Anna-Maija Hallaksela

Diversity of Norway spruce needle endophytes in various mixed and pure Norway spruce stands

Norway spruce needles were sampled from two series of stand areas located in southern Finland. Both series consisted of five sampling areas in mature managed stands and one in a mature virgin stand. The proportion of spruce varied from 8 to 100% of the basal tree area and the major other species were pubescent birch and Scots pine. From each sampling area (some of which consisted of several sites) 40 mature spruces were randomly chosen and healthy looking needles of the third age class were sampled from heights of 5-8 m and incubated on water agar for isolation of endophytic fungi.

Toxic Bacillus pumilus from indoor air, Recycled Paper Pulp,Norway Spruce, Food Poisoning Outbreaks and Clinical Samples

Forty-four B. pumilus isolates of food poisoning, clinical, environmental and industrial origins were investigated for toxin production using the boar spermatozoan motility assay, previously shown to be a sensitive method for detecting non-protein toxins from B. cereus and B. licheniformis. The three toxic isolates originated from live tree, indoor air and recycled paper pulp and were more toxic than the previously described food poisoning isolates of B. licheniformis, whereas the B. pumilus food poisoning and clinical isolates were lower in toxicity. The type strain also produced inhibitory substances. The toxic substances were insensitive to heat (100 degrees C, 20 min), to pH 2 or pH 10 and to digestion with pronase. The substances were readily soluble in methanol and chloroform, but less soluble in toluene. Exposure of boar spermatozoa to 1-10 microg ml(-1) (EC50) of methanol soluble substance from the four strains disrupted the plasma membrane permeability barrier, induced abnormalities in the postacrosomal sheath, collapsed the mitochondrial and suppressed cytoplasmic NAD reduction. No change was observed in human peripheral blood lymphocytes exposed to concentrations of B. pumilus extract that affected spermatozoa. The toxin producing isolates were 99.4 to 99.6% similar in 16SrDNA (500 bp) to the type strain and could not be distinguished from the 41 non-toxic isolates by biochemical properties or whole cell fatty acid composition.

Fungal diversity in Norway spruce: a case study

The majority of microbes living on forest trees are still unnamed and our knowledge of their species richness is vague. This paper describes the fungal diversity of the above ground parts of a 61 year old Norway spruce tree lacking visible signs of damage or disease. The problem involved with identification of the fungi to named species was circumvented by classifying them into operational chemotaxonomic units (OCTUs) by using their combined fatty acid and sterol profiles (FAST-profiles). The variation of these units was chosen to correspond to a within-species-variation determined for several morphologically defined taxonomic species occurring on Norway spruce. Ninety-nine OCTUs were identified from 666 fungal isolates obtained. Bacteria were found only occasionally from the inner bark samples (three isolates) and from needles (five isolates). Models describing the accumulation of OCTUs against the number of samples taken were used for extrapolation of the total number of fungal OCTUs in the above ground parts of the tree. Our results suggest that an undamaged, apparently healthy Norway spruce, harbours in its above ground parts nearly two hundred fungal species. The majority were estimated to be needle epiphytes. At the large forest area scale, the species richness may be one order of magnitude higher.

Stem discoloration of planted silver birch

The spread of discoloration in planted silver birch (Betula pendula Roth.) stems was investigated in 137 stands in Finland and the role of microbes was investigated in 71 stands. The colour changes noted in the pith of planted silver birches (18–65 years old) were very common but restricted and did not show unacceptable quality or reduction in veneer yield over 30 years. The horizontal spread of discoloration was generally less than 4 cm. The mean vertical spread of pith discoloration in planted silver birch trees was 3.2 m to 5.3 m in 30‐year‐old and younger stems, respectively. Dead and broken branches seem to be the main reason for harmful stem discoloration. The most common microbial community found in discoloured xylem includes primary invaders such as the non‐decaying fungi, Phialophora fastigiata, Phialemonium‐spp. and yeast‐like fungi and Enterobacter‐ and Pseudomonas‐type bacteria.

Direct analysis of ribosomal DNA in denaturing gradients: application on the effects of Phlebiopsis gigantea treatment on fungal communities of conifer stumps.

The aim of this study was to test the usefulness of direct PCR-amplification in analysing fungal diversity in stumps. The analysis was conducted on stumps treated against Heterobasidion spp. using a commercial formulation of Phlebiopsis gigantea (Rotstop), and carried out using denaturing gradient gel electrophoresis (DGGE) of small subunit (SSU) ribosomal DNA (rDNA) fragments PCR-amplified directly from wood DNA samples using two separate fungus-specific primer pairs. On average, two (range 0-9) different amplification products were observed by DGGE in single wood samples of approximately 500 mm3. The PCR products were classified into operational taxonomical unit (OTU) groups based on their DGGE mobility. Six OTUs could be affiliated with a known species based on a reference fungal collection of 37 species: Heterobasidion annosum, H. parviporum. Hypholoma capnoides, P. gigantea, Resinicium bicolor and Stereum sanguinolentum. Sequence analyses did not give further identifications. OTU profiles from old (6 yr-old) and fresh (1-year-old) Scots pine and Norway spruce stumps from treated and untreated forest plots were compared statistically, and some significant differences were observed in the species composition between the treated and untreated plots. However, the frequency of most of the OTUs did not seem to be affected, and the treatment did not seem to have reduced the overall level of fungal diversity. Based on these results, direct PCR-amplification seems to be useful in analyses of fungal communities in decaying conifer stumps.

A chemotaxonomical method based on FAST-profiles for the determination of phenotypic diversity of spruce needle endophytic fungi

Assessment of diversity (including both species richness and intraspecific variation) within a habitat may often be difficult when morphological criteria are used for identification and classification of isolates. Here we describe how combined fatty acid and sterol profiles (FAST-profiles) can be used for classification of fungal isolates into FAST-groups (i.e. operational chemotaxonomic units) according to a defined upper variation limit. Data analysis includes a two stage statistical approach. First, the isolates are grouped into taxonomic species using a discriminant model based on well identified ‘model’ isolates. These species groups together with one group that includes isolates which could not be identified with the discriminant model are further divided into operational chemotaxonomic units using a FAST-profile mismatch threshold. This method is demonstrated with Norway spruce needle endophytes.

Relationship between Prosthemium betulinum and Pleomassaria siparia

Prosthemium betulinum and Pleomassaria siparia are found in the same sites on birch branches. Single spore cultures of P. siparia produced conidia similar to those of P. betulinum. Restriction analysis of 18S rDNA did not differentiate between P. betulinum and P. siparia. Random amplified microsatellite fingerprints showed a high amount of variation, which was not explained by the origin of cultures. We conclude that P. betulinum is the anamorphic state of P. siparia.