text:
Serviços Personalizados
Links relacionados
- Citado por Google
- Similares em Google
South African Journal of Science
versão On-line ISSN 1996-7489
versão impressa ISSN 0038-2353
S. Afr. j. sci. vol.104 no.7-8 Pretoria Set./Ago. 2008
Ectomycorrhizas in association with Pinus patula in Sabie, South Africa
G.L. HawleyI; A.F.S. TaylorII; J.F. DamesI, *
IDepartment of Biochemistry, Microbiology and Biotechnology, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa
IIDepartment of Mycology and Forest Pathology, Swedish University of Agricultural Science, Box 7026, S-750 07 Uppsala, Sweden
Forestry is an economically important industry in South Africa, involving extensive exotic plantations of Eucalyptus, Pinus and Acacia species. These tree species have fungal associations, such as ectomycorrhizas, that have become locally naturalized. The forestry industry is increasingly faced with problems of long-term sustainability, increasing soil acidity and depletion of soil nutrients. It is, therefore, essential that the fundamental importance of the ectomycorrhizal (ECM) symbioses in the nutrient cycling, growth, health and survival of these tree species should not be ignored. Research on the species diversity of ECM fungi associated with forestry plant species has been hampered by the difficulty of identifying the fungi involved in the symbiosis. This investigation focused on the ECM fungi associated with Pinus patula (Schlecht. et Cham.) grown in managed plantations in the Sabie region, Mpumalanga province, South Africa. ECM roots were morphotyped and DNA was extracted. The internal transcribed spacer (ITS) region was amplified using the ITS 1F and ITS 4 primers. The sequences were BLASTed using the GenBank and UNITE databases. Twenty-seven extractions were successfully amplified representing 17 different morphotypes. Of the 27 sequences, 21 were identified as ECM fungi and, from the BLAST results, eleven different ECM species could be identified. Selected ECM root types were morphologically and anatomically described according to root morphology, mantle structure, specialized hyphae and rhizomorph arrangement. Seven dominant field types were described and identified as two Amanita species, Scleroderma citrinum, a suilloid species, Thelephora terrestris, a tometelloid species and one resembled an Albatrellus species.
Forestry in South Africa is extensive, covering approximately 1.3 million hectares,1 and is planted mainly with exotic species such as Pinus, Acacia and Eucalyptus. Naturalized ectomycorrhizal (ECM) fungi have been found associating with these tree species, and are necessary for successful tree establishment in managed plantations.2 The diversity and associated mechanisms of the symbiosis in a South African context have received only limited attention. Doidge3 published the last comprehensive list of fungi in South Africa in 1950, which included ECM fungal species from the genera Amanita, Boletus, Cantharellus, Lactarius, Scleroderma, Rhizopogon and Russula.
Previous attempts to document the ECM fungal species diversity occurring in Pinus plantations were conducted using sporocarp surveys. Lundquist4 produced a good listing of all the fungi associated with the genus Pinus, but it is limited in its information as there is no reference to the mycorrhizal status of the fungi. The list is based on the sporocarp deposits in the National Collection of Fungi, which is biased against undetectable ECM fungi below-ground, or non-sporulating fungi. Van der Westhuizen and Eicker5 recorded 21 Basidiomycota ECM fungi from sporocarp data, basing the mycorrhizal status on current understandings of genera and species known to form ectomycorrhizas. Although above-ground diversity confirms the presence of species, it is understood that above- and below-ground species diversity and frequency may differ considerably, given the micro-niches and dynamics within the soil.6,7 It is also possible for fungal species that do not form sporocarps and hypogeous or resupinate sporocarps to go undetected.
In South Africa, research on the below-ground diversity at ECM root tip level is limited. Dames et al.8 characterized two ECM morphotypes from field samples growing in association with Pinus patula (Schlecht. et Cham.), identifying them as Scleroderma citrinum Pers. and Boletus pinicola Rea. Carlson9,10 observed a response of ECM fungal species associated with P. patula to acidic precipitation. Carlson9,10 detected a change in morphotype composition and concluded that there was no decrease in root colonization.
Traditional morphological characterization of ECM root tips is regarded as an important identification technique. Currently, six confirmed ECM fungal species, including the descriptions by Dames et al.8 associated with P. patula, have been characterized. Amanita muscaria (L) Hook., Laccaria laccata (Scop.) Fr., Suillus brevipes (Peck) Kuntze and Thelephora terrestris Ehrh. were characterized by Mohan et al.11,12 after inoculating nursery seedlings with sporocarp material and harvesting the roots after five months.
Molecular and DNA amplification techniques have been increasingly used to resolve or aid a number of systematic and ecological challenges associated with the identification of ECM fungi,13 such as species phylogeny and delineation from field samples as well as allowing for fast and accurate identification, and therefore allowing processing of large community/population sampling. Molecular techniques have proved useful, especially when morphological data are non-informative or not sufficiently refined. Molecular techniques that have been employed included restriction fragment length polymorphism,1416 DNA probing,15 DNA sequencing of the large ribosomal subunit and internal transcribed spacer regions. The molecular data derived from field samples can then be compared to known data (restriction patterns or sequences) from identified sporocarp material or by comparison with sequences submitted to large online databases.
The forestry industry in South Africa is facing increasing problems of soil nutrient depletion, soil acidity due to pollution, and legislation of land used for forestry. Licensing is required, limiting the expansion of land for forestry plantation (National Water Act, No. 36 of 199817). The industry is therefore faced with having to strategically manage the current plantations and to render the soils re-useable as a sustained resource. No long-term strategy has been devised to develop and maintain soil microbial health or to maximize nutrient availability. Khalil et al.2 reported that, in South Africa, P. patula survival and establishment after transplanting was dependent on ECM establishment.
Continued ECM research in South Africa will depend on comprehensive records of fungal species diversity recorded from both above- and below-ground collections to determine their population dynamics. These records should rely on both morphological and molecular techniques to determine above- and below-ground species identification and description. The motivation was to create an initial database of ECM fungal species associating with P. patula species grown in managed plantations in South Africa. Characterizing root tip material in terms of their DNA sequences and pairing these with classical morphological descriptions was carried out to achieve this.
Study site description
Two Pinus patula plantations, Brooklands and Driekop, were selected around Sabie in Mpumalanga province, which supports an intensive forestry industry. The sites are approximately 50 km apart. The climate of this area is subtropical, experiencing mild winters (6°C minimum to 27°C maximum as the daily average) and hot, humid summers (18°C minimum to 30°C maximum as the daily average) with a mean annual rainfall of 736 mm.18 One compartment was selected from each plantation.
The Brooklands plantation site, compartment E23A (25°11.2'S, 30°43.5'E), was planted in 1983 as a second rotation P. patula stand, at an altitude of 1410 m. The Driekop plantation site, compartment C25 (25°02.9'S, 30°53.4'E), was planted in 2000, with plots of P. patula, P. elliotii and P. taeda. This site is situated at an altitude of 1600 m. Only the P. patula plots were sampled for this study. The mature (Brooklands) and young (Driekop) compartments were chosen such that successive ECM fungal species could be represented.
Sampling of root material
Soil cores (diameter 5 cm) were taken from selected plots in each of the two compartments. These were taken approximately 1 m from the base of the tree. The corer extracted soil to a depth of 20 cm, and the root-containing soil was placed in sealed plastic bags and kept at 4°C until processed.
Approximately 4045 cores were collected during each event from Brooklands and Driekop, respectively. Five sampling events were conducted in June 2002, December 2002, April 2003, December 2003 and March 2005. All the roots were washed clean of any soil and organic matter using a 2-mm sieve and were inspected under a dissecting microscope (Leica S4E). Where possible, the material was observed fresh or preserved in 50% ethanol for future examination.19 ECM root tips exhibiting similar morphological characteristics of colour, branching type and mantle surface texture were morphotyped.20 These were coded according to the characteristics and stored in 50% ethanol to maintain the integrity of the fungal DNA.
Molecular analysis
Extraction of DNA from ECM root tips. DNA sequencing was conducted at the end of 2002 and in mid-2003 at the Swedish University of Agricultural Science, Uppsala. DNA was extracted from individual tips with 26 representatives of each morphotype using a modified CTAB method.21 Single individual root tips were homogenized in 600 µm 2% CTAB solution and incubated at 65°C for 30 minutes. Proteinase K (20 µm, 20 mM) was added to the root material before incubation, to degrade any proteins, which could inhibit the polymerase chain reaction (PCR). The DNA was precipitated with isopropanol and the pellet was resuspended in 50 µm autoclaved milli-Q water.
The primer pair used in the PCR reaction were ITS1F22 and ITS4,23 such that amplification was not limited to the Basidiomycota, as members of Ascomycota and Zygomycota are also known to form ECM.19,21,24 High Fidelity Taq polymerase (ExpandTM High Fidelity PCR System, Boehringer Mannheim, Germany) was used for the PCR reaction. The PCR amplification reactions contained both positive and negative controls, of which the DNA template contained a positively amplified fungal ITS region and autoclaved milli-Q water, respectively. The PCR reaction medium contained, in percentage volumes, 41.8% sterile milli-Q water, 10% 10× reaction buffer (MgCl2), 10% dNTPs (2 mM of each base), 6.5% 25 mM MgCl2, 3% 10 µM of each primer and 0.75% 3.5 U/µl Taq polymerase, and was conducted with the following temperature cycles: 94°C for 2 min; 94°C for 60 s, 50°C for 30 s, 72°C for 60 s for 35 cycles; final extension at 72°C for 7 min. Products of the PCR reactions were fractionated on a 2% agarose gel. Double bands that were observed on the gel were considered as contamination or co-amplification of more than one species, and were discarded.
Successful amplifications were cleaned with a PCR Purification Kit (Qiagen Cat. No. 28004) and re-amplified using a Terminator Ready Reaction Mix (BigDYE V3.1 ABI, U.S.A.) with both primers separately and sequenced using the ABI 310 DNA sequencer (Perkin Elmer). The forward and reverse sequences were aligned using the Vector NTI suite package (InforMax) and edited. The sequences obtained in this study for the different ECM fungal species were deposited in the GenBank25 database and accession numbers obtained. Non-mycorrhizal fungi were submitted to GenBank.25
Sequence BLAST analysis. The consensus sequences were submitted to the GenBank25 and UNITE26 databases and compared with similar sequence references, for identification. Identity percentages were used to compare sequences within genotypic groups. In this study, the preferential cut-off for species inclusion was 98%, below which the sequences were considered to represent different species.
From Table 1 and Figs AD (in supplementary material online at www.sajs.co.za), it is evident that a number of different morphotypes resulted in the same species alignment result. As a result of differing sequence lengths, BLAST alignment results can be misleading by misaligning with closely related species. To confirm the taxonomic position of the ECM root tip sequences generated, similarity trees were derived. This was accomplished by downloading several known sequences from GenBank25 and UNITE26 (Table 1), and sequences generated from sporocarp collections,27 together with the ECM root tip sequences, which were aligned in ClustalW28 and visualized in Treeview (Win32, 1.6.6, 2001). The ITS region, although useful for species distinction, should not be used for higher-order resolution as the variation between different genera, particularly in the ITS 1 and ITS 2 regions, would not allow for homologous base-pair alignment. Species within the same genus were therefore aligned and analysed with appropriate outgroup species from sister genera.
Morphological and anatomical description
Selected representatives of each morphotype were used for further morphological and anatomical description. The key morphological features that were examined included root tip branching, shape of branches, mantle colour and surface texture, presence of rhizomorphs and emanating hyphae, as described by Agerer.20 Anatomical characteristics that were observed included detailed records of mantle morphology, characteristics of emanating hyphae and rhizomorphs and structure of the Hartig net in terms of hyphal arrangement, hyphal dimensions, presence of specialized cells and mantle layering.11,12, 2931 Mantle sections were peeled from whole roots, mounted in lactoglycerol32 or water, and examined for surface hyphal arrangement and key distinguishing anatomical features, including pigmented and specialized hyphae. ECM root descriptions were based on the examination of 710 root tips for each morphotype.
Sectioning, staining and photography
Cross- and longitudinal sections were examined to characterize various layers of hyphal arrangement and determine the presence of specialized cells.20 The sectioning of root material was conducted by mounting individual root tips of each morphotype in 2% glycerol.32 Cross- and longitudinal sections, approximately 50 µm thick, were prepared using a freeze microtome (Mectron model 150, Frigistor). The resulting sections were placed in a drop of water in 1.5 ml Eppendorf tubes. The lactoglycerol trypan blue staining procedure32,33 for whole root staining was followed and modified by extending the hydrogen peroxide soak until darker sections were bleached. During the course of staining, the samples were centrifuged at 5000 rpm for 30 s if root sections became suspended. Sections were slide mounted in lactoglycerol and anatomical characteristics were examined using a compound microscope (Olympus BX50).
ECM morphological characteristics were examined after immersion of samples in water, using a dissecting microscope (Wild Photomakroskop M400) and photographed on 100 ASA daylight slide film. Anatomical characteristics were examined using bright field microscopy (Olympus BX50) and photographed on 100 ASA black and white film.
Molecular analysis
The ECM morphotype sequences were BLAST analysed in relation to the GenBank25 and UNITE26 databases. The results given in Table 1 indicate incongruence within morphotypes. Different species are evident within a single morphotype and a number of different morphotypes constitute the same genotypic species.
For example, results showed that Thelephora terrestris was represented by several different morphotypes: Partial white B, Black A, Fwhite, Yelsil C and Yelsil A (Fig. A). Fwhite was aligned with Thelephora coerulea. The morphotype sequences that aligned with Thelephora species were aligned with Tomentella and Thelephora species downloaded from UNITE.26 In the derived tree the morphotype Fwhite was grouped together with Thelephora penicillata, indicating that the alignment with T. caryophylla was potentially inaccurate. All the other morphotypesYelsil A, Yelsil C, Partial white B and Black Agroup with Thelephora terrestris at basal nodes.
Xerocomus badius was represented by morphotypes BSY 2, Beige and Dull orange. The taxonomic position of the Xerocomus morphotypes was investigated by aligning the sequences together with species of Boletus and Suillus (Fig. B). The morphotypes Dull orange, BSY2 and Beige formed a monophyletic group with X. badius (UNITE). All other BSY morphotypesGrey-pink, Orange and White Aaligned with Amanita spissa (Table 1).
The genus Scleroderma aligned poorly to both morphotypes White B and Yelsil B, with expectation values of e-164 and e-128, respectively (Table 1). Albatrellus ovinus was represented by morphotype Grey. The similarity tree of the three genera, Russula, Lactarius and Albatrellus (Fig. C), confirmed the position of the morphotype Grey within the genus Albatrellus. DSY aligned well with Clavulina cristata (Fig. D).
White C and Partial white A were aligned with Hymenoscyphus sp. The morphotype Beige/white resulted in a strong alignment with Heterobasidion annosum on the GenBank database. Discrepancies between the database BLAST results, e.g. for Tuberculate and White C, are the result of missing species in either database and the limitation of the UNITE database containing only ECM fungal species.
Morphological description
Many of the ECM morphotypes were rare or infrequent and were therefore not selected for detailed description. From the Brooklands plots the dominant morphotypes White, BSY, Orange and Grey were selected. Three dominant morphotypes from Driekop, Black, Yelsil and Tuberculate, were characterized. Of the above types, White, Orange, Yelsil, Black and Tuberculate were common between the two sites, but varied in frequency of occurrence.27 The appearance and descriptions of each morphotype are presented in Figs 17.
Morphotype White
Sequence matches to this morphotype were Hymenoscyphus sp., Amanita spissa and Scleroderma bovista (Table 1). Morphotype White was derived from the Brooklands study site and was recorded on 33% of all the ECM root tips that were examined.27
Root tip morphology
Root tip branching was dichotomous, long and straight (Fig. 1A) and the dimensions of the last order axis tips ranged from 0.51.2 mm in length and from 0.250.3 mm in diameter. The mantle surface consisted of loosely arranged stringy hyphae that trap air, making the mantle hydrophobic, and giving the root tips a silvery sheen. The mantle was white, bruising dull yellow with no soil attachments on the mantle. Frequent rhizomorphs emanating from the mantle were white to light yellow/ opaque, and had smooth margins with few hyphal branches. Emanating hyphae were found along the length of the colonized root.
Anatomical morphology
The mantle was composed of two plectenchymatous layers (Type A20). The outer layer was loosely arranged (Fig. 1D) with hyphal dimensions of 2.93.9 µm in width and 14.634.0 µm in length. The inner layer was more compact, 2.93.9 µm in width and 9.721 µm in length. Rhizomorphs were observed to be thicker in diameter at branching points, forming nodia and internodia (Fig. 1C). The diameter of the rhizomorph ranged from 34.038.8 µm. Emanating hyphae were clamped (Fig. 1B) and were 2.54.9 µm in width and 43.658.2 µm in length from septum to septum. The Hartig net was in a palmetti arrangement20 that developed from branching hyphae aligned alongside each other to form a single layer between the cells (Fig. 1E). The Hartig net hyphae penetrated 1 or 2 layers of the root cortical cells.
Morphotype BSY
The morphotype BSY was represented by two species, Xerocomus badius (see Fig. B in supplementary material online) and Amanita spissa (Table 1), based on sequence data. BSY was described from the Brooklands study site and was noted on 21% of all the ECM root tips that were recorded from there.27
Root tip morphology
Branching of the root tips was coralloid to irregularly pinnate (Fig. 2A), with the last order axis ranging from 22.5 mm in length and 0.5 mm in diameter. The mantle was a dirty yellow in colour with a slight sheen, indicating hydrophobic properties. The ramification was straight and the mantle surface texture smooth to reticulate. Soil particles covered the entire root, concentrating in the base of the root branches. Rhizomorphs were dark yellow to tan in colour, but infrequent.
Anatomical morphology
Emanating hyphae were infrequent, and were septate at the base of attachment (Fig. 2C). The outer mantle was arranged in a loose delicate plectenchymatous hyphal net, with hyphal dimensions of 1.82.9 µm in width and 1011.6 µm in length. The inner mantle consisted of strongly angular pseudoparenchymatous cells (Fig. 2D) (Type L20), 610 µm in width and 911 µm in length. The rhizomorphs were highly differentiated (Type F20). There appeared to be an inner thin-walled tube surrounded by shorter thick-walled hyphae (Fig. 2E). Thick-walled cystidia were observed on the outer mantle and the outermost cells of the rhizomorph (Fig. 2B, E). The Hartig net was in a palmetti arrangement20 and penetrated 1 to 2 layers of the cortical cells.
Morphotype Black
Two fungal species, Thelephora terrestris (Fig. A in supplementary material online) and Phialocephala fortinii (Table 1), were identified from sequences of the Black morphotype. Morphotype Black was described from the Driekop study site and was recorded on 7.5% of all the ECM root tips examined.27
Root tip morphology
Branching of the root tips was unramified to short dichotomous and straight (Fig. 3A). The last order axis was 0.50.8 mm in length and 0.30.4 mm in diameter. The mantle was highly pigmented (Fig. 3A) and black in colour. The texture of the mantle surface was reticulate to woolly and was formed by emanating hyphae that then extended straight off the mantle to form spine-like structures. Soil particles were held between emanating hyphae. No rhizomorphs were observed, but emanating hyphae were found along the length of the ECM root tip, concentrating around younger, acropetal areas. Emanating hyphae formed fan-like morphologies.
Anatomical morphology
Emanating hyphae were frequent, dark and clamped. Cystidia were observed attached to the mantle by a globular base (Fig. 3B). Spine-like hyphae emanated from the globular structure, which was separated from the base by septa. The spine-like hyphae measured 75 µm in length from septum to septum and 3 µm in diameter. The mantle consisted of one layer of irregularly shaped transitional hyphae (Type H20) from plectenchymatous to pseudoparenchymatous arrangement (Fig. 3C). The hyphal dimensions in this layer were 48 µm in width and 710 µm in length. The Hartig net formed a typical palmetti arrangement20 and penetrated 3 or 4 layers of the root cortical cells.
Morphotype Orange
Morphotype Orange was identified from the ITS sequence as Amanita spissa (Table 1). Approximately 8.4% of all the ECM root tips examined from the Brooklands study sited were colonized by morphotype Orange.27
Root tip morphology
The branching associated with the Orange morphotype was short dichotomous to coralloid, and straight (Fig. 4A). The length and diameter of the last order axis were 0.50.7 mm and 0.20.5 mm, respectively. The mantle surface was smooth with a few hyaline emanating hyphae. The mantle was bright orange in colour, dulling to a darker orange with age. No rhizomorphs were observed.
Anatomical morphology
The outer mantle layer consisted of a delicate hyphal net, the hyphae ranging from 57 µm in width and 1520 µm in length calculated from septum to septum. The net covered a middle mantle layer of pseudoparenchymatous cells with an angular configuration (Type P20) and hyphal dimensions of 815 µm in width and 1418 µm in length (Fig. 4B). The inner layer was pseudoparenchymatous to plectenchymatous (Type H20), in another net-like arrangement with hyphal dimensions of 35 µm width and 1025 µm length. Cystidia (Fig. 4C) with what appeared to be a pigment at the tip were observed. The Hartig net, in palmetti arrangement,20 penetrated 3 or 4 layers of cortical cells (Fig. 4D).
Morphotype Yelsil
Two species of ECM fungi were identified within this morphotype from the sequence identification, namely Thelephora terrestris (Fig. A) and a Scleroderma species (Table 1). The morphotype Yelsil was described from the Driekop study site and colonized 9.4% of all the ECM root tips examined at the site.27
Root tip morphology
The root of the Yelsil morphotype was a luminescent lemon-yellow that branched dichotomously (Fig. 5A). The last order axis was 0.71.2 mm in length and 0.40.5 mm in width. The mantle was smooth with a few short, emanating hyphae.
Anatomical morphology
Three layers were observed in the mantle of the Yelsil morphotype. The outer layer is arranged in a loose plectenchymatous network with clamped emanating hyphae (Fig. 5D). The hyphal dimensions of the outer mantle layer were 23 µm in width and 2340 µm in length measured between septa. Infrequent flask-shaped cystidia were observed on the outer mantle (Fig. 5B). The middle layer was tightly plectenchymatous (Type B20) and the hyphae were observed to be 34 µm in width and 1015 µm in length (Fig. 5C). The inner layer was pseudoparenchymatous (Fig. 5E) (Type M20), with hyphal dimensions of 47 µm in width and 810 µm in length. Rhizomorphs were not observed. The Hartig net penetrated 2 layers of the root cortical cells.
Morphotype Grey
This morphotype was identified as Albatrellus ovinus from the ITS sequence BLAST (Fig. C). The morphotype Grey was found only on approximately 2% of the ECM root tips examined in the Brooklands site.27
Root tip morphology
The Grey morphotype was dichotomously branched (Fig. 6A) with straight unramified ends. The length and width of the last order branches were 0.60.7 mm and 0.250.3 mm, respectively. The mantle surface was smooth to slightly woolly. Soil particles were attached to the length of the mantle.
Anatomical morphology
Two layers were observed in the mantle. The outer mantle layer has a plectenchymatous arrangement with squarrose branching (Fig. 6C) (Type E20), with hyphal length and width ranging from 1520 µm and 34 µm, respectively. The inner mantle was plectenchymatous with scattered clusters of rounded hyphae (Type K20) (Fig. 6D) and had hyphal dimensions of 79 µm in length and 57 µm in width. No rhizomorphs were observed. Emanating hyphae were clamped and long (Fig. 6B), ranging between 6075 µm in length from septum to septum and 34 µm in diameter. The Hartig net was in a palmetti arrangement,20 penetrating 2 layers of the root cortical cells.
Morphotype Tuberculate
The Tuberculate morphotype did not amplify as an ECM fungus (Table 1) and was therefore not identified from the sequence data. This morphotype was encountered frequently at the Driekop study site and colonized approximately 9% of the ECM root tips examined there.27
Root tip morphology
The Tuberculate morphotype was greyish-white in colour (Fig. 7A) and was densely coralloid (Fig. 7B), becoming dark grey with age. The tubercle-like aggregations were roughly oval in shape and measured between 23.5 mm in width and 35 mm in length. The mantle was smooth to slightly reticulate.
Anatomical morphology
The mantle hyphae protruded perpendicularly to the root surface (Fig. 7D) in a plectenchymatous hymeniform arrangement (Type I20), 5.810.6 µm in width and 18.424.5 µm in length. The hyphal ends contained frequent amounts of oil droplets (Fig. 7C). From a surface view of the mantle, the hyphae appeared round (Fig. 7E). The hyphae of the mantle between the branches were tightly arranged and plectenchymatous (Fig. 7F). The rhizomorphs were observed as a mass of emanating hyphae with thickened hyphal walls. The Hartig net was in a typical palmetti arrangement,20 penetrating 4 to 5 layers of the root cortical cells.
Molecular analysis
The BLAST alignments of ECM morphotypes (Table 1) resulted in interesting matches.
Discrepancies between the databases for two of the morphotype sequences, Tuberculate and White C, can be explained by the lack of particular species in either database. In the case of Tuberculate, the GenBank BLAST25 retrieved a non-ECM fungus, therefore the purely ectomycorrhizal UNITE26 database would not result in the same species. The Tuberculate sequence was aligned, on GenBank, with Scleroconidioma sphagnicola, which is a fungal pathogen that has been found in the moss Sphagnum fuscum.34 The alignment with S. sphagnicola indicated that the fungal DNA amplified was not from an ECM fungus but potentially that of a co-habiting fungus.
BLAST alignments of several morphotypes led to further investigation of Phialocephala fortinii and Hymenoscyphus ericae (Table 1). The latter represents a number of closely related taxa that form an aggregate; it was previously thought that these fungi were primarily ericoid mycorrhizas.35 Strains from this aggregate have recently been shown to produce ECM associations with Betula pubescens, Pi cea abies and Pinus sylvestris, and ericoid mycorrhizas with Vaccinium vitis-idaea.36 Villarreal-Ruiz et al.37 showed that the same strain of H. ericae can form both ECM and ericoid mycorrhizas with P. sylvestris and Vaccinium myrtillus, respectively. The results from the matches retrieved from GenBank of morphotype White C and Partial white A (see Table 2 in supplementary material online) match the morphotype sequences of Meliniomyces variabilis with 100% and 99% identities, respectively. M. variabilis forms part of the Rhizoscyphus ericae (= Hymenoscyphus ericae) aggregate and has recently been allocated to it own genus.38 The mycorrhizal status of this genus its unknown.38 The presence of two morphotypes for this species could be a result of different stages of colonization. The Partial white morphotype could represent only partial colonization of the root tip.
The Partial white C morphotype found at Driekop and the Black B morphotype found at Brooklands (Table 1) both have a BLAST match to Phialocephala fortinii on GenBank. The UNITE database produced a relatively poor match to H. ericae (92%). With a 98% identity to sequences from GenBank (Table 1), these morphotypes were identified as P. fortinii. This species is a dark septate endophyte (DSE), found globally, colonizing numerous plant species.The functional biology of DSEs, and whether they are pathogenic or symbiotic, is uncertain.39 The mechanism by which the hyphae colonize the root was examined by Yu et al.39 and found to be similar to that of arbuscular mycorrhizal fungi. Additionally, this genus is closely related to the ericoid ectomycorrhizal genus Hymenoscyphus.35
Five morphotypes, Black A, Fwhite, Yelsil C, Yelsil A and Partial white B, were matched to Thelephora species, a resupinate/ bracket ectomycorrhizal fungus (Fig. A). Fwhite was poorly aligned (93%) with a different species, T. coerulea, according to the UNITE database, which corresponds with the inconclusive BLAST result from GenBank. Comparisons between the alignments of all the morphotypes and their taxonomic position (Fig. A) indicate that Fwhite is a different species, grouping with Thelephora penicillata. The presence of T. terrestris can be confirmed by above-ground sporocarp collections27 and has been documented both in the field and in nurseries in South Africa.2,4
Xerocomus badius was represented by three morphotypes (Fig. B) according to the concurring results of both GenBank and UNITE databases. The morphotypes Dull Orange and Beige resulted in strong alignments with X. badius, with 99% identity. BSY2 aligned poorly with X. badius (94%), suggesting that this morphotype is closely related, but not the same species. The taxonomic placement of this morphotype did, however, match within the X. badius group in the derived tree (Fig. B), indicating that more-rigorous analysis is required to determine the taxonomic species.
The Grey morphotype was strongly aligned with Albatrellus ovinus on the GenBank and UNITE databases (Fig. C), with 99% and 98% identities, respectively. The taxonomic position of this morphotype as A. ovinus was confirmed in the derived tree (Fig. C online), where this morphotype grouped with a known sequence of this species. However, no sporocarp collections of this species have ever been recorded in South Africa. Morphotype DSY was tentatively identified as Clavulina cristata, as the ITS sequence aligned with this species in relation to GenBank and UNITE sequences, 96% and 95% identity, respectively. The species' position was verified in the derived tree (Fig. D online), where DSY grouped with known sequences of C. cristata.
Four morphotypes have been identified as Amanita spissa from the GenBank and UNITE databases: Pink, White A, BSY 1, 3, 4 and 5, BSY and Orange (Table 1). BLAST alignments of morphotypes BSY4 and White A resulted in lower percentage identities (97% and 98%, respectively, from UNITE) to A. spissa. GenBank gave a match of A. rubescens with White A. This mismatch between the two databases may be a result of misidentified sequence material being deposited in them. It is important to note that all three of these species are closely related and have been placed in the Sect Validae (Fr.) Quél.40 Within the genus, the ITS region displays high rates of divergence that do not allow proper species distinction. For this reason, the ITS regions may not be sufficiently informative for identification to species level. The LSU region, which is more conserved, is currently used in the literature for the phylogenetic analysis of this genus.40 Amanita spissa and A. rubescens have been recorded by Lundquist4 and Van der Westhuizen and Eicker5 in Pinus stands in South Africa.
The morphotypes White B and Yelsil B were poorly aligned with Scleroderma bovista on the GenBank and UNITE databases (94% and 9092%, respectively; Table 1), which indicates that the identity of the ECM fungi falls within the genus, but could potentially be a different species. Sporocarps of S. citrinum were found at high frequency at the Brooklands study site,27 confirming the above statement.
The morphotype Beige/white displayed a fair alignment (97% identity) on GenBank, with Heterobasidion annosum (Table 1). This species is a wide-ranging, basidiomycetous plant pathogen that has been studied at an infected site of Pinus sylvestris and Betula pendula,41 and is considered to be highly infective over extended periods of time. The fungi colonize fresh plant tissue normally exposed as stumps during felling and infects surrounding plants through the rhizosphere. The consequences of infection that are experienced are reduced wood quality, growth losses and mortality of Pinus sylvestris.42 No record of this fungal infection has been made in South Africa.
It is clear that there is some genotypic variation within morphotypes (e.g. the BSY group), and yet some conformity between different morphotypes (e.g. Pink, BSY and Orange all resolve as Amanita spissa) (Table 2). Mah et al.43 observed similar results in a study where 22 genotypes were derived from eight morphotypes. A genotypic analysis of morphotypes found by Erland et al.44 recorded eight species within six morphotypic groups. Phenotypic variations may be a result of the morphological changes of the ECM root tip that occur during the ageing process. Mantle colour may change or may be altered in different environmental conditions. However, Sakakibara et al.45 found an average 93% genotypic conformity with the eight morphotypes examined and employed techniques of anatomical analysis which, although more accurate, may be impractical for large-scale population surveys. A possible 11 species may be identified from the BLAST results of the root tip DNA sequences examined in this study.
Morphological descriptions
The most informative features of the ECM root tip morphology observed were the cystidia and rhizomorphs (when present) and patterns discernible in the mantle layers. Dichotomous branching of the root was not a unique distinguishing feature, which may have been due to the natural occurrence of dichotomous branching of Pinus roots in the abscence of ECM fungi.46 This study described seven morphotypes from P. patula plantations in Sabie.
The morphotype White was identified as Scleroderma citrinum according to the colour atlas key,20 in that clamps were present (Fig. 1B) and contact-clamps and contact septa were lacking. There are, however, differences in the root ramification. The morphotype examined in this study branched dichotomously (Fig. 1A), whereas the branching presented in the colour atlas is monopodial-pyramidal. The White morphotype is similar to morphotype WITS 01, also described as S. citrinum, by Dames et al.,8 from P. patula, but differs in the colour of the rhizomorph. Dames et al.8 found the rhizomorphs to be yellow, but notes that the variation in colour could be attributed to age. The white ECM root identified by Mohan et al.,12 from P. patula as Amanita muscaria (L.) Hook, was compared with the morphotype in this study, but differed in that the specimen in this investigation contained clamp connections and lacked tannin cells in the mantle slide mounts.
The mantle morphology of morphotype BSY is remarkably similar to Orange in that the mantle has an angular pseudoparenchymatous hyphal arrangement in the middle to inner layer. This hyphal arrangement led to the comparison with Amanita citrina in the colour atlas.20 Features that were common between the BSY and A. citrina were an outer plectenchymatous hyphal arrangement and a strongly angular pseudoparenchymatous hyphal layer (Fig. 2D). The rhizomorphs also displayed a common differentiation consisting of a large inner tube (Fig. 2E). The morphotypes differed in the mantle layer, where an inner pseudoparenchymatous layer was described for A. citrina. Also, the hyphae in the outer mantle of the ECM morphotype described in this study consisted of frequent cystidia and hyphae with thickened cell walls (Fig. 2B), which are not observed with A. citrina. This indicates that BSY is possibly a different species of the same genus.
The Black morphotype was identified as a tomentelloid ECM fungus. Keying out to genus or species was not possible. Comparisons with Tomentella species were made, although occasional specimens of Cenococcum geophilum were observed, however, which were characterized by stellate pseudoparenchymatous mantles.47 Other, rarely found, black root tip types were comparable to Hymenoscyphus ericae and were characterized by plectenchymatous hyphal arrangement in the mantle.48 The similar morphologies of these black root tips may result in morphotyping inaccuracies. The morphotype Black in this study did compare with Tomentella albomarginata in that emanating hyphae extended from globular hyphae in the mantle (Fig. 3B). Although not all the mantle layers described from T. albomarginata49 correlate with the current morphotype, a similar pattern of irregular, transitional pseudoparenchymatous to plectenchymatous type hyphae were comparable.
The strong angularity of the hyphae in the pseudoparenchymatous mantle layer was comparable with the morphotype Orange and Amanita genera in the colour atlas.20 Although there is only one Amanita species illustrated, A. citrina,50 the key to mycobionts of Pinus, presents two Amanita species. A comparison of mantle structure between A. citrina and morphotype Orange can be made. Both species share loose, ring-like plectenchymatous surface hyphae (Fig. 4C) and a definite angular pseudoparenchymatous middle layer (Fig. 4B), as well as clamped emanating hyphae. The only differences that were noted between morphotype Orange and A. citrina were the root colour, the lack of emanating hyphal structures, and the presence of cystidia associated with Orange. The deduction therefore is that morphotype Orange is not A. citrina, but possibly a species within the same genus.
The morphotype Yelsil was keyed out in the colour atlas20 as Thelephora terrestris and was compared with the photographic illustration provided. The description of T. terrestris, in association with Picea abies (L.) Karst.,51 differed little from the root tip in this study in terms of mantle morphology. The root ramification was the only differing feature in that the morphotype described in this study branched dichotomously as opposed to monopodial-pinnate, but this may be a result of the dichotomous root branching exhibited in Pinus species, as discussed earlier. The Yelsil morphotype characterized in this study was also comparable to the T. terrestris described on P. patula by Mohan et al.11 T. terrestris is a common and widely distributed ECM fungus and known as an aggressive colonizer, especially in nurseries in South Africa, the presence of which is therefore not surprising.52
The morphotype Grey is not represented in the Pinus key in the colour atlas.20 It was, nevertheless, compared to the Albatrellus ovinus (Schaeff.: Fr.) Kotl. & Pouzar plate.53 Similarities and dissimilarities were noted. Both specimens were grey in colour, and were described as having a loose layer of emanating hyphae (Fig. 6C). In both cases the outer mantle was plectenchymatous, the arrangement becoming more tightly packed towards the inner mantle (Fig. 6D). The difference between them was the presence of cystidia, which were not reported from the Grey morphotype in our study. The branching reported in association with Picea abies53 was represented as monopodial, where the present description associated with Pinus patula was dichotomously branched. The species identification of this ECM fungal morphotype is therefore inconclusive. Albatrellus species have not been recorded in South Africa, and therefore there are no sporocarp data to confirm or provide insight into the presence of this species.
The morphotype Tuberculate was keyed out as Rhizopogon roseolus (Corda) TM Fries, in that the ECM branching was tightly coralloid (Fig. 7B), the emanating hyphae contained oil droplets (Fig. 7C) and were not embedded in gelatinous matrix, the mycorrhizal system was smooth and clamps were absent.20 The morphotype may also have been a tubercle-forming Suillus species, as ECM roots formed by this genus are commonly tuberculate, e.g. S. pictus.54,55
Concluding remarks
The characterization of seven morphotypes has led to the identification of Scleroderma citrinum, two Amanita species (morphotypes BSY and Orange), a tomentelloid species, Thelephora terrestris, and a possible suilloid and Albatrellus species. ECM root morphologies documented in this investigation can be used to direct future ECM root studies in South Africa, creating a database of recorded South African species with which to consult. With the exception of Albatrellus, all the genera discussedAmanita, Scleroderma, Suillus and Thelephoraare well represented by several species in South Africa and have been recorded in association with Pinus species.4,5 It is necessary, though, to emphasize that root tip morphology can be highly variable, especially with regard to the age of ECM root tips, the stage of colonization, as well as geographic differences within the same species.
The identification and characterization of the ECM fungi occurring in South Africa is by no means complete, with only seven morphotypes having been described. The other morphotypes observed were infrequent or rare and are indicative of greater fungal species diversity than previously suspected.27 Morphological characterization of ECM roots is necessary for further understanding within this field and may be enhanced by ECM synthesis from sporocarp material or by tracing ECM via rhizomorphs and hyphae from the sporocarp to the root.
We thank Kata Ihrmark for her guidance and for sharing knowledge that was crucial for the molecular analysis of all the fungal samples. We would also like to acknowledge funding from SIDA and the NRF, whose support provided the means to achieve the research presented.
1. Smit W. and Pitcher M. (2003). A case study on ensuring sustainable management of planted forests: The economic, social and environmental role of commercial plantations in South Africa. Ensuring SRM session. UNFF Intersessional Experts Meeting on the Role of Planted Forests in Sustainable Forest Management, 2430 March 2003, New Zealand, Paper 15. [ Links ]
2. Khalil S., Labuschagne N. and Wingfield M.J. (1999). Pinus patula establishment problem associated with poor ectomycorrhizal development in previously cultivated soils. S. Afr. For. J. 186, 5965. [ Links ]
3. Doidge E. (1950). The South African Fungi and Lichens to the end of 1945. Bothalia 5, 11094. [ Links ]
4. Lundquist J.E. (1986). Fungi associated with Pinus in South Africa, Part 1. The Transvaal. Suid-Afrikaanse Bosboutydskrif 138, 113. [ Links ]
5. Van der Westhuizen G.C.A. and Eicker A. (1987). Some fungal symbionts of ectotrophic mycorrhizae of pines in South Africa. S. Afr. For. J. 143, 2024. [ Links ]
6. Dahlberg A. (2001). Community ecology of ectomycorrhizal fungi: an advancing interdisciplinary field. New Phytol. 150, 555562. [ Links ]
7. Jonsson L., Dahlberg A. Nilsson M. Zackrisson O. and Kårén O. (1999). Ectomycorrhizal fungal communities in late-successional Swedish boreal forests, and their composition following wildfire. Mol. Ecol. 8, 205215. [ Links ]
8. Dames J.F., Straker C.J. and Scholes M. (1999). Ecological and anatomical characterisation of some Pinus patula ectomycorrhizas from Mpumalanga, South Africa. Mycorrhiza 9, 924. [ Links ]
9. Carlson C. (1992). Effects of acidic precipitation on calcium and magnesium uptake by Pinus patula. M.Sc. thesis, University of the Witwatersrand, Johannesburg. [ Links ]
10. Carlson C. (1994). The influence of fertilisation on ectomycorrhizal colonisation of Pinus patula roots. Suid-Afrikaanse Bosboutydskrif 171, 16. [ Links ]
11. Mohan V., Natarajan K. and Ingleby K. (1993). Anatomical studies on ectomycorrhizas. I, The ectomycorrhizas produced by Thelephora terrestris on Pinus patula. Mycorrhiza 3, 3942. [ Links ]
12. Mohan V., Natarajan K. and Ingleby K. (1993). Anatomical studies on ectomycorrhizas. II, The ectomycorrhizas produced by Amanita muscaria, Laccaria laccata and Suillus brevipes on Pinus patula. Mycorrhiza 3, 4349. [ Links ]
13. Bruns T.D. and Gardes M. (1993). Molecular tools for the identification of ectomycorrhizal fungi taxon specific oligonucleotide probes for suilloid fungi. Mol. Ecol. 2, 233242. [ Links ]
14. Kårén O., Högberg N., Dahlberg A., Jonsson L. and Nylund J. (1997). Inter- and intraspecific variation in the ITS region of rDNA of ectomycorrhizal fungi in Fennoscandia as detected by endonuclease analysis. New Phytol. 136, 313325. [ Links ]
15. Martin F., Delaruelle C. and Ivory M. (1998). Genetic variability in intergenic spacers of ribosomal DNA in Pisolithus isolates associated with pine, eucalyptus and Afzelia in lowland Kenyan forests. New Phytol. 139, 341352. [ Links ]
16. Timonen S., Tammi H. and Sen R. (1997). Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCRRFLP analyses. New Phytol. 135, 313323. [ Links ]
17. Chamberlain D., Essop H., Hougaard C., Malherbe S. and Walker R. (2005). G:nensis part III: Technical note and appendices. Online: www.forestry.co.za/ fsa/ViewFile?file_id=91 [ Links ]
18. Weather SA. Online: www.weathersa.co.za/Climat/Climatestats/Nelspruit Stats.jspweather SA [ Links ]
19. Brundrett M., Bougherm N. Dell B. Grovem T. and Malajczuk N. (1996). Working with Mycorrhizas in Forestry and Agriculture. Australian Centre for International Agricultural Research, Canberra. [ Links ]
20. Agerer R. (19872002). Colour Atlas of Ectomycorrhizae. Einhorn-Verlag, Schwäbisch Gmünd, Germany. [ Links ]
21. Zeze A., Ophel-Kellerm K. and Smith S. (1998). DNA isolation and development of molecular tools for identification and detection of VA mycorrhizal fungi. In Mycorrhizal Manual, ed. A. Varma, pp. 373385. Springer, Berlin. [ Links ]
22. Gardes M. and Bruns T.D. (1993). ITS primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. Mol. Ecol. 2, 113118. [ Links ]
23. White T.J., Bruns T., Lee S. and Taylor J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications, eds. M.A. Innis, D.H. Gelfand J.J. Shinsky T.J. White, pp 315322. Academic Press, San Diego. [ Links ]
24. Carlile M.J., Watkinson S. Gooday G.W. and Watkinson S.C. (2001). The Fungi, 2nd edn. Academic Press, London. [ Links ]
25. Altschul S.F., Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. and Lipman D. (1997). Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 33893402. [ Links ]
26. Kõljalg U., Larsson K-H. Abarenkov K. Nilsson R.H. Alexander I.J. Eberhardt U. Erland S. Hoiland K. Kjoller R. Larsson E. Pennanen T. Sen R. Taylor A.F.S. Tedersoo L. Vrålstad T. and Ursing B.M. (2005). UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi. New Phytol. 166, 10631068. [ Links ]
27. Hawley G.L. (2008). Ectomycorrhizal characterisation, species diversity and community dynamics of Pinus patula Schelcht. et Cham. plantations. Ph.D. thesis, Rhodes University, Grahamstown. [ Links ]
28. Chenna R., Sugawara H. Koike T. Lopez R. Gibson T.J. Higgins D.G. and Thompson J.D. (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic Acids Res. 31, 3497500. [ Links ]
29. Taylor A.F.S. and Alexander I.J. (1989). Ectomycorrhizal synthesis with an isolate of Russula aeruginea. Mycol. Res. 92, 103107. [ Links ]
30. Massicotte H.B., Melville L.H. Peterson R.L. and Molina R. (1999). Biology of the ectomycorrhizal fungal genus, Rhizopogon. IV. Comparative morphology and anatomy of ectomycorrhizas synthesized between several Rhizopogon species on Ponderosa pine (Pinus ponderosa). New Phytol. 142, 355370. [ Links ]
31. Kraigher H., Agerer R. and Javornik B. (1995). Ectomycorrhizae of Lactarius lignyotus on Norway spruce, characterisation by anatomical and molecular tools. Mycorrhiza 5, 175180. [ Links ]
32. Smith S.E. and Dickson S. (1997). VA Mycorrhizas: Basic Research Techniques. Cooperative Research Centre for Soil and Land Management, Adelaide. [ Links ]
33. Koske R.E. and Gemma J.N. (1989). A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92, 486489. [ Links ]
34. Tsuneda A., Chen M.H. and Currah R.S. (2001). Characteristics of a disease of Sphagnum fuscum caused by Scleroconidioma sphagnicola. Can. J. Bot. 79, 1217 1224. [ Links ]
35. Grünig C.R. (2003). Population biology of the tree-root endophyte Phialocephala fortinii. Ph.D. thesis (Natural Sciences), Swiss Federal Institute of Technology, Zürich. [ Links ]
36. Vrålstad T., Schumacher T. and Taylor A.F.S. (2002). Mycorrhizal synthesis between fungal strains of the Hymenoscyphus ericae aggregate and potential ectomycorrhizal and ericoid hosts. New. Phytol. 153, 143152. [ Links ]
37. Villareal-Ruiz L., Anderson I.C. and Alexander I.J. (2004). Interaction between an isolate from the Hymenoscyphus ericae aggregate and roots of Pinus and Vaccinium. New Phytol. 164, 183192. [ Links ]
38. Hambleton S. and Sigler L. (2005). Meliniomyces, a new anamorph genus for root-associated fungi with phylogenetic affinities to Rhizoscyphus ericae (= Hymenoscyphus ericae), Leotiomycetes. Stud. Mycol. 53, 127. [ Links ]
39. Yu T., Nassuth A. and Peterson R.L. (2001). Characterisation of the interaction between the dark septate fungus Phialocephala fortinii and Asparagus officinalis roots. Can. J. Microbiol. 47, 741753. [ Links ]
40. Weiβ M., Yang Z.L. and Oberwinkler F. (1998). Molecular phylogenetic studies in the genus Amanita. Can. J. Bot. 76, 11701179. [ Links ]
41. Lygis V., Vasiliauskas R. and Stenlid J. (2004). Planting Betula pendula on pine sites infested by Heterobasidion annosum: disease transfer, silvicultural evaluation, and community of wood-inhabiting fungi. Can. J. For. Res. 34, 120130. [ Links ]
42. Pukkula T., Möykkynen T., Thor M., Rönnberg J. and Stenlid J. (2005). Modelling infection and spread of Heterobasidion annosum in even-aged Fennoscandian conifer stands. Can. J. For. Res. 35, 7484. [ Links ]
43. MahK., Tackaberry L.E., Egger K.N. and Massicotte H.B. (2001). The impacts of broadcast burning after clear-cutting on the diversity of ectomycorrhizal fungi associated with hybrid spruce seedlings in central British Columbia. Can. J. For. Res. 31, 224235. [ Links ]
44. Erland S., Jonsson T., Mahmood S. and Finlay R.D. (1999). Below-ground ectomycorrhizal community structure in tow Picea abies forests in southern Sweden. Scand. J. For. Res. 14, 209217. [ Links ]
45. Sakakibara S.M., Jones M.D., Gillespie M., Hagerman S.M., Forrest M.E., Simard S.E. and Durall D.M. (2002). A comparison of ectomycorrhiza identification based on morphotyping and PCRRFLP analysis. Mycol. Res. 106, 868878. [ Links ]
46. Kaska D.D., Myllylä R. and Cooper J.B. (1999). Auxin transport inhibitors act through ethylene to regulate dichotomous branching of lateral root meristems in pine. New Phytol. 142, 4958. [ Links ]
47. Agerer R. and Gronbach E. (1988). Cenococcum geophilum. In Colour Atlas of Ectomycorrhizae, ed. R. Agerer, plate 11. Einhorn-Verlag, Schwäbisch Gmünd, Germany. [ Links ]
48. Brand F., Gronbach E. and Taylor A.F.S. (1992). Hymenoscyphus ericae. In Colour Atlas of Ectomycorrhizae, ed. R. Agerer, plate 73. Einhorn-Verlag, Schwäbisch Gmünd, Germany. [ Links ]
49. Agerer R. (1996). Tomentella albomarginata. In Colour Atlas of Ectomycorrhizae, ed. R. Agerer, plate 111. Einhorn-Verlag, Schwäbisch Gmünd, Germany. [ Links ]
50. Mleczko P. (2002). Amanita citrina. In Colour Atlas of Ectomycorrhizae, ed. R. Agerer, plate 159. Einhorn-Verlag, Schwäbisch Gmünd, Germany. [ Links ]
51. Agerer R. and Weiss M. (1990). Thelephora terrestris. In Colour Atlas of Ectomycorrhizae, ed. R. Agerer, plate 48. Einhorn-Verlag, Schwäbisch Gmünd, Germany. [ Links ]
52. Van Greuning J. and van der Westhuizen G. (1990). An ectomycorrhizal fungus of pine seedlings in an Eastern Transvaal Nursery. Suid-Afrikaanse Bosboutydskrif 155, 14. [ Links ]
53. Agerer R. (1996). Albatrellus ovinus. In Colour Atlas of Ectomycorrhizae, ed. R. Agerer, plate 91. Einhorn-Verlag, Schwäbisch Gmünd, Germany. [ Links ]
54. Olsson P.A., Münzenberger B. Mahmood S. and Erland S. (2000). Molecular and anatomical evidence for a three-way association between Pinus sylvestris and the ectomycorrhizal fungi Suillus bovinus and Gomphidius roseus. Mycol. Res. 104, 13721378. [ Links ]
55. Randall B.L. and Grand L.F. (1986). Morphology and possible mycobionts (Suillus pictus) of a tuberculate ectomycorrhiza on Pinus strobus. Can. J. Bot. 64, 21822191. [ Links ]
Received 13 April 2007. Accepted 5 July 2008.
This article is accompanied by a supplementary table and figures online at www.sajs.co.za
* Author for correspondence. E-mail: firstname.lastname@example.com
Hawley G.L., Taylor A.F.S. and Dames J.F (2008). Ectomycorrhizas in association with Pinus patulain Sabie, South Africa. S. Afr. J. Sci.104, 273283.


text:
Lil Chucky Interview On The Breakfast Club: Charlamagne Asks If He Ever Kissed Baby Or Wayne, Virginity, Teaching How To Throw That D Dance & More
The Breakfast Club Interviews Lil Chuck
Please click the "Report" button below if the video on this page is not working properly.
Comments will only be displayed by verified emails. No Spamming or Racism.

subset: commoncrawl


text:
AUER Packaging's special ESD compartments enable you to subdivide your ESD Euro containers in any way you want. The practical dividers can be custom-made for you upon request. Please note that the special compartments can only be configured in the online shop.
- Electrically conductive material
- Custom-made
- Precisely pre-cut connector system
- Matched to EURO dimensions


text:
By BEN WALKER
AP Baseball Writer
NEW YORK (AP) _ Alex Rodriguez drew loud boos in his return home and the New York Yankees overcame Miguel Cabrera's crushing home run off Mariano Rivera, beating Detroit 4-3 in 10 innings Friday night and snapping the Tigers' 12-game winning streak.
Cabrera temporarily saved the Tigers by hitting a two-out, two-run homer in the ninth off Rivera that made it 3-all. The reigning Triple Crown winner connected while still hobbling after apparently fouling a ball off himself earlier in the at-bat.
Brett Gardner grounded an RBI single with the bases loaded and two outs in the 10th to end New York's four-game losing streak.
The AL Central-leading Tigers were trying for their best string since 1934, when they tied the team record of 14 straight wins. That run ended across the street at the old Yankee Stadium when Babe Ruth and Lou Gehrig each drove in runs to down Detroit.
Gardner singled just past Cabrera's dive at third base. Al Alburquerque (2-2) took the loss and Shawn Kelley (3-1) got the win.
Austin Jackson got three doubles and four hits overall for Detroit. His one-out double began Detroit's comeback in the ninth.
With some scattered fans wearing No. 13 jerseys, Rodriguez finished 0 for 4 in his first game at Yankee Stadium since last October. The sellout crowd of 46,545 let him have it the whole night, from the moment the lineups were announced to the time he looked at a slider for his third strikeout of the game.
A-Rod made his season debut Monday in Chicago against the White Sox, hours after Major League Baseball handed him a 211-game drug suspension. The three-time AL MVP can play until there is a decision on his appeal, likely after the season.
Jayson Nix replaced Rodriguez in the top of the ninth. Nix walked to start the 10th and scored the winning run.
Cabrera delivered a most dramatic hit an inning earlier.
After a foul ball left him hopping, Cabrera got a checkup from manager Jim Leyland. Fact is, Cabrera almost didn't make it that far _ moments before that, first baseman Lyle Overbay reached over a railing and barely missed catching Cabrera's foul pop that would've ended the game.
But Cabrera regrouped and lined a 2-2 pitch over the wall in dead center field, slowly making his way around the bases and silencing the crowd. He drove in Detroit's first three runs, and now has 34 homers and 108 RBIs.
For Rivera, it was his second straight failure _ the first time that happened since 2011 for the career saves leader. Rivera has blown four in 39 chances this year, including a ninth-inning miss Wednesday night in Chicago against the White Sox.
Yankees starter Ivan Nova allowed one run in seven innings. He wiggled out of a jam in the first, striking out Victor Martinez and Andy Dirks to leave the bases loaded.
Nova cruised until Jackson led off the seventh with a double. Torii Hunter was then called out on strikes and objected to the call by Triple-A fill-in umpire Will Little.
After Cabrera followed with an RBI single, Little ejected Tigers pitcher Max Scherzer for hollering from the dugout.
Alfonso Soriano had an RBI grounder in the first and Robinson Cano lined a two-run double in the third off Rick Porcello that put the Yankees ahead 3-0.
NOTES: There was a 47-minute rain delay at the start. … Detroit C Alex Avila sat out, a day after he was hit in the mask by a foul ball and left the game. He was checked for a possible concussion and got medical clearance to play. … Tigers RHP Anibal Sanchez (9-7, 2.58) starts Saturday against RHP Phil Hughes (4-10, 4.87). … The Yankees wore their batting practice hats with the white bills. They did it to honor their partnership with the David C. Koch Foundation, New Era caps and the fight against cancer. … Cabrera was 0 for 5 lifetime vs. Rivera before homering. … Ichiro Suzuki ran into the RF wall after catching Cabrera's drive in the third.
(© Copyright 2013 The Associated Press. All Rights Reserved.)


text:
STTG Q2 2013 Reader Satisfaction Survey
With the 2nd Quarter of 2013 now behind us, it is time to take a break from our daily market recaps to conduct our next StockTradingToGo (STTG) Reader Satisfaction Survey.
For newer readers, our quarterly surveys are only three questions and it is extremely important to participate.
Your feedback ensures we continue to deliver the best possible commentary and analysis each evening, so please help us keep our free blog great and take the survey now,
Thank you in advance for sharing your opinion. Market Recaps will resume tomorrow (Thursday).


text:
Payless Shoes
Find great prices and shoes at a Payless store near you
Payless ShoeSource has been your family's shoe store for more than 60 years. With footwear for women, men, girls, and boys, you'll find something for everyone at Payless.com or a Payless ShoeSource store near you at one of our thousands of locations.
Our selection of shoes, boots, sandals, and kids' shoes are available at the lowest prices you'll find at any shoe store. Our customers can always find additional Payless deals and coupons with online discounts, and by joining our Payless Rewards program to get exclusive updates and insider news about our biggest sales! For more special offers, join the Payless text program today.


text:
What Do We Know About Lana Del Rey's 'Ultraviolence?'
Is pop music's somber sweetheart Lana Del Rey about to pull a Beyonce?
Her sophomore set, 'Ultraviolence,' feels like it is shrouded in secrecy. With a new billboard featuring Del Rey's pretty face and the album title landing in Brooklyn this week, sources are buzzing about a single, reportedly dubbed 'West Coast,' dropping in surprise fashion, possibly this week, as well. But the truth is, we don't know. Not much has been released in terms of official information from the singer's camp.
There are rumors of a May 1 release date, which is right around the corner, so the fact that the signage was seen in Brooklyn suggests that a new LDR album -- likely full of doom and gloom love anthems -- is on its merry way.
Lord (not Lorde, of course) knows that all sorts of older and unfinished LDR tracks leaked left and right after the release of her debut, 'Born to Die.'
Check out the LDR banner on a brick building in the 7-1-8 below, which was tweeted out.
Usually, those types of promo billboards serve a purpose and have a release date and other concrete information posted on them, alerting fans to the coming of an album, since not everyone is plugged in 24/7. Or are they?
What we do know is that LDR has booked a Coachella gig this weekend and next, and that she has a tour on the horizon, something she did not do in support of 'Born to Die,' which lead to endless questions about her ability to perform live and pull off her material.
Artists usually tour to promote something, you know, like a new album, and it would make sense to use the time in front of fans to inform them of what's on the docket and to debut some new tracks.
It does seem that Lana and her team are keeping information under wraps. They are not as covert as Beyonce, but…
…why so much silence?
We don't know the release date or the single date. We just know that 'Ultraviolence' is soon upon us. We hope. Bring on another bout of summertime sadness.


text:
Jan. 17, 2008
CAMBRIDGE, Mass. - MIT baseball announced its preliminary schedule for 2008 featuring 36 games against 19 opponents from nine different conferences. This year's slate of games promises to be challenging as 11 of MIT's opponents had winning records in 2007 with nine of them collecting an impressive 20 wins or more. Also highlighting the schedule are four games against opponents that qualified for the 2007 NCAA Tournament and three versus teams ranked in the 2007 ABCA/Collegiate Baseball Division III Preseason National Poll.
The Engineers open things up on Saturday, March 15, when they travel to upstate New York to face Vassar College. Adding a little intrigue to the match-up, MIT head coach Andy Barlow spent nine years as the head coach of the Brewers and led Vassar to its first winning season in 2003. The doubleheader will mark the first meeting between the two teams and will be the home opener for Vassar at their newly constructed Prentiss Field.
The home opener for MIT will be on March 18, as the Engineers welcome Little East opponent Rhode Island College to Briggs field. The Anchorman will be a tough challenge as they are coming off a 21-14 season. The following day, MIT plays another non-conference home game versus Wentworth Institute of Technology. Despite having campuses that are only minutes apart, these schools where unable to play in 2007 due to two rainouts. That Saturday, MIT begins its NEWMAC portion of the schedule with a home doubleheader against Babson College. Last year, MIT swept the Beavers at Govoni Field, but lost an elimination game in the NEWMAC Tournament. Heading into this season the NEWMAC is rated as the toughest conference in New England according to the Massey Ratings. The Massey Ratings, similar to a Ratings Percentage System (RPI), are calculated using strength of schedule and scoring margin.
Immediately following the conference doubleheader against Babson, MIT will travel to Arizona to take part in the Greater Phoenix Invitational where they will play seven games in five days. First up for the Engineers will be No. 19 Southern Maine. The nationally ranked Huskies are a perennial NCAA qualifier. Also scheduled during the trip will be doubleheaders versus Gustavus Adolphus and Colby as well as single nine-inning games against Middlebury and Castleton State. In the past two years, MIT has beaten several NCAA Tournament teams during the week-long trip including the 2006 NCAA Division III National Champions Wisconsin-Whitewater.
Upon returning home, MIT will continue to play games at a rapid pace. After enjoying a much needed rest/travel day on the 29th, MIT will play host to Newbury College on the 30th of March. Following this doubleheader, the Engineers will have completed a stretch where they will have played 11 games in nine days.
On April 1, MIT plays an important regional non-conference game against Brandeis. The last two years these two teams have locked up in classic pitching duels with Brandeis coming out on top, 3-2, in both occasions. After playing Coast Guard in a single, nine-inning home game on the 3rd, MIT will travel to No. 13 Wheaton in a rematch of the 2007 NEWMAC Semifinal game. MIT will be looking to exact revenge on the Lyons, who defeated the Engineers on three separate occasions last year on the way to earning the NEWMAC's automatic bid to the NCAA Tournament.
Beginning with the doubleheader against Wheaton, MIT begins an important stretch of conference play against Clark, WPI, Coast Guard, and Springfield. Also mixed in over these two weeks are non-conference tilts against Eastern Nazarene and Elms. On the 20th of April, MIT will travel to NESCAC foe Wesleyan University in the Engineers' final games before the NEWMAC Tournament. Last season, MIT had an impressive 5-2 record versus NESCAC opponents.
The NEWMAC Tournament will have a new twist this spring as the conference has implemented a new playoff format. Teams seeded #1 through #6 in the regular season will now play single nine-inning games on April 23 and 24. If the Engineers can advance past the first two days of the tournament, they will earn their program-record third consecutive appearance in the NEWMAC Semifinals. The semifinal and final rounds are set for April 26-27.
The last week of the regular season will see MIT play non-conference games against some quality regional opponents. After playing single nine-inning games against Worcester State and UMass-Boston, the Engineers will host 2007 NCAA Tournament participant Williams College in a doubleheader on May 3. Last season, the Ephs captured the NESCAC post-season title and concluded the season with a 27-10 record. The Engineers have defeated Williams in each of the past two seasons, and last year's 12-7 win by MIT snapped what had been an impressive seven-game winning streak. The next day, Suffolk comes to MIT looking to break a three-game losing streak against the Engineers. The regular season comes to a close on May 6 with a road game against MASCAC opponent Fitchburg State.
In two of the last three seasons, the conclusion of the regular season has been an exciting time for the Engineers as they have qualified for the ECAC Tournament. This season, MIT will look to make its first appearance in the NCAA Tournament since 1974. This year's NCAA New England Regionals are scheduled to begin on May 14 and will continue through the 18th. The Division III World Series, to be played at Fox Cities Stadium in Appleton, Wisconsin, is slated to begin on the 23rd with a champion to be determined on the 27th.


text:
Visit our main website at www.StDavids.com
Post Truh-mat-ick Head-ak
byCynthia M. Johnson, MA
A post-traumatic headache is a common symptom following injury or trauma to the head and neck.
Copyright © Nucleus Medical Media, Inc.
Post-traumatic headache occurs after a trauma to the head and neck. The headache may be caused by:
There are different types of headaches. Common post-traumatic headache types include:
The headache may also include psychiatric, behavioral, or social factors.
Your chance of a getting a post-traumatic headache is increased if you have had a history of head injuries.
A post-traumatic headache may occur right after the injury or as the injury is healing. Symptoms may include:
You will be asked about your symptoms, medical history, and any recent injuries. A physical exam will be done. The diagnosis is based on the exam and history. You may be referred to a doctor who specializes in the brain and nervous system if the headache persists, there are changes on the exam, or it is severe.
You will also be asked about the frequency and pattern of your headaches. To help provide answers, you may consider keeping a diary of:
Images may be taken of your brain, head, and neck to look for signs of injury. This can be done with:
Talk with your doctor about the best treatment plan for you. Options include:
Your doctor may advise medications to help manage pain. Medication options may include:
Head and neck injuries can take some time to heal. It is important to follow your doctor's advice and adjust activities as needed. Complete rest is rarely needed.
Other home care steps may include ice and massage.
Stress-reduction techniques and stress-management techniques may also help decrease or manage pain.
To help reduce your chance of getting a head and neck injury, take these steps:
Family Doctor—American Academy of Family Physicians
National Headache Foundation
Canadian Association of Emergency Physicians
Health Canada
Finkel A. Concussion and post-traumatic headache. American Headache Association website. Available at: http://www.americanheadachesociety.org/assets/1/7/Alan_Finkel_-_Concussion_and_PTH.pdf. Accessed January 14, 2015.
Headache. EBSCO DynaMed website. Available at: http://www.ebscohost.com/dynamed. Updated June 24, 2014.
Headaches after head injuries—post-traumatic headaches. Brainline.org website. Available at: http://www.brainline.org/content/2008/12/headaches-after-head-injuries-8212-post-traumatic-headaches.html. Accessed January 14, 2015.
Headaches after traumatic brain injury. Model Systems Knowledge Translation Center website. Available at: http://www.msktc.org/tbi/factsheets/Headaches-After-Traumatic-Brain-Injury. Accessed January 14, 2015.
Lane JC, Arciniegas DB. Post-traumatic headache. Curr Treat Options Neurol. 2002 Jan;4(1):89-104.
Lenaerts ME, Couch JR. Posttraumatic headache. Curr Treat Options Neurol. 2004;6:507-517.
Marcus DA. Disability and chronic post-traumatic headache. Headache: Journal of Head & Face Pain. 2003;43:117-121.
Post-traumatic headache. National Headache Foundation website. Available at: http://www.headaches.org/education/Headache_Topic_Sheets/Post_Traumatic_Headache. Accessed January 14, 2015.
Sheedy J, Harvey E, et al. Emergency department assessment of mild traumatic brain injury and the prediction of post-concussive symptoms in a 3-month prospective study. J Head Trauma Rehabil. 2009;24(5):333-343.
Last reviewed January 2015 by Michael Woods, MD
Please be aware that this information is provided to supplement the care provided by your physician. It is neither intended nor implied to be a substitute for professional medical advice. CALL YOUR HEALTHCARE PROVIDER IMMEDIATELY IF YOU THINK YOU MAY HAVE A MEDICAL EMERGENCY. Always seek the advice of your physician or other qualified health provider prior to starting any new treatment or with any questions you may have regarding a medical condition.
Copyright © EBSCO Publishing. All rights reserved.
Your Care Team, the right expertise at the right time. Our Physicians Our Care Team
Acute inpatient rehabilitation provides you the intensity and level of care you need for your road to recovery. Learn More
Privacy Policy Patient Rights Terms & Conditions Remote Gateway Access Joint Commission


text:
A small church in Gainesville, Fla., (ironically called Dove World Outreach Center) has announced plans to burn copies of the Quran on Sept. 11, in vengeful commemoration of the terrorist attacks on New York and Washington nine years ago. This news item happened to reach me while I was attending a conference in Berlin, Germany - and for that reason it struck me with special force.
Earlier that same day, I had stood in a public square known as the Bebelplatz, just across the street from Berlin's Humboldt University. In the middle of the Bebelplatz there is a translucent panel embedded in the pavement. Looking down through the glass, you see below ground many rows of white bookshelves, all completely empty.
This simple but moving memorial is designed to recall the events of May 10, 1933, when Nazi propaganda minister Joseph Goebbels instigated a book-burning to destroy books by Jewish authors, along with other ideologically "incorrect" works. With the eager participation of the SS and the Hitler Youth, more than 20,000 books were burned in the Bebelplatz on that infamous day, in the shadow of Germany's pre-eminent university.
Nearby stands a plaque containing the words of the German poet Heinrich Heine: "Dort, wo man Bücher verbrennt, verbrennt man am Ende auch Menschen" ("There, where they burn books, they will in the end burn people.") Heine wrote those words in 1820. One hundred and thirteen years later, his prophecy began coming true, and Heine's own works were among those burned by the Nazis.
Having just witnessed Berlin's rueful monument of public contrition, I was dismayed to hear the report of the planned book-burning in Florida - not least because I fear this planned event is a sign and symptom of the angry temper of our times.
Many Christian leaders in the United States promptly disavowed and deplored the spiteful plans of the tiny Florida church. Richard Land, director of the Southern Baptist Convention's Ethics and Religious Liberty Commission, called the plan "appalling, disgusting and brainless."
News reports gave several reasons for such disapproval: Burning Muslim books is contrary to America's own ideals of freedom of speech and religion; such actions will exacerbate tensions between Christians and Muslims around the world; if Christians burn the Quran, we may soon see Muslims burning Bibles. (The last point seems to be a pragmatist permutation of the Golden Rule: Don't do something bad to somebody else, because if you do, they may retaliate.)
But in fact followers of Jesus have deeper reasons not to burn the books of their enemies, reasons integral to their own faith.
The apostle Paul, struggling against opponents of his gospel in the city of Corinth, insisted that "the weapons we fight with are not the weapons of the world." Rather than resorting to violence, he sought to "demolish arguments" and "captivate every thought" through open statement of the truth.
For him, to use coercive or deceptive means would be to succumb to the forces he was opposing. His message could be defended only by clear, peaceful proclamation of the word. As Angel Nuñez of the National Hispanic Christian Leadership Conference observed, "The greatest weapon a Christian has is godly love."
Similarly, the Gospel of Luke tells a story about Jesus' response to a Samaritan village that rejected him and his followers. His disciples James and John asked, "Lord, do you want us to command fire to come down from heaven and consume them?" But Jesus rebuked them and said (according to some ancient manuscripts of Luke's Gospel), "No, you don't know what spirit you belong to" (Luke 9:51-56).
I fear that my Christian brothers and sisters in the Dove World Outreach Center, like James and John, do not know what spirit they belong to. If they burn the Quran, they will be acting in the spirit of Goebbels. History has taught us where that leads. But if they listen to Jesus, they will learn that his way is not to call down fire against enemies. Instead, his way is to commend the gospel through open statement of the truth - to act in a spirit of patience and generosity, to return good for evil, to pray for those who hurt us. That is the spirit of Jesus.
I pray that the citizens of Gainesville will not someday in the future need to build a memorial like the one in Berlin's Bebelplatz.
Richard B. Hays is the dean of Duke Divinity School.
Thursday, September 23, 2010
WWJD? Not burn the Quran
Subscribe to: Post Comments (Atom)


text:
Minnehaha County, Communities Receive Block Grants for Projects
PIERRE, S.D. (AP) - Minnehaha County and five communities in South Dakota have received federal grants for a variety of projects.
Gov. Dennis Daugaard has announced that Brookings, De Smet, Flandreau, Fort Pierre, Twin Brooks and Minnehaha County will receive more than $1.7 million to assist with projects totaling more than $7 million.
The funding will cover a variety of projects, including creating or improving Boys and Girls Clubs, building a wellness center and improving sanitary sewer systems.
The Community Development Block Grant is a reimbursement program, and communities receive funds as work is finished.
The federal Department of Housing and Urban Development funds the CDBG Program, and the Governor's Office of Economic Development administers it.
Copyright 2012 by The Associated Press. All rights reserved. Material may not be redistributed.


text:
DAMASCUS, Syria, March 13 (UPI) -- Two million children, at risk for malnutrition and disease, are caught in the civil war between Syrian troops and rebels, Save the Children said Wednesday.
In its report, "Childhood Under Fire," released to mark two-year anniversary of violence in Syria, Save the Children details the impact of the conflict on children, finding many struggle to find enough to eat; live in barns, parks and caves, and can't attend school because teachers have fled and schools have been attacked.
The Save the Children report, citing research by Bahcesehir University in Turkey, said one in three children reported being hit, kicked or fired upon.
"Childhood under Fire" also reported how some young boys were used by armed groups while some girls were married off early to "protect" them from a perceived threat of sexual violence, the organization based in Westport, Conn., said.
"For millions of Syrian children, the innocence of childhood has been replaced by the cruel realities of trying to survive this vicious war," said Carolyn Miles, Save the Children president and chief executive officer. "Many are now living out in the open, struggling to find enough to eat, without the right medicine if they become sick or injured. We cannot allow this to continue unchecked; the lives of too many children are at stake."
Save the Children, which is providing humanitarian aid in Syria and neighboring countries, has called for all parties to the conflict to allow safe access to populations in need and to ensure all is being done to end the fighting.
Save the Children has planned vigils in 21 countries Thursday to mark two years of conflict in Syria, as well as a virtual vigil.


text:
JACKSONVILLE — The Jaguars' stated free-agent philosophy since the current administration arrived in January 2013: Sign players in their mid-20s and avoid having any guaranteed money beyond the second year of the contract.
That approach hasn't worked out, evident by the Jaguars' 15-48 record during that span.
The new standard should be to acquire more experienced players .
General manager Dave Caldwell should see no other alternative; 2017 will be the final year of his contract. The Jaguars will have salary-cap space (as much as $70 million) to fortify the pass rush, offensive line and tight-end spots. Maybe that means pursuing players who aren't yet available but hit...


text:
We use cookies to ensure that we give you the best experience on our website. If you continue without changing your browser settings, we'll assume that you are happy to receive all cookies on the Knight Frank website.
Find out more about how Knight Frank uses cookies
The report provides expert insight from our international network, highlights recent market performance, buyer demand and features a selection of prime properties being marketed by Knight Frank.
For more information contact:


text:
SDSU Basketball Tickets Selling at Record Level
BROOKINGS, SD -- Following NCAA Tournament runs by the both the Jackrabbit men's and women's basketball teams, season ticket sales are at an all-time high at South Dakota State University.
As of Sept. 19, 1,565 full season tickets have been sold for the 2012-13 season, far surpassing the previous record that was set last year with 1,348 season tickets.
"Our fans truly understand that a home-court advantage starts in the stands," said Director of Athletics Justin Sell. "Our student-athletes feed off the energy and noise created in Frost Arena, and we thank everyone for making our court the best environment in the Summit League."
Season tickets are still available, and can be purchased by calling the Jackrabbit Ticket Office at 1-866-GO JACKS (465-2257) or by logging on to JackrabbitTickets.com. Full-season packages start at only $9 per game.
Courtesy: South Dakota State University


text:
Emily Dickinson
Emily Dickinson (10 December 1830 – 15 May 1886 / Amherst / Massachusetts)
» Click to list general statistics » OR, select a date :
You Said That I
You said that I "was Great"—one Day—
Then "Great" it be—if that please Thee—
Or Small—or any size at all—
Nay—I'm the size suit Thee—
read more >>
- Hits: Number of visitors who read the poem on the given date.
- Posts: Number of visitors who posted the poem to a friend (via e-mail) on the given date.
- Dates that have "0" hits may not be shown on the list.
- Dates, poems and poets that have "0" hits may not be shown on the list. Statistics are generated daily.


text:
relets is a valid word in this word list. For a definition, see the external dictionary links below.
The word "relets" uses 6 letters: E E L R S T.
Direct anagrams of relets:
Words formed by adding one letter before or after relets (in bold), or to eelrst in any order:
a - elaters realest relates reslate stealer b - belters trebles c - tercels f - reflets telfers g - reglets h - shelter i - leister retiles sterile k - kelters kestrel skelter l - retells tellers m - melters remelts resmelt smelter n - nestler relents o - soleret p - pelters petrels respelt spelter s - streels tressel t - letters settler sterlet trestle v - svelter w - swelter welters wrestle y - restyle tersely z - seltzer
Shorter words found within relets:
eel eels el els else er ere ers erst es ester et lee leer leers lees leet leets lest let lets re ree reel reels rees reest relet res reset rest ret rete rets see seel seer sel ser sere set sleet steel steer stele stere tee teel teels tees tel tele teles tels terse tree trees
List shorter words within relets, sorted by length
Words formed from any letters in relets, plus an optional blank or existing letter
Try a search for relets in these online resources (some words may not be found):
Wiktionary - OneLook Dictionaries - Merriam-Webster - Google Search
Each search will normally open in a new window.
List all words starting with relets, words containing relets or words ending with relets
All words formed from relets by changing one letter
Other words with the same letter pairs: re el le et ts
Browse words starting with relets by next letter
Previous word in list: relet
Next word in list: reletter
Some random words: weskit eucaine uintahite teds phaeton seeing mho
This is not a dictionary, it's a word game wordfinder.
- Help and FAQ - Examples - Home
Privacy and Cookies Policy - Share - © Copyright 2004-2019 Morewords.com - 26.229mS


text:
|Olney, Md.
|High School:
|Freestyle, IM
|Sport Management
2011-12: Embarks on her second collegiate swimming campaign ... gained outstanding experience as a rookie ... is a strong student-athlete who can power through the water ... committed to consistent improvement ... a hard-working youngster who will look to turn that work into greater success as a sophomore.
2010-11: Swam well in her first season with the Spartans … at her first CAC championships, recorded times of 5:27.43 in the 500 free, a 18:54.40 in the 1650 free, and a 5:02.13 in the 400 IM … part of York's third-place 800 freestyle relay squad as she helped the group to a time of 8:19.33 ... also, posted times of 1:02.77 in the 100 free and a 2:09.17 in the 200 free at the Marymount/W&J meet on November 6 … registered times of 32.68 in the 50 backstroke and a 1:17.03 in the 100 backstroke at the Rowan Cup on November 20.
Personal: Is a sport management major ... is the daughter of Brian and Maura Hinkle ... born on July 6, 1992 ... swam on the club level for the Flying Gull Aquatic Club ... names her parents as the people she most admires ... hobbies include hanging out with friends, going to the beach, and watching television ... favorite athletes are Natalie Coughlin and Alex Ovechkin ... chose York because of the "beautiful campus, nice size, and friendly people" ... lists her most significant athletic achievement as making METROs in high school and posting her best times ... her most memorable athletic moment while at York is taking 10th in the 500 and 1650 freestyle races at her first CAC Championships while also helping York to a third-place finish in the 800 freestyle relay ... and after graduation she plans to have a successful career and travel the world.


text:
TaxationJuly 11, 2005
Here's one for any fellow countrymen who intend to work with companies in the United States.
Live in Canada? Run a small web design studio or development business? Looking to work with a company based in the US? Got hit with IRS paperwork? There's good news.
First and foremost though, the legal stuff: I am not a lawyer, this can not be construed as legal advice, and I can't guarantee the accuracy of these statements. This is for informational purposes only. You'll want to consult with a lawyer who specializes in US tax law to analyze your own situation, as there are bound to be various specifics that need the attention of a professional. If printed, this article must be printed in its entirety and feature this notice.
As the owner of a small Canadian design studio, I frequently take on contracts with US-based businesses. The flow of money across the border is obviously important to the governments involved, as it's taxable income. I've consulted with US tax lawyers on this issue to make sure I'm on solid legal ground, and I'm simply passing on what I've been able to interpret about what I found out during these consultations. Again, it's simply my interpretation, not genuine legal advice. (Yes, it's tricky to know when to quit with the disclaimers…)
When initiating a new contract, the foreign company I'm working with usually passes along paperwork that needs to be completed before any payment can be issued. The larger the company, the more likely it is to happen. The paperwork usually consists of an IRS form W8-BEN (PDF link), along with any internal forms their accounting department requires. The latter are for the company's own records, and a necessary hoop to jump through. What interests us here is the W8-BEN. This is where it gets tricky.
The accounting department is usually working on the basis of local tax laws, whereas in your case, if you're working on Canadian soil, those laws may not apply. (The laws are different for Canadian citizens working on US soil, but I still have yet to figure out how THAT works.) The important point to note here is that the international US-Canada tax treaty supersedes any local law where there's overlap or potential conflict. Getting your hands on a copy, and then deciphering it, though, is no easy task. Again for information purposes only, here's an online version which may or may not be accurate. If you can find all the articles that apply to you, and then actually understand their implications, you'll be fine. Unfortunately, for pretty much all of us who didn't study law in school, doing so means hiring a lawyer.
Essentially, the Article relevant to the situation in which you and I find ourselves is Article VII. When working for hire, you're on the hook for paying taxes only to the country in which you reside. Sorry, that does mean your US income is taxable by the Canadian government just the same as any Canadian income, and you are responsible for filing your own return. But on the bright side, no, you don't have to pay tax on it twice. Canada has tax jurisdiction, and you are only accountable to the Canada Revenue Agency. (And if you have a GST number you probably know this already, but it's relevant to point out here that you shouldn't be charging GST when billing foreign companies.)
So what about the paperwork? You aren't required to do it. But that comes with a caveat: convincing the foreign accounting department of that may be difficult, if they don't have anyone on staff familiar with the tax treaty. This article may provide you with a bit of negotiating power when dealing with them. Though you don't need to, you may find it less burdensome simply to keep a completed W8-BEN on hand and send it in when requested, instead of arguing out the specifics every time. There's no real harm in doing so, other than the fact that a foreign government agency has a clear trail of your income. (And considering your clients will be reporting the payments, they probably do anyway.)
The W8-BEN does exist for a reason though; the case we've been looking at here depends on a few things:
- You must reside in, and perform all services in Canada.
- The compensation is received strictly for work for hire, and not for other services, or forms of royalties, capital gains, or other types of income.
In the case of other types of income (royalties became relevant to me last year, for example), the W8-BEN is a way to claim additional treaty benefits. In very few instances will you actually have to pay the IRS anything, but there are various other reasons why you may need to fill out the form to make sure a percentage of your income isn't withheld.
When a W8-BEN is required, unfortunately you first need to apply for a US taxpayer number, whether that's an Employer Identification Number (EIN) or an Individual Taxpayer Identification Number (ITIN). If you own a business, you have the ability to call the IRS directly and get an EIN for your business; expect the number to arrive by mail within a month. If you apply for an ITIN, the wait will take a lot longer and you must visit a US consulate (or visit the country itself) to have your application notarized by an approved US notary. Considering the narrow window the notary was available at my own local consulate (M,W,F, 1:30-4:30pm only) and the long wait for approval, I would highly recommend trying for an EIN first if at all possible.
I'll leave you with some parting advice. If you think you might find yourself in a situation where any of this advice is relevant, consult with a tax lawyer now. Bring a laptop or a notepad and write down everything you possibly can during that consultation. The time and effort you save will more than offset the few hundred dollars an hour; researching this information on your own will not be worth the effort, and misunderstandings can lead to litigation, even if they're honest mistakes.
Any comments below are solely the opinion of the poster, and can not be construed as legal advice.
I think you can file for an EIN online as well and get it in less than a week. However, I'm not sure this works for Canadians… Perhaps just US Companies.
I think everyone is too scared to post comments
Thanks for the advice.
Er, yes, I did indeed mean consulate. Thanks for pointing that out.
OMG - and I thought EU VAT regulations were complex…
i think dave's legal counsel did a number on him. he's spewing legal-speak out of every orifice. ;) however, cheers for the tax tip for the canucks.
Thanks for the article. Being a Canadian I appreciate the advice from experience and I should be seeking legal advice once the time comes.
Just to clarify, did you mean that you have to visit a US consulate to get an ITIN? (The embassy is fine for those living in Ottawa, but it would be a big pain for everyone else.)


text:
The thing is, UVA will provide more opportunities for public interest than Tulane. Public Interest positions where you have the ability to make a real impact are just as competitive as BIGLAW jobs.
Ok. So Im not arguing either side. However, I want to point out something that may have been overlooked
summer positions.A few months ago while discussing transferring to a higher ranked school Googler (http://www.lawschooldiscussion.org/prelaw/index.php/topic,51296.0.html) provided an example of a friend who has applied for positions in the NYC area but got no call backs. After the friend transferred to Cornell in July and reapplied for the same positions, they received some positive responses. Now keep in mind that well paying summer positions can net you tens of thousands.
I'm a rankings whore on the inside, but I don't let it make my decisions for me. I'll be at Wash U next year on a full scholarship (yay for the Olin Fellowship!) although I probably could have gone to a better ranked school with 3.65/169/very strong ECs. (I planned to apply to Fordham, Duke, Penn, Harvard, Yale, NYU, Columbia, and BU as well, but after finding out about my $25/year SOL award in October I only ended up applying to Wash U, NYU and Columbia)I honestly applied to NYU and Columbia because of their rank/prestige and location, not because I thought they were a good fit. I think deep down I was just hoping I'd be able to say I turned one of them down, because I figured I'd end up at Wash U either way. (At the moment I haven't heard from either, so probably bad news. However, doesn't really bug me)Sometimes it bugs me when people trash Wash U because it really is a great school, but who cares what others think?


text:
HANG TIME HEADQUARTERS — Blake Griffin said Lob City was dead.
He lied.
And that might actually be a good thing for the Los Angeles Clippers.
That obituary highlighted here and elsewhere at the start of training camp was a tad bit premature. The Clippers remain a highlight show waiting to happen under Doc Rivers, courtesy of Griffin and plays like this one (sorry Rudy Gobert). I'm not complaining, mind you. I loved everything about the Clippers' Lob City routine. When you are a team in the midst of the transformation the Clippers are in right now, a clear-cut identity is a good thing to get a handle on.
And when you possess the parts the Clippers do — Griffin, a masterful architect in Chris Paul, a fellow high-flyer in big man DeAndre Jordan and others — playing to those strengths, at least offensively, makes perfect sense.
The Clippers have the personnel to run several different styles. Versatile talents like Jamal Crawford, Jared Dudley, J.J. Redick (who made his preseason debut in Wednesday night's win over the Utah Jazz), Matt Barnes, Ryan Hollins, Byron Mullens, Darren Collison and others give Rivers an assortment of player to choose from on any given night.
Continuing the Lob City movement would be no problem, if that's what Rivers wanted to do. Rivers, of course, has already made it clear he has something else in mind. He wants to upgrade the toughness of this crew and make sure they are don't fall into the trap of thinking highlight plays will deliver them deep into the playoffs.
It's going to take more than a month to teach, preach and truly embed his philosophy into the collective psyche of this bunch.
But Paul is the ideal leader to spearhead Rivers' effort. He bought in immediately, before free agency began in July. So Rivers has already solidified the initial bond with the principles needed to run whatever system he needs. Griffin and Jordan are his biggest projects and, according to Ramona Shelburne of ESPNLosAngeles.com, they're all in as well:
"Actions speak louder than words. You can say this or that, but then you get out there and it doesn't really happen," Griffin said when asked why he has responded to Rivers' approach so quickly. "I think that's the biggest reason. He says it, and then you see it in action."
As the senior vice president of basketball operations, Rivers has the power to trade any of his players. That's something of a double-edged sword for a coach. That power isn't always a good thing. But in Rivers' case, so far at least, it has helped his relationships with the two players who are crucial to the Clippers' championship aspirations.
From the jump, he looked both in the eye and said he believed in them.
"As soon as [Rivers] got here he told me, 'We're going to do it here with you,' " Jordan said. "He looked me in my eyes and I knew he was telling the truth.
"I respected that on a different level once he told me that up front."
It was exactly what he needed to hear after two seasons of feeling insecure about his place in the organization's plans, and a summer of reading his name in trade rumors.
"This summer, I just felt like he had to be here for us to be what we want to be," Rivers said. "And he's done it. He's really played terrific basketball. His defense has been unbelievable.
"You guys can't hear his talk, but I can. His talk on the floor has been terrific. I didn't know DeAndre so I didn't know if he could or would do that. But he's been just off the charts."
All of that precious Ubuntu-esque chatter can't obscure the obvious. The Clippers, at least through the preseason, are still relying on the same things they did before Rivers arrived. Lob City is alive and kicking.
Chances are the Clippers will need to lean on that familiar mode of operation at times throughout this season, even as they pledge to continue digging that early grave for Lob City!