Endophytic fungi species composition in healthy needles of Austrian pine (Pinus nigra Arn.) was investigated at eight locations in Slovenia. Results from October 1994 and January 1995 isolations were compared with analyses of macronutrients, sulphur and lead content of the needles. About 80 species of microfungi were revealed. From the observations and measurements described it was not possible to discriminate between the effect of environmental factors and effect of air pollutants. The pollutants load of all measured elements is low and the environmental factors are very diverse. From the dendogram of isolation frequencies it seems that the age of the tree affects the species composition and frequencies of endophytes to the greatest extent.

Three principal approaches are used in our laboratory to analyze plant genomes: (i) The construction of high density molecular maps: We have established a refined microdissection procedure to construct chromosomal and subchromosomal libraries. Synchronized meristematic root tip tissue from which metaphase spreads can be prepared with a novel dropspread technique in combination with nl-scale PCR allowed the cloning of DNA of 10 or less chromosomes or chromosome segments. The construction of high-resolution maps from discrete genome regions can greatly facilitate genetic fingerprinting, gene isolation and QTL studies. (ii) Synteny analysis: The Aegilops-based deletion mapping system in wheat with an array of deleted chromosome parts and chromosome-specific RFLP markers has lead to the construction of a high density physical consensus map of wheat. The integration of wheat, barley and oat RFLP markers proves the colinearity between the wheat A-, B- and D-genomes, the H- genome of barley and the E-genome of Agropyron. (iii) Gene mapping in situ and chromosomes at high resolution: For the sensitivity enhancement of fluorescence in situ hybridization (FISH), the efficient preparation of plant chromosomes for high resolution scanning electron microscopy, mapping of low-copy sequences, and for comparative in situ hybridization a modified drop technique for plant protoplasts was developed. A tandemly amplified repetitive sequence element from microdissected barley chromosomes has enabled the karyotyping of single Gramineae genomes in a single step. These sequences are also useful for simultaneous double or triple hybridization experiments in an attempt to localize new sequence on specific chromosomes or chromosome segments. The physical mapping of the Sec-1 locus has been refined on the satellite of chromosome 1R of rye, and the syntenic locus on barley chromosome 1H identified. A method was developed for in situ hybrization and signal detection at high resolution using a field emission scanning electron microscope and a backscattered electron detector. Colloidal gold particles were localized on chromosome structures resembling the 30 mm fibre. Comparatively, an rDNA probe was located in the secondary constriction and highly compact adjacent regions of barley chromosomes.

Annual research has proven that the level of the so called natural cytogenetic damage to Norway spruce in Slovenia has been correctly estimated at 8. The results of cytogenetic bioindication calculated in accordance with this level show that average cytogenetic damage in the period from 1985 to 1992 enable us to determine environmental influences on the expression of cytogenetic damage.

Preliminary results and methodology related to the use of the fluorescence in situ hybridization technique for detection of plants hybrids are presented. Total genomic DNA probe preparation and the non-isotopic biotin probe labelling technique is presented. The use of an excess of unlabelled competitor DNA from a related plant species gives more constant results in determining whole plant genomes with in situ experiments on plant hybrids. Preliminary results of flourescence in situ hybridization for detecting alien rye chromatin and localisation on metaphase chromosomes and interphase nuclei in the bread wheat cultivar Yugoslavia are described. The methodology for detection of interspecies hybrids of common and sessile oak with flourescence in situ hybridization is given. The advantege of the use of total genomic biotin labelled probes for in situ hybridization is that it requires less effort than preparing species specific probes and the ease of interpretation of the results. The technique of fluorescence in situ hybridization is less species than other cytological techniques for higher plants and almost the same methodology can be used for detection of oak hybrids. The extraction of a high quantity of species specific DNA of high quality and probe preparation and labelling are the most important step.

Forest soil was sampled on two different observation plots by following well described methodology. Sampling sites had been chosen in place with typical soil conditions for the observed forest site. To make sampling easier some natural forest site characteristics were considered. Mg, Zn and Cd were determined by atomic absorption spectroscopy in all samples. Results were statistically evaluated and different sources of variance were estimated. It was also estimated whether the number of samples was sufficient for our requirement for accuracy of analytical results.

This article deals with use of several bioindication methods in forest decline studies within an international TEMPUS project carried out in Slovenia. The aim of the project was to introduce and connect different bioindication methods in order to improve methods and qualify the research staff working in this research field in Slovenia. The following bioindication methods were introduced or adapted for forest decline research in Slovenia: biochemical and physiological markers of air pollution / stress impact on forest trees (Graz Univ.: photosynthetic pigments, thiols, ascorbic acid, protective enzyme systems; Cambridge Univ.: plant hormones; Munich Univ.: mineral nutrition of forest trees); epiphytic lichens as indicators of air quality on forest sites (Graz Univ., Ljubljana Univ); determination of types of ectomycorrhizae as indicators of forest soil condition (Munich Univ., Ljubljana Univ.). The significance and practical use of bioindication methods in environmental studies is also discussed.

Tissue specific localization of soluble secondary metabolites was demonstrated in ectomycorrhizae and uninfected roots of Picea abies, Larix decidua, Pinus sylvestris and Abies alba. Compounds were analysed by HPLC and by histochemical methods. Specific distribution and age-dependent changes were observed with several metabolites. For catechin corresponding patterns were found within the cortex of all investigated conifers. Levels of this metabolite were highest within the apical cortex cells and decreased on aging of the tissue. In spruce contents of catechin showed evident seasonal changes. Stilbene glucosides were detected within the pericycle of spruce roots and as well the phloem tissue of larch with levels increasing during root development. In larch two anthocyanins accumulated in the calyptra. In older tissues of larch roots larixin-glucosid and quercetin-rhamnosid were detected, the latter with highest levels in the phloem tissue. For cell wall bound ferulic acid increasing levels could be found concomitant with the development of the root.

To use alterations to epicuticular wax morphology as a successful tool in forest damage research, however, it is necessary to distinguish environmental influence and artefacts from common pollution effects. This paper gives a short review about alterations to epicuticular wax morphology of spruce needles.

The Forest Research Plot was chosen on a typical site for the Pokljuka plateau. It is covered by a supposedly autochthonous old Norway spruce stand with rejuvenation centres. Data on the physiological parameters can be used as representation values for a subalpine Norway spruce stand. The studies include: standard site and dendrological parameters, physiological parameters (foliar analysis, antioxidants, hormones and aminoacids in spruce needs), genetical and biochemical analysis (isoenzymes and molecular markers), microbiological activity (acid phosphatase, rate of decomposition, number and occurrence of fungal decomposers of wood and litter) and studies of symbionts and pathogens of Norway spruce (mapping of fruitbodies of mycorrhizal fungi, types of ectomycorrhizae, endophytes, epiphytes - lichens and phytopathological surveys). All physiological and ecological studies are planned to be repeated at certain time periods in order to understand better the functioning of the forest ecosystems on Pokljuka.

Distribution patterns of ectomycorrhizal types and length density of finest roots were investigated in three comparable Scots pine ecosystems located along a deposition gradient of air pollutants in the new states of Germany. The plots Rosa, located near Bitterfeld and Halle, had received high depositions loads of SO2, NOX and alkaline fly ashes due to chemical industries and brown coal fired power plants in the former German Democratic Republic. Taura, located near Leipzig, was moderately affected by air pollutants. The background site Neuglobsow is located in a remonte area north of Berlin. All the three plots were dominated by only a few mycorrhizal types. Mycorrhizal projection area of these types was highest in Neuglobsow and strongly reduced at the polluted plots. The distribution patterns of mycorrhizal types differed also qualitatively. Length density of finest roots was lower in the humus layer of Neuglobsow than in the humus layer of Rosa and Taura. The results are correlated to high nutrient inputs and high rates of N-mineralization at the impacted plots.