe or Asia (2). In May of 2008, we observed what appeared to be P. seminiperda on seeds from seed bank samples collected in Turkey. Evidence of disease was observed as macroscopic black stromata protruding from the seed. The characteristic club-shaped stromata were collected from a Taeniatherum caput-medusae seed near Pamukkale, Turkey and six Bromus tectorum seeds in Love Valley near Goreme, Turkey. An additional collection from a single undispersed B. tectorum seed was obtained from Perissa, Greece. Identity of the pathogen was tentatively established by evaluating morphological characteristics for nine isolates in V8 agar culture. After 4 days of incubation at 20°C with a 12-h photoperiod, the cultures produced white mycelium. Following wounding, the mycelium produced black, club-shaped stromata (2 to 8 × 0.4 to 0.9 mm) in a radial pattern. These produced branched conidiophores bearing crescent-shaped, multicellular conidia (79 to 125 μm long). These attributes are consistent with those of the anamorph of P. seminiperda as described by Shoemaker (4) and Campbell et al. (1). The teleomorph was not observed. The identity of the isolates as P. seminiperda was confirmed with ribosomal DNA internal transcribed spacer (ITS) genetic sequencing analysis. ITS sequences obtained were identical to sequences for North American haplotypes of this species. Four of the Love Valley isolates, (representative isolate: GQ168725, BPI 879142, NRRL 54032) matched the HTA haplotype (GQ168724), while the other four (representative isolate: GQ168736, BPI 879143, NRRL 54033) matched the HTJ haplotype (GQ168735). The isolate from Perissa, Greece (GQ168728, BPI 879144, NRRL 54034) matched the HTC haplotype (GQ168727). Pathogenicity of several Love Valley isolates was confirmed by producing conidia in culture, dusting nondormant B. tectorum seeds with 0.003 g of conidial inoculum per 50 seeds, and incubating for 14 days at 10/20°C with a 12-h photoperiod. Stromata developed on >90% of inoculated seeds and mortality as high as 34% was observed. Morphological similarities combined with ITS sequence data provide conclusive evidence that P. seminiperda occurs in Turkey and Greece. The discovery of this pathogen in these countries indicates that it may be widespread in Eurasia and that it could have arrived in North America on seeds of B. tectorum (3) rather than representing a novel pathogen for this important weed in its North American range.

A preliminary molecular historical biogeography of Caragana (Leguminosae) based on ITS and trnL-F DNA sequence data

The nrDNA internal transcribed spacer (ITS) and cpDNA trnL-F internal spacer (IGS) sequence data of Caragana eight species and one outgroup Halimodendron halodendron, was employed to reconstruct a phylogenetic tree, then the area relationship was analyzed by means of component analysis (CA), Brooks parsimony analysis (BPA), and dispersal-vicariance analysis (DIVA), six areas were selected from two divided distributions of East Asia and Tethys in Caragana. The phylogenetic tree indicated that there were three distinctive groups, which were attributed to some morphological characters, first with pinnate foliage and deciduous rachis, second with palmate foliage and persistent sclerotic stick rachis, and third with pinnate foliage and persistent sclerotic stick rachis. The results of CA and BPA illustrated general area relationships. An explicit area relationship should be Altai-Sayan, Far East NE China and North China (Hengduan Mountains). DIVA recognized several explanatory vicariance and dispersal events. As the scenario of Caragana distribution pattern, it looks like the vicariance versus dispersal plays more important role. In vicaraince, there are not only the isolated far-distance vicariance, but also the adjacent vicariance especially a vicariance between Hengduan Mountains and North China.

Phylogenetic relationships in the tribes Schizopetaleae and Thelypodieae (Brassicaceae) based on nuclear ribosomal ITS region and plastidndhF DNA sequences

Sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region and plastid gene ndhF of 95 species, represented by 147 accessions, were used to determine the tribal limits, monophyly status, and phylogenetic intra-tribal relationships of genera within the New World tribe Schizopetaleae (formerly Thelypodieae; Brassicaceae). Maximum parsimony, Bayesian, and maximum-likelihood analyses all support the separate recognition of three distinct and generally well-supported clades. The largest corresponds to the North–South American Thelypodieae (27 genera: Catadysia , Caulanthus , Chaunanthus , Chilocardamum , Chlorocrambe , Coelophragmus , Dictyophragmus , Dryopetalon , Englerocharis , Hesperidanthus , Mostacillastrum , Neuontobotrys , Polypsecadium , Pringlea , Pterygiosperma , Romanschulzia , Sibara , Sibaropsis , Stanleya , Streptanthella , Streptanthus , Thelypodiopsis , Thelypodium , Thysanocarpus , Warea , Weberbauera , and Werdermannia ). The remaining six South American Schizopetaleae genera were divided into two clades: SCHIZ I of four genera ( Aschersoniodoxa , Brayopsis , Eudema , and Onuris ) and SCHIZ II Schizopetaleae s. str.: Mathewsia , and Schizopetalon ). Resolution within the Thelypodieae clade was limited in both the ITS- and ndhF-based phylogenies. Based on ITS sequence data, elements of the Old World tribe Brassiceae were sister to the Thelypodieae, whereas the ndhF data strongly supported the Sisymbrieae as sister to the Thelypodieae, and the Brassiceae as sister to both tribes. Sister groups to the Schizopetaleae clades I and II were not clearly resolved. Morphological and cytological data support the separation of these three clades. In SCHIZ II, the sepals are always erect to form a closed tube, and the trichomes are mostly dendritic, although other types also occur. In both the Thelypodieae and SCHIZ I clades, the sepals are variously oriented but never form a closed tube, whereas the trichomes are either absent, or simple, forked, or rarely dendritic. The latter clade differs from both SCHIZ II and Thelypodieae by lacking trichomes or rarely having forked ones and by having exclusively basal, entire leaves. Thelypodieae circumscription should be restricted in future to the Thelypodieae clade, Schizopetaleae circumscription to the SCHIZ II clade, while a new tribe is needed for the SCHIZ I clade.

Phylogenetic relationships and generic delimitation in Inuleae subtribe Inulinae (Asteraceae) based on ITS and cpDNA sequence data.

Phylogenetic relationships in Inuleae subtribe Inulinae (Asteraceae) were investigated. DNA sequence data from three chloroplast regions (ndhF, trnL-F and psbA-trnH) and the nuclear ribosomal internal transcribed spacer (ITS) region were analysed separately and in combination using parsimony and Bayesian inference. A total of 163 ingroup taxa were included, of which 60 were sampled for all four markers. Conflicts between chloroplast and nuclear data were assessed using partitioned Bremer support (PBS). Rather than averaging PBS over several trees from constrained searches, individual trees were considered by evaluating PBS ranges. Criteria to be used in the detection of a significant conflict between data partitions are proposed. Three nodes in the total data tree were found to encompass significant conflict that could result from ancient hybridization. Neither of the large, heterogeneous and widespread genera Inula and Pulicaria is monophyletic. A monophyletic group ("the Inula complex") that comprises all species of Inula includes also Telekia, Carpesium, Chrysophthalmum, Rhanteriopsis, Amblyocarpum, and Pentanema sensu stricto. Two species of Pentanema were found to be closer to Blumea (including Blumeopsis and Merrittia) and Caesulia. The monophyletic "Pulicaria complex" includes all taxa with heteromorphic pappus. Within this group, Francoeuria is distinct from Pulicaria and merits recognition as a separate genus. © The Willi Hennig Society 2009.

First European Report of a Leaf Spot on Common Ragweed (Ambrosia artemisiifolia) Caused by a Phoma sp.

Common ragweed, native to North America, has recently become invasive in some parts of Europe. In Hungary, it has become the most widespread agricultural weed species and the most important producer of allergenic pollen since the 1990s. During surveys for its fungal plant pathogens to be evaluated as potential biological control agents (1), ragweed plants exhibiting necrotic spots on the leaves and stems were repeatedly found in Heves and Vas counties in Hungary in September 2004 and 2006. Numerous globose and ostiolate pycnidia, 68 to 115 μm in diameter, containing hyaline, unicellular conidia, 3 to 8 μm long, were found in necrotic tissues. On the basis of these characteristics, the fungus was identified as a Phoma sp., and 21 isolates were obtained on Czapek-Dox medium supplemented with 2% malt and 0.5% tetracycline in 2004 and 2006. Two well-sporulating isolates, designated Ph-5 and Ph-17, were selected for further studies. DNA was extracted from mycelium with a Qiagen DNeasy Plant Kit (Hilden, Germany) and the rDNA internal transcribed spacer (ITS) sequences were amplified and determined as described by Szentiványi et al. (2). The ITS sequences were identical in these two isolates and were 97 to 98% similar to those of Didymella bryoniae (anamorph Phoma cucurbitacearum), a pathogen of cucurbits, and also to those of other Phoma spp. No ITS sequences identical to those determined in Phoma isolates Ph-5 and Ph-17 were found in GenBank. Sequence data were deposited in GenBank (No. FJ794609). To test the pathogenicity of Ph-17 grown on Czapek-Dox medium with 2% malt, a 2 to 6 × 105 conidia/ml aqueous suspension was used to inoculate 2-month-old potted ragweed plants and 1-month-old cucumber cv. Rajnai fürtös, bottle gourd (Lagenaria leucantha) cv. Minibottle, and watermelon (Citrullus lanatus) cv. Sugar Baby, which were all grown from seeds in a greenhouse. Plants were kept in transparent plastic chambers for 6 weeks. Five pots with one to three plants each were used for each plant species tested and the experiment was carried out twice. Noninoculated plants, two pots with one to three individuals for each species kept in the same way, served as controls. Necrotic spots with pycnidia developed on 38 to 47% of the leaves of all inoculated ragweed plants 18 to 25 days after inoculation, whereas all the cucurbitaceous plants tested, as well as the control ragweed plants, did not develop disease symptoms. Although the Phoma isolate Ph-17 was, based on ITS sequence data, closely related to D. bryoniae, it was not pathogenic to cucurbits. The pathogen was reisolated from two diseased ragweed plants. Several Phoma spp. strains were isolated from Ambrosia artemisiifolia in the United States and Canada (3,4), but to our knowledge, none were isolated outside North America. One of the strains has already been used as a potential biological control agent of ragweed in Canada, but then lost its virulence in culture (3). The biocontrol potential of the Hungarian Phoma sp. isolate Ph-17 against A. artemisiifolia is currently being investigated. To our knowledge, this is the first report of a Phoma sp. on A. artemisiifolia in Europe.

First Report of Powdery Mildew Caused by Golovinomyces cichoracearum on Crotalaria juncea ('Tropic Sun' Sunn hemp).

Crotalaria juncea L. (Fabaceae), commonly known as sunn hemp, is a subtropical annual legume grown in the United States as a cover crop that improves soil quality, provides nitrogen, suppresses weeds and nematodes, and adds organic matter to soils. In Florida, sunn hemp is a warm- and short-season cover crop that is typically planted in June and cut and incorporated into soil in September. In 2008, powdery mildew was observed on sunn hemp in a research field in Hastings, FL. This disease is important because it has the potential to impact the health and quality of sunn hemp, and this particular powdery mildew can infect cucurbits that are grown in north Florida from late summer to fall. Fungal growth appeared as typical white, powdery mildew colonies initially seen on upper leaf surfaces, especially along the midvein of infected leaves, but moving to undersides as disease progressed; petioles and floral parts were disease free. As disease progressed, colonies enlarged and coalesced to cover the entire leaf surface; heavily infected leaves senesced and abscised. Infection was primarily seen on the lower, more mature leaves of plants and not on the top 0.6 m (2 feet) of the plant. Mycelia produced white accumulations of conidiophores and conidia. Hyphae were superficial with papillate appressoria and produced conidiophores with cylindrical foot cells that measured 48.5 × 10.0 μm (mean of 100 foot cell measurements) and short chains of conidia. Conidia were hyaline, short-cylindrical to ovoid, lacked fibrosin bodies, borne in chains, had sinuate edge lines with other immature conidia, and measured 22.5 to 40.0 (mean = 29.85 μm) × 12.5 to 20.0 μm (mean = 15.55 μm). The teleomorph was not observed. The nuclear rDNA internal transcribed spacer (ITS) regions were amplified by PCR, using universal primers ITS1 and ITS4, and sequenced (GenBank Accession No. FJ479803). On the basis of morphological characteristics of the asexual, imperfect state that are consistent with published reports of Golovinomyces cichoracearum (2) and ITS sequence data that indicated 100% homology with G. cichoracearum from Helianthus annus (GenBank Accession No. AB077679), this powdery mildew was identified as caused by G. cichoracearum of the classification Golovinomyces Clade III (3). Pathogenicity was confirmed by gently pressing disease leaves onto leaves of healthy C. juncea plants. Inoculated plants were placed into plastic bags containing moist paper towels to maintain high humidity. The temperature was maintained at 24°C, and after 2 days, powdery mildew colonies developed in a manner consistent with symptoms observed under field conditions. A powdery mildew on Crotalaria was previously identified as caused by Microsphaera diffusa Cooke & Peck (1). To our knowledge, this is the first report of G. cichoracearum causing powdery mildew on C. juncea.

Molecular evidence for the hybridity ofIlex × wandoensis and the phylogenetic study of KoreanIlex based on ITS sequence data

In this study, internal transcribed spacer (ITS) regions within the nuclear ribosomal DNA of KoreanIlex were analyzed in order to investigate any molecular evidence thatI. × wandoensis could serve as a putative hybrid betweenI. cornuta andI. integra. We also sought to clearly identify taxonomic relationships and problems caused by consecutive external morphological characters among taxa in the genus. We sequenced 20 clones fromI. × wandoensis and found these individuals displayed intra-genomic polymorphisms within ITS regions. The analysis of the clones clearly demonstrated the presence of discrete sequences from bothI. cornuta andI. integra, thereby confirmingI. × wandoensis is a species that was formed by crossingI. cornuta andI. integra. Lastly, the subgenusIlex, which contains the evergreen species, failed to form a monophyletic group in a strict consensus tree that was prepared based upon ITS regions.I. crenata var.microphylla in the subgenus KoreaIlex, which has presented taxonomic problems previously, formed an independent clade within the consensus tree, thereby showing distinction fromI. crenata.) Genetic discontinuity ofI. macropoda andI. Macropoda for.pseudo-macropoda individuals couldn't be confirmed.

Intraspecific and within-isolate sequence variation in the ITS rRNA gene region ofPythium mercurialesp. nov. (Pythiaceae)

Sixteen Pythium isolates from diverse hosts and locations, which showed similarities in their morphology and sequences of the internal transcribed spacer (ITS) region of their rRNA gene, were investigated. As opposed to the generally accepted view, within single isolates ITS sequence variations were consistently found mostly as part of a tract of identical bases (A-T) within ITS1, and of GT or GTTT repeats within the ITS2 sequence. Thirty-one different ITS sequences obtained from 39 cloned ITS products from the 16 isolates showed high sequence and length polymorphisms within and between isolates. However, in a phylogenetic analysis, they formed a cluster distinct from those of other Pythium species. Additional sequencing of two nuclear genes (elongation factor 1 alpha and beta-tubulin) and one mitochondrial gene (nadh1) revealed high levels of heterozygosity as well as polymorphism within and between isolates, with some isolates possessing two or more alleles for each of the nuclear genes. In contrast to the observed variation in the ITS and other gene areas, all isolates were phenotypically similar. Pythium mercuriale sp. nov. (Pythiaceae) is characterized by forming thin-walled chlamydospores, subglobose to obovoid, papillate sporangia proliferating internally and smooth-walled oogonia surrounded by multiple antheridia. Maximum likelihood phylogenetic analyses based on both ITS and beta-tubulin sequence data place P. mercuriale in a clade between Pythium and Phytophthora.