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Find video protocols related to scientific articles indexed in Pubmed.
Hymenoscyphus pseudoalbidus, the causal agent of European ash dieback.
Mol. Plant Pathol.
PUBLISHED: 10-07-2013
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The ascomycete Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea) causes a lethal disease known as ash dieback on Fraxinus excelsior and Fraxinus angustifolia in Europe. The pathogen was probably introduced from East Asia and the disease emerged in Poland in the early 1990s; the subsequent epidemic is spreading to the entire native distribution range of the host trees. This pathogen profile represents a comprehensive review of the state of research from the discovery of the pathogen and points out knowledge gaps and research needs.
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Mitigation of antagonistic effects on plant growth due to root co-colonization by dark septate endophytes and ectomycorrhiza.
Environ Microbiol Rep
PUBLISHED: 07-18-2013
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Dark septate endophytes (DSE) are very common root colonizers of woody plant species. Ascomycetes of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) are the main representatives of DSE fungi in forest ecosystems. PAC and mycorrhizal fungi share the same habitat, but interactions among PAC, mycorrhizal fungi and plants are poorly understood. We compared the effects of single and dual inoculation of Norway spruce seedlings with PAC and the ectomycorrhizal (ECM) fungus Hebeloma crustuliniforme on host growth, degree of mycorrhization and density of endophytic PAC biomass. Single colonization by H.?crustuliniforme or PAC significantly reduced plant biomass. Dual colonization reduced or neutralized plant growth depression caused by single fungal colonization. The degree of mycorrhization was independent on PAC colonization, and mycorrhization significantly reduced endophytic PAC biomass. Plant biomass of dually colonized plants positively correlated with PAC biomass. These results demonstrate the ability of dual inoculation of PAC and H.?crustuliniforme to neutralize plant growth depression caused by single fungal inoculation. Our explanations of enhanced plant growth in dually inoculated plants are the inhibition of PAC during root colonization by the ECM mantle and ECM-mediated access to plant growth-promoting nutrients resulting from the mineralization of the potting medium by PAC.
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Resistance to Dutch elm disease reduces presence of xylem endophytic fungi in Elms (Ulmus spp.).
PLoS ONE
PUBLISHED: 01-16-2013
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Efforts to introduce pathogen resistance into landscape tree species by breeding may have unintended consequences for fungal diversity. To address this issue, we compared the frequency and diversity of endophytic fungi and defensive phenolic metabolites in elm (Ulmus spp.) trees with genotypes known to differ in resistance to Dutch elm disease. Our results indicate that resistant U. minor and U. pumila genotypes exhibit a lower frequency and diversity of fungal endophytes in the xylem than susceptible U. minor genotypes. However, resistant and susceptible genotypes showed a similar frequency and diversity of endophytes in the leaves and bark. The resistant and susceptible genotypes could be discriminated on the basis of the phenolic profile of the xylem, but not on basis of phenolics in the leaves or bark. As the Dutch elm disease pathogen develops within xylem tissues, the defensive chemistry of resistant elm genotypes thus appears to be one of the factors that may limit colonization by both the pathogen and endophytes. We discuss a potential trade-off between the benefits of breeding resistance into tree species, versus concomitant losses of fungal endophytes and the ecosystem services they provide.
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Host species and strain combination determine growth reduction of spruce and birch seedlings colonized by root-associated dark septate endophytes.
Environ. Microbiol.
PUBLISHED: 12-29-2011
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Interactions of Betula pendula and Picea abies with dark septate endophytes of the Phialocephala fortinii-Acephala applanata species complex (PAC) were studied. PAC are ubiquitous fungal root symbionts of many woody plant species but their ecological role is largely unknown. Sterile birch and spruce seedlings in monoculture and mixed culture were exposed to four PAC strains, added either singularly or paired in all possible combinations at 18°C and 23°C. Plant and fungal biomass was determined after 4 months. The most significant factors were strain and host combination. One of the strains significantly reduced biomass gain of spruce but not of birch. Plant biomass was negatively correlated with total endophytic fungal biomass in half of the strain - plant combinations. Endophytic PAC biomass was four times higher in spruce (? 40 mg g(-1) drw) than in birch (? 10 mg g(-1) drw). Competition between strains was strain-dependent with some strains significantly reducing colonization density of other strains, and, thus, attenuating adverse effects of pathogenic strains on plant growth in some strain - plant combinations. Biomass gain of spruce but not of birch was significantly reduced at higher temperature. In conclusion, host, fungal genotype, colonization density and presence of a competing PAC strain were the main determining factors for plant growth.
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Negative effects on survival and performance of Norway spruce seedlings colonized by dark septate root endophytes are primarily isolate-dependent.
Environ. Microbiol.
PUBLISHED: 08-04-2011
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Root endophytes are common and genetically highly diverse suggesting important ecological roles. Yet, relative to above-ground endophytes, little is known about them. Dark septate endophytic fungi of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC) are ubiquitous root colonizers of conifers and Ericaceae, but their ecological function is largely unknown. Responses of Norway spruce seedlings of two seed provenances to inoculations with isolates of four PAC species were studied in vitro. In addition, isolates of Phialocephala subalpina from two populations within and one outside the natural range of Norway spruce were also included to study the effect of the geographic origin of P. subalpina on host response. The interaction of PAC with Norway spruce ranged from neutral to highly virulent and was primarily isolate-dependent. Variation in virulence was much higher within than among species, nonetheless only isolates of P. subalpina were highly virulent. Disease caused by P. subalpina genotypes from the native range of Norway spruce was more severe than that induced by genotypes from outside the natural distribution of Norway spruce. Virulence was not correlated with the phylogenetic relatedness of the isolates but was positively correlated with the extent of fungal colonization as measured by quantitative real-time PCR.
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Microsatellite-based quantification method to estimate biomass of endophytic Phialocephala species in strain mixtures.
Microb. Ecol.
PUBLISHED: 01-22-2011
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Fungi of the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC) are ubiquitous endophytic colonizers of tree roots in which they form genotypically diverse communities. Measurement of the colonization density of each of the fungal colonizers is a prerequisite to study the ecology of these communities. Up to now, there is no method readily available for the quantification of PAC strains co-colonizing the same root. The new DNA quantification method presented here is based on the amplification of microsatellites by competitive polymerase chain reaction (PCR). The method proved to be suitable to detect and quantify at least two strains within one single sample by the addition of a known amount of mycelium of a reference strain before DNA extraction. The method exploits the correlation between the reference/target ratio of light emitted during microsatellite detection (peak ratio) and the reference/target ratio of mycelial weights to determine the biomass of the target strain. Hence, calibration curves were obtained by linear regression of the peak ratios on the weight ratios for different mixtures of reference and target strains. The slopes of the calibration curves and the coefficients of determination were close to 1, indicating that peak ratios are good predictors of weight ratios. Estimates of fungal biomass in mycelial test mixtures of known composition laid within the 95% prediction interval and deviated on average by 16% (maximally 50%) from the true biomass. On average, 3-6% of the root biomass of Norway spruce seedlings consisted of mycelial biomass of either one of two inoculated PAC strains. Biomass estimates obtained by real-time quantitative PCR were correlated with the estimates obtained by the microsatellite-based method, but variation between the two estimates from the same root was high in some samples. The microsatellite-based DNA quantification method described here is currently the best method for strainwise estimation of endophytic biomass of PAC fungi in small root samples.
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Suitability of quantitative real-time PCR to estimate the biomass of fungal root endophytes.
Appl. Environ. Microbiol.
PUBLISHED: 07-02-2010
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A nested single-copy locus-based quantitative PCR (qPCR) assay and a multicopy locus-based qPCR assay were developed to estimate endophytic biomass of fungal root symbionts belonging to the Phialocephala fortinii sensu lato-Acephala applanata species complex (PAC). Both assays were suitable for estimation of endophytic biomass, but the nested assay was more sensitive and specific for PAC. For mycelia grown in liquid cultures, the correlation between dry weight and DNA amount was strong and statistically significant for all three examined strains, allowing accurate prediction of fungal biomass by qPCR. For mycelia colonizing cellophane or Norway spruce roots, correlation between biomass estimated by qPCR and microscopy was strain dependent and was affected by the abundance of microsclerotia. Fungal biomass estimated by qPCR and microscopy correlated well for one strain with poor microsclerotia formation but not for two strains with high microsclerotia formation. The accuracy of qPCR measurement is constrained by the variability of cell volumes, while the accuracy of microscopy can be hampered by overlapping fungal structures and lack of specificity for PAC. Nevertheless, qPCR is preferable because it is highly specific for PAC and less time-consuming than quantification by microscopy. There is currently no better method than qPCR-based quantification using calibration curves obtained from pure mycelia to predict PAC biomass in substrates. In this study, the DNA amount of A. applanata extracted from 15 mm of Norway spruce fine root segments (mean diameter, 610 microm) varied between 0.3 and 45.5 ng, which corresponds to a PAC biomass of 5.1 +/- 4.5 microg (estimate +/- 95% prediction interval) and 418 +/- 264 microg.
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Phylogeny of Phaeomollisia piceae gen. sp. nov.: a dark, septate, conifer-needle endophyte and its relationships to Phialocephala and Acephala.
Mycol. Res.
PUBLISHED: 04-08-2009
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Dark, septate endophytes (DSE) were isolated from roots and needles of dwarf Picea abies and from roots of Vaccinium spp. growing on a permafrost site in the Jura Mountains in Switzerland. Two of the isolates sporulated after incubation for more than one year at 4 degrees C. One of them was a hitherto undescribed helotialean ascomycete Phaeomollisia piceae gen. sp. nov., the other was a new species of Phialocephala, P. glacialis sp. nov. Both species are closely related to DSE of the Phialocephala fortinii s. lat.-Acephala applanata species complex (PAC) as revealed by phylogenetic analyses of the ITS and 18S rDNA regions. Morphologically dissimilar fungi, such as Vibrissea and Loramyces species, are phylogenetically also closely linked to the new species and the PAC. Cadophora lagerbergii and C. (Phialophora) botulispora are moved to Phialocephala because Phialocephala dimorphospora and P. repens are the closest relatives. Several Mollisia species were closely related to the new species and the PAC according to ITS sequence comparisons. One DSE from needles of Abies alba and one from shoots of Castanea sativa formed Cystodendron anamorphs in culture. Their identical 18S sequences and almost identical ITS sequences indicated Mollisia species as closest relatives, suggesting that Mollisia species are highly euryoecious.
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The ectomycorrhizal morphotype Pinirhiza sclerotia is formed by Acephala macrosclerotiorum sp. nov., a close relative of Phialocephala fortinii.
Mycorrhiza
PUBLISHED: 03-17-2009
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Relatively few ectomycorrhizal fungal species are known to form sclerotia. Usually, sclerotia are initiated at the extraradical mycelium. In this study, we present anatomical and ultrastructural evidence for the formation of sclerotia directly in the hyphal mantle of the mycorrhizal morphotype Pinirhiza sclerotia. A dark-pigmented fungal strain was isolated from Pinirhiza sclerotia and identified by molecular tools as Acephala macrosclerotiorum sp. nov., a close relative of Phialocephala fortinii s.l. As dark septate fungi are known to be mostly endophytic, resyntheses with Pinus sylvestris and A. macrosclerotiorum as well as Populus tremula x Populus tremuloides and A. macrosclerotiorum or P. fortinii s.l. were performed under axenic conditions. No mycorrhizas were found when hybrid aspen was inoculated with A. macrosclerotiorum or P. fortinii. However, A. macrosclerotiorum formed true ectomycorrhizas in vitro with P. sylvestris. Anatomical and ultrastructural features of this ectomycorrhiza are presented. The natural and synthesized ectomycorrhizal morphotypes were identical and characterized by a thin hyphal mantle that bore sclerotia in a later ontogenetic stage. The Hartig net was well-developed and grew up to the endodermis. To our knowledge, this is the first evidence at the anatomical and ultrastructural level that a close relative of P. fortinii s.l. forms true ectomycorrhizas with a coniferous host.
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Cryptic speciation and community structure of Herpotrichia juniperi, the causal agent of brown felt blight of conifers.
Mycol. Res.
PUBLISHED: 01-22-2009
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Conifer twigs showing brown felt blight were collected along 100-m long transects at the timberline in the Swiss Alps and single-hyphal-tip cultures were prepared. Forty-seven of the sequenced 48 strains were Herpotrichia juniperi based on sequence comparisons of the internal transcribed spacers (ITS). A non-sporulating strain was tentatively identified as another, undescribed Herpotrichia species. Herpotrichia coulteri was not isolated. Most strains were from Juniperus communis var. saxatilis, the rest from Picea abies and Pinus mugo. Each twig was colonized by a different genotype as revealed by ISSR-PCR fingerprinting. More than one clone was present on some needles and twigs. Thus, importance of vegetative mycelial growth for dispersal seems to be limited to the spread of the disease to twigs of the same tree or of immediately adjacent trees, and, consequently, dispersal occurs mainly by ascospores. The H. juniperi strains could be assigned to five distinct groups based on the ISSR-PCR data. The strains from P. abies formed one of these groups but the other groups did not correlate with either host, transect or position along the transects. Multi-locus analysis based on beta-tubulin, elongation factor 1-alpha and ITS sequences confirmed the subdivision into five groups. Population differentiation among groups was distinct with N(ST) values varying between 0.545 and 0.895. H. juniperi seems to be composed of several cryptic species, one of them specific to P. abies.
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Skin sensitization to p-phenylenediamine: the diverging roles of oxidation and N-acetylation for dendritic cell activation and the immune response.
J. Invest. Dermatol.
PUBLISHED: 01-07-2009
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Skin is a target of allergic reactions to aromatic amine hair dye precursors, such as p-phenylenediamine (PPD). As conversion of PPD on or in the skin is expected to be required for the induction of allergic contact dermatitis, we analyzed the role of oxidation and N-acetylation as major transformation steps. PPD and its oxidative and N-acetylated derivatives were tested for their sensitizing potential in vitro using a dendritic cell (DC) activation assay and in vivo using the local lymph node assay (LLNA). PPD did not induce relevant DC activation but induced a positive LLNA response. In contrast, DC activation was obtained when PPD was chemically pre-oxidized or after air oxygen exposure. Under both conditions, the potent sensitizing PPD oxidation product Bandrowskis base was identified along with other di- and trimeric species, indicating that PPD oxidation products provide an effective immune stimulation (danger signal). In contrast mono- and diacetylated PPD did not induce DC activation or a positive LLNA response. We conclude that dermal N-acetylation of PPD competes with the formation of oxidized PPD whereas skin exposure conditions allowing auto-oxidation, as in the LLNA, provide an effective danger signal necessary to induce skin sensitization to PPD.
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Mycorrhiza reduces adverse effects of dark septate endophytes (DSE) on growth of conifers.
PLoS ONE
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Mycorrhizal roots are frequently colonized by fungi of the Phialocephala fortinii s.l.-Acephala applanata species complex (PAC). These ascomycetes are common and widespread colonizers of tree roots. Some PAC strains reduce growth increments of their hosts but are beneficial in protecting roots against pathogens. Nothing is known about the effects of PAC on mycorrhizal fungi and the PAC-mycorrhiza association on plant growth, even though these two fungal groups occur closely together in natural habitats. We expect reduced colonization rates and reduced negative effects of PAC on host plants if roots are co-colonized by an ectomycorrhizal fungus (ECM). Depending on the temperature regime interactions among the partners in this tripartite ECM-PAC-plant system might also change. To test our hypotheses, effects of four PAC genotypes (two pathogenic and two non-pathogenic on the Norway spruce), mycorrhization by Laccaria bicolor (strain S238N) and two temperature regimes (19°C and 25°C) on the biomass of the Douglas-fir (Pseudotsuga menziesii) and Norway spruce (Picea abies) seedlings were studied. Mycorrhization compensated the adverse effects of PAC on the growth of the Norway spruce at both temperatures. The growth of the Douglas-fir was not influenced either by PAC or mycorrhization at 19°C, but at 25°C mycorrhization had a similar protective effect as in the Norway spruce. The compensatory effects probably rely on the reduction of the PAC-colonization density by mycorrhizae. Temperature and the PAC strain only had a differential effect on the biomass of the Norway spruce but not on the Douglas-fir. Higher temperature reduced mycorrhization of both hosts. We conclude that ectomycorrhizae form physical and/or physiological barriers against PAC leading to reduced PAC-colonization of the roots. Additionally, our results indicate that global warming could cause a general decrease of mycorrhization making primary roots more accessible to other symbionts and pathogens.
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Do colonization by dark septate endophytes and elevated temperature affect pathogenicity of oomycetes?
FEMS Microbiol. Ecol.
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Phialocephala subalpina is one of the most frequent dark septate root endophytes in tree roots but its function in forest ecosystems is largely unknown. A full-factorial infection experiment was performed, using six P. subalpina isolates, two pathogenic oomycetes (Phytophthora plurivora [syn. Phytophthora citricola s.l.] and Elongisporangium undulatum [syn. Pythium undulatum]) and two temperature regimes (17.9 and 21.6 °C) to examine the ability of P. subalpina to protect Norway spruce seedlings against root pathogens. Seedling survival, disease intensity and seedling growth were affected by P. subalpina genotype, temperature and pathogen species. Some P. subalpina isolates effectively reduced mortality and disease intensity caused by the two pathogens. Elevated temperature adversely affected seedling growth but did not aggravate the effect of the pathogens. Elongisporangium undulatum but not P. plurivora significantly reduced plant growth. Colonization density of P. subalpina measured by quantitative PCR was not affected by temperature or the presence of the pathogens. In conclusion, P. subalpina confers an indirect benefit to its host and might therefore be tolerated in natural ecosystems, despite negative effects on plant health and plant growth.
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Season and tissue type affect fungal endophyte communities of the Indian medicinal plant Tinospora cordifolia more strongly than geographic location.
Microb. Ecol.
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A total of 1,151 endophytic fungal isolates representing 29 taxa were isolated from symptom-less, surface-sterilized segments of stem, leaf, petiole, and root of Tinospora cordifolia which had been collected at three locations differing in air pollution in India (Ramnagar, Banaras Hindu University, Maruadih) during three seasons (summer, monsoon, winter). Endophytes were most abundant in leaf tissues (29.38% of all isolates), followed by stem (18.16%), petiole (10.11%), and root segments (6.27%). The frequency of colonization (CF) varied more strongly among tissue type and season than location. CF was maximal during monsoon followed by winter and minimal during summer. A species each of Guignardia and Acremonium could only be isolated from leaves, whereas all other species occurred in at least two tissue types. Penicillium spp. were dominant (12.62% of all isolates), followed by Colletotrichum spp. (11.8%), Cladosporium spp. (8.9%), Chaetomium globosum (8.1%), Curvularia spp. (7.6%), and Alternaria alternata (6.8%). Species richness, evenness, and the Shannon-Wiener diversity index followed the same pattern as the CF with the tissue type and the season having the greatest effect on these indices, suggesting that tissue type and season are more influential than geography. Dissimilarity of endophyte communities in regards to species composition was highest among seasons. Colletotrichum linicola occurred almost exclusively in winter, Fusarium oxysporum only in winter and summer but never during monsoon and Curvularia lunata only in winter and during monsoon but never in summer. Emissions of NO(2), SO(2), and suspended particulate matter were negatively correlated with the CF. Ozone did not have any effect. The frequency of most species declined with increasing pollution, but some showed an opposite trend (e.g., Aspergillus flavus). Five unnamed taxa (sterile mycelia) were identified as Aspergillus tubingensis, Colletotrichum crassipes, Botryosphaeria rhodina, Aspergillus sydowii, and Pseudofusicoccum violaceum, using molecular tools. Fifteen of the 29 endophyte taxa exhibited antibacterial activity. B. rhodina (JQ031157) and C. globosum showed activity against all bacterial human pathogens tested, with the former showing higher activity than the latter.
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