Thomas N. Sieber

Hymenoscyphus pseudoalbidus, the causal agent of European ash dieback

UNLABELLED 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. TAXONOMY Members of the genus Hymenoscyphus (Helotiales, Leotiomycetidae, Leotiomycetes, Ascomycota) are small discomycetes which form their ascomata on dead plant material. A phylogeny based on the internal transcribed spacers (ITSs) of the rDNA indicated the avirulent Hymenoscyphus albidus, a species native to Europe, as the closest relative of H. pseudoalbidus. SYMPTOMS Hymenoscyphus pseudoalbidus causes necrotic lesions on leaves, twigs and stems, eventually leading to wilting and dieback of girdled shoots. Bark lesions are characterized by a typical dark- to cinnamon-brown discoloration. LIFE CYCLE Hymenoscyphus pseudoalbidus is heterothallic and reproduces sexually on ash petioles in the litter once a year. Ascospores are wind dispersed and infect ash leaves during the summer. The asexual spores only serve as spermatia. TOOLS AND TECHNIQUES The most important techniques for fungal handling, such as detection, isolation, culturing, storage, crossing and ascocarp production, are briefly described. MANAGEMENT Once the disease is established, management is hardly possible. The occurrence of a small fraction of partially tolerant trees constitutes hope for resistance breeding in the future. Healthy-looking trees should be preserved.

Endophytic fungi in twigs of healthy and diseased Norway spruce and white fir

Almost all 5- to 6-yr-old twigs of healthy and diseased white fir ( Abies alba ) and Norway spruce ( Picea abies ) were colonized by at least one fungal taxon. The endophyte populations of both tree species were very similar but some populations differed significantly with site, possibly due to climatic and ecological differences. Significant differences in endophyte populations between healthy and diseased trees could only be detected at one site for Norway spruce with respect to Sirodothis sp. and Phomopsis occulta . Air pollutants are suspected as possible causes of changes in endophyte populations.

Fungal associations of serially washed healthy non-mycorrhizal roots of Picea abies

The mycoflora of serially washed healthy fine roots of Norway spruce ( Picea abies ) from six climatically and edaphically different sites in Southern Bavaria was examined. More than 120 taxa were detected. Mycelium radicis atrovirens α was most frequently isolated and occurred in up to 70% of roots. Three main types of fungal associations were identified among sites. Soil pH and altitude had a decisive influence on the type of association. Fungal associations on roots in alkaline soil at low altitudes were dominated by Cylindrocarpon destructans with Cryptosporiopsis sp., Idriella lunata, Mortierella alpina, M. minutissima and Mucor hiemalis f. hiemalis as the other major components. A second type of fungal association formed on roots in peat. Trichoderma viride was the most typical representative of this association, although M. radicis atrovirens α was more frequent. M. radicis atrovirens α was also the dominant fungus of the third association, which formed in both acidic soil and alkaline soil at high altitude. The possible relationships between M. radicis atrovirens α or C. destructans and Norway spruce are discussed.

Characterisation of dark septate endophytic fungi (DSE) using inter-simple-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplification

Suitability and reproducibility of ISSR-PCR to find strain-specific and taxon specific markers for strains of the tree-root endophytes Phialocephala fortinii and ’Type 1’, a non-sporulating dematiaceous mycelium, were examined. The results were compared with data previously generated by isozyme analyses. P. fortinii and ’Type 1’ are two DSE taxa and are abundant colonisers of coniferous forest-tree roots in the North Temperate zone. ’Type 1’ was never observed to sporulate in pure culture but is well defined by its cultural characteristics. DNA of 14 strains per taxon was amplified with three short, 17-18-nucleotide-long, tandemly-repeated primers (CCA, CGA, ACA) with two (CCA) or three degenerated bases at their 5’-ends. The resulting DNA products were separated by agarose gel electrophoresis. The bands were scored for absence/presence, respectively, and the binary matrix subjected to multiple correspondence analysis (MCA). ISSR-PCR was found to be highly reproducible since amplification of DNA from several single-hyphal-tip cultures of the same strain resulted in identical banding patterns. Eighty-five (92.4%) of the 92 DNA fragments were polymorphic. The fragments ranged from 320 to 4100 bp. ISSR-PCR was found to be a powerful tool to find strain-specific and taxon-specific markers. Each strain showed a unique banding pattern and diagnostic bands for the two taxa could be identified. ISSR-PCR data correlated neither with the geographical nor the host origin of the strains. The strains grouped into similar clusters independently of whether MCA was performed with ISSR-PCR or isozyme data. However, ISSR-PCR allowed the differentiation of strains with the same allozyme phenotype.

Simulated acid rain affects birch leaf endophyte populations.

Endophytes were frequently isolated from mountain birch (Betula pubescens var. tortuosa (Ledeb.) Nyman) leaves at a subarctic site where natural air pollution is low. We tested whether simulated acid rain had any influence on the occurrence of endophytes. Dry controls with only ambient rain and irrigated controls treated with spring water of pH 6 were compared with acid treatments at pH 3 and pH 4, prepared by adding both sulphuric and nitric acids. Treatments began in 1985 and leaf samples were taken twice during the summer of 1992. Leaves were surface sterilized, five leaf disks from each leaf placed on malt extract agar, and growing colonies were counted and identified. The most frequently isolated endophyte from birch leaves was a Fusicladium anamorph of Venturia sp. (88% of all the isolates in July and 75% of all the isolates in August), followed by a sterile mycelium and Melanconium sp. The number of endophytes isolated and the species number increased from July to August. Endophytes were most frequently isolated from the basal part of the midrib. The percentage of colonization by endophytes was similar in short and long shoots. More endophytes were isolated from leaves of branches taken at 1 m height than at 2 m height. The stronger acid rain treatment (pH 3) reduced by approximately 25% the number of isolated endophytes in August. Treatments did not have any effect on species composition of endophyte assemblages in birch leaves.

Molecular and phenotypic description of the widespread root symbiont Acephala applanata gen. et sp. nov., formerly known as dark-septate endophyte Type 1

Acephala applanata gen. et sp. nov. is described. A. applanata is a dark-septate endophyte (DSE) of conifer roots and belongs to the Phialocephala fortinii species complex. Several genetic markers, including isozymes, inter-simple-sequence-repeat (ISSR) fingerprints, single-copy restriction fragment length polymorphisms (RFLP) and sequences of the internal transcribed spacers (ITS), let us unambiguously separate isolates of A. applanata from isolates of P. fortinii s.l. and other dark-septate endophytes. Alleles at four RFLP loci and two fixed nucleotides in the ITS region were diagnostic for A. applanata. One of the fixed nucleotides resulted in the addition of an Afa I restriction site. PCR amplification with primers prITS4 and the newly developed primer PF-ITS_F (ACT CTG AAT GTT AGT GAT GTC TGA GT) and restriction digestion with Afa I yielded three fragments (203 bp, 117 bp, 56 bp) in A. applanata but only two (260 bp and 117 bp) in P. fortinii s.l. Population differentiation (GST) between A. applanata and other cryptic species of P fortinii was pronounced, and the index of association (IA) did not deviate significantly from zero, showing that recombination occurs or had occurred in A. applanata. Although isolates of A. applanata never were observed to sporulate, it can be distinguished morphologically from P fortinii s.l. by the scarcity of aerial mycelium, significantly slower growth and denser mycelium on cellophane overlaid on water agar. These phenotypic characteristics, combined with diagnostic RFLP alleles and/or PCR-RFLP of the ITS fragment with the fixed Afa I restriction site, unequivocally allow identification of A. applanata.

Negative effects on survival and performance of Norway spruce seedlings colonized by dark septate root endophytes are primarily isolate-dependent

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.

Assemblages of endophytic fungi in coppice shoots of Castanea sativa

Young healthy coppice shoots of Castanea sativa were collected at two sites, one south and one north of the Alps in Switzerland. The surface-sterilized shoots were incubated under two different dry...

Spatial distribution of dark septate endophytes in a confined forest plot

In the present study we investigated the abundance and spatial distribution of dark septate root endophytes (DSE) in a 3 x 3 m plot in a spruce stand ( Picea abies ). A total of 144 DSE isolates were obtained by means of a hierarchical sampling design. Most roots were colonised, as DSE were isolated from 81.7% of root segments. ISSR-PCR fingerprinting was used to identify 21 unique ISSR types. Dominant types were isolated from adjacent points that covered an area of up to 6.8 m 2 of the study plot, and ISSR types were intermingled extensively. Frequency of isolation of the different ISSR types was uneven with two dominant types that accounted for 38% and 28% of all DSE isolates, respectively. Seven DSE strains representing six different ISSR types were identified as Phialocephala fortinii based on the morphology of fertile conidiophores and/or ITS 1 and 2 sequence comparisons.

Season and Tissue Type Affect Fungal Endophyte Communities of the Indian Medicinal Plant Tinospora cordifolia More Strongly than Geographic Location

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 NO2, SO2, 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.