Comparative studies on ectomycorrhizae synthesized with various in vitro techniques using Picea abies and two Hebeloma species

Summary Various in vitro synthesis techniques with Picea abies and two Hebeloma species showed that structures of the mantle and Hartig net of synthesized ectomycorrhizae within the given two fungus species are stable. However, thickness of mantle, and penetration depth and number of hyphal cell rows between cortical cells of the Hartig net are dependent on techniques and substrates. Porous glass balls as substrate in the Erlenmeyer technique seem to suppress or delay mantle and Hartig net formation. With the other techniques (growth pouch, open cuvette, Erlenmeyer with a vermiculite-peat moss mixture) development of the mantle is simultaneous with or shortly in advance of Hartig net formation. The ectomycorrhizae of the two tested Hebeloma species are similar and cannot be morphologically differentiated by the in vitro techniques used.     

Characterization of Picea abies (L.) Karst. ectomycorrhizas: discrepancy between classification according to macroscopic versus microscopic features

Summary In a 3-year study of ectomycorrhizal diversity in 2 Norway spruce stands in Switzerland the macroscopically classified ectomycorrhizal types were analyzed microscopically to compare the results of the 2 approaches. A total of 22 ectomycorrhizal types were macroscopically classified in the 2 stands. Microscopic investigations — particularly of mantle structures — resulted in the identification of 7 additional types to give a total of 29. These additional types resulted from separation of macroscopically identical types occurring on both stands (type-pairs) at the microscopic level. The problematic nature of characterization and classification of ectomycorrhizas is discussed.     

A new Late-glacial site with Picea abies in the northern Apennine foothills: an exception to the model of glacial refugia of trees

We describe a new palaeobotanical site at Bubano quarry on the easternmost Po plain, northern Italy. Pollen and macrofossils from river and marsh sediments demonstrate the occurrence of Picea in a Pinus sylvestris forest growing in a radius of some tens of kilometres south of the sedimentation place, at the beginning of the Late-glacial interstadial. The Late-glacial and Holocene history of Picea in the northern Apennines is reconstructed on the basis of the palaeobotanical record. The sharp climatic continentality increase eastwards across the northern Apennines from the Tyrrhenian to the Adriatic coast is considered significant for the survival of Picea during the Late-glacial. The most critical phase of survival is related to the moisture changes and consequent Abies competition associated with the last glacial-interglacial transition and the early Holocene. The residual spruce populations expanded during the middle Holocene. The history of Picea in the northern Apennines is a case of ineffective interglacial spread of tree populations from pre-existing stands of LGM (Last Glacial Maximum) and Late-glacial age.     

Analysis of the distribution of marker classes in a genetic linkage map: a case study in Norway spruce (Picea abies karst)

In order to analyze the large-scale structure of the genome of Norway spruce (Picea abies Karst.), a pseudo-testcross genetic linkage map was built using markers of six different types, belonging to the low (amplified fragment length polymorphisms, simple sequence repeats) or high (sequence-specific amplified polymorphisms, inter-retrotransposon amplified polymorphisms) copy-number fraction of the genome, and including expressed region-derived markers (expressed sequence tag polymorphisms). Twenty seven and 23 linkage groups of at least four markers were obtained for the female and the male parent maps, respectively. A subset of these linkage groups coalesced into 13 bi-parental linkage groups through markers shared between the two maps. This map was used to investigate the frequency of each marker type over chromosomes and the distribution of marker types relative to each other, using autocorrelation techniques. Our results show that, while the composition of chromosomes is homogeneous, low- and high-copy-number markers tend to occupy separate regions of the linkage groups, and that expressed sequences are preferentially associated with microsatellites and separated from retrotransposons. These results indicate that the spatial structure of Norway spruce chromosomes is not homogeneous. Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

Proportion of fungal mantle,cortex and stele of ectomycorrhizas in Picea abies (L.) Karst. in different soils and site conditions

Anatomical variability of ectomycorrhizal short roots in Norway spruce (Picea abies (L.) Karst) stands was investigated in five stands differing in site quality class (I^a–V) and soil type. Ten root samples per stand were randomly collected from the forest floor and the subsequent 20 cm soil layer. Thin transverse or axial sections (5 m) of randomly taken short roots were examined by light microscopy (AXIOPHOT; magnification 200–800×). All analyzed short root tips of Norway spruce in different stands were colonized by ectomycorrhizal fungi. Thickness of the mantle (T_mantle) and cortex (T_cortex) and diameter of the root (D_root) were measured in four crossing radial directions for transverse sections and in two radial directions for axial sections. The proportions of the mantle (PS_mantle), cortex (PS_cortex) and stele (PS_stele) of the root cross-sectional area (CSA) were calculated. Mean T_mantle and T_cortex varied from 16.5 ± 0.6 to 29 ± 1.3 m and 83.9 ± 1.7 to 108.4 ± 2.4 m, respectively; significant differences between stands were found. The number of cell rows in the cortex (4–6) did not vary between different stands, thus thickness of cortex depended on cell size. Mean PS_mantle, PS_cortex and PS_stele of the root CSA varied from 17.7 to 28.1%, from 58.9 to 66.9%, and from 13.4 to 15.8%, respectively. No differences between stands were revealed in mean CSA ratio of cortex and stele. It can be concluded that irrespective of big differences in soil and site conditions, including the influence of fungal symbionts, a 4:1 relation on a CSA basis between the cortex and stele is inherent to Norway spruce.     

Patterns and mechanisms of transpiration in a large subalpine Norway spruce (Picea abies (L.) Karst.)

In situ water relations of a large subalpine Norway spruce (Picea abies) were analyzed by simultaneous measurements of sap flow at different crown positions. In the diurnal scale, transpiration varied greatly, both spatially and temporally. Over longer periods, however, different parts of the crown transpired in fairly constant proportions. The average estimated transpiration was about 3.5 times greater in the upper than in the lower half and decreased 1.6-fold from south to north. Water intercepted from rain, fog and dew buffered and significantly decreased the transpiration. The effect was strongest in those parts which were least coupled to the free atmosphere. The top of the crown seemed to experience a regular shortage of water shortly after starting transpiration, when it was forced to switch from internal reserves to sources in the soil. Further, lower branches then started transpiring, which may have led them to compete for the water. An enhanced nocturnal sap flow during warm and dry winds (Foehn) indicated that the tree also transpired at night. Shaded twigs had more capacity to intercept water externally than twigs in the sun. The significance of the crown structure for interaction with water in both liquid and vapour phases is discussed.     

Effect of nitrogen on drought strain and nutrient uptake in Norway spruce Picea abies (L.) Karst.) trees

A field lysimeter study was established with the aim of investigating the effect of nitrogen availability upon drought strain in Norway spruce trees. Forest soil (Typic Udipsamment) was filled in lysimeters 1 m in diameter and 1 m deep. Small trees of Norway spruce from five different clones were planted in the lysimeters. Roofs under the canopy of the trees ensured full control of water and nutrient input. Three levels of nitrogen were given to the trees during five years; ambient rainwater, and five and fifteen times this N concentration, respectively. Additional N was given as NH₄NO₃ in irrigation water. Mean annual N-addition during the five years corresponded to 5, 27 and 82 kg per ha and year for the three treatments, respectively. During the third and fifth growth season drought was artificially induced. In addition to a watered control, two levels of drought were applied, representing water deprivation for 2 and 3 months, respectively, in 1990 and 3 and 4 months, respectively in 1992. A higher water consumption in the nitrogen fertilized trees during the droughts resulted in a significantly lower pre-dawn shoot water potential compared to the trees receiving ambient rain N. The interaction between drought and nitrogen fertilization was clear also for photosynthesis and transpiration. A decrease in height- and diameter increment caused by drought was most pronounced in the 82 kg N ha^–1 yr^–1 treatment. A water strain integral showed a strong positive correlation to the needle biomass of the trees. Foliar concentrations of several nutrients decreased significantly with increasing drought strain in the trees. Concentration of potassium and boron were especially low and visual symptoms of deficiency occurred.     

Vertical distribution of an ectomycorrhizal community in upper soil horizons of a young Norway spruce (Picea abies [L.] Karst.) stand of the Bavarian Limestone Alps

The vertical niche differentiation of genera of ectomycorrhiza (ECM) was assessed in a 17-year-old Norway spruce (Picea abies [L.] Karst.) plantation on a mountainous dolomitic site (1,050 m above sea level) of the Bavarian Limestone Alps. We determined ECM anatomotypes, recorded the abundance of corresponding ECM root tips and classified them into groups of ECM exploration types, which refer to the organisation and the extent of their extramatrical mycelia. The abundance of ECM was highest in the organic soil layers, compared to the mineral soil horizon. The ordination of the ECM communities and of the exploration types revealed segregation related to soil horizon properties. While Cenococcum geophilum preferred the organic soil layers, Lactarius spp., Tomentella spp. and Craterellus tubaeformis were generally most abundant in the mineral soil horizons. Cenococcum geophilum was the predominant species, possibly based on enhanced competitiveness under the prevailing site conditions. The short-distance exploration types (e.g. C. geophilum) preferentially colonised the organic soil layer, whereas the contact types (e.g. most of the Tomentella spp., C. tubaeformis) together with medium-distance types (e.g. Amphinema byssoides) were primarily associated with the underlying A-horizons. Therefore, the soil horizons had an important effect on the distribution of ECM and on their community structure. The spatial niche differentiation of ECM genera and exploration types is discussed in regard to specific physico-chemical properties of soil horizon and the assumed ecophysiological strategies of ECM.     

Comparative investigations on the differentiation of the endodermis and the development of the Hartig net in mycorrhizae of Picea abies and Larix decidua

Summary The differentiation of the endodermis of mycorrhizal roots of Picea abies and Larix decidua was investigated by means of light and transmission electron microscopy and with fluorescence techniques. The initiation and differentiation of the Hartig net were recorded. Differences between the two tree species were found, as were differences between the two tree species and angiosperms. The Casparian band developed immediately after the origin of endodermal cells from the meristem in mycorrhizae of both tree species. In L. decidua only the primary endodermis was present in most mycorrhizal laterals. The secondary structure of the endodermis was restricted to main roots and proximal parts of larch mycorrhizae. In P. abies mycorrhizae, however, the secondary stage of the endodermis developed soon after the primary endodermis and was characterized by regular alternation of short, active passage cells and elongated, rapidly degenerating cells, the inner surface of which was covered by a thick suberin layer. Hartig net development started in P. abies short roots only after the differentiation of endodermis into the secondary stage, whereas in L. decidua, the Hartig net was already initiated at the primary endodermal stage. Differences were specific for tree species.     

Stomatal patchiness in conifers: experiments with Picea abies (L.) Karst. and Abies alba Mill.

Summary Strong evidence for the occurrence of pronounced stomatal patchiness in needles of Picea abies (L.) Karst. and Abies alba Mill. was found using various indirect methods. Anatomical investigations revealed a septate leaf anatomy for both species, a phenomenon expected if a patchy distribution of stomatal aperture is present. Calculation of some photosynthetic characteristics (e.g. carboxylation efficiency) from gas exchange measurements is shown to be markedly affected by the patchy distribution of stomatal apertures on the needles. The importance of stomatal patchiness in connection with air pollution related forest decline symptoms as well as an hypothesis suggesting a possible role of the phenomenon as a protective mechanism against photoinhibition are discussed.     

Changes in root growth patterns of (Picea abies) spruce roots by inoculation with an ectomycorrhizal fungusPisolithus tinctorius and jasmonic acid treatment

Symbiosis between fungi and plant roots forming a mycorrhiza involves extensive interactions at the molecular level between both partners. The role of plant hormones in the regulation of mycorrhizal infection is not known to involve jasmonates. Their endogenous levels increase during pathogen attack; however, little has been done on their involvement in mycorrhizae. In our recent work, root growth patterns of 2-month-old spruce seedlings after inoculation withPisolithus tinctorius and/or jasmonic acid (JA) treatment were studied using a paper-sandwich technique. Changes in root length, the degree of branching, presence and length of root hairs, and infection parameters were followed using a stereomicroscope. The first mycorrhizal contact of hyphae with roots was significantly accelerated upon treatment with 0.5 M JA. Interactions between root hairs and fungal hyphae were seen by scanning electron microscopy. The multiplication of root hairs of non-mycorrhized seedlings treated with 5.0 M JA and changes of the root surface were observed by the same technique.     

Fine-root morphology and uptake of 32P and 35S in a Norway spruce (Picea abies (L.) Karst.) stand subjected to various nutrient and water supplies

An investigation of fine (< 1 mm in diameter) and small (1–2 mm in diameter) roots in the organic soil layer was carried out in a Norway spruce forest stand with different treatments of water and nutrients, including control (C); ammonium sulphate application (NS); nitrogen-free fertilization (V); irrigation with liquid fertilization (a complete nutrient solution) (IF); NS followed by artificial drought (ND); V followed by artificial drought (VD). In order to evaluate the vitality and function of the fine roots, the following approaches were used: i) classification of fine roots, based on morphological characteristics; ii) nutrient uptake bioassay, using ³²P-phosphate and ³⁵S-sulphate; iii) nutrient concentration in fine roots and its relation to nutrient uptake. The NS treatment showed effects on the fine and small roots, with a decrease in amount of living roots, and a decrease in the total amount of fine and small roots. The VD treatment resulted in increased amounts of living small roots, while the ND treatment showed the opposite, as compared with the V and NS treatments, respectively. The uptake of P was negatively related to the P supply, with a higher P uptake for C and NS fine roots than for IF and V fine roots. The specific root length (SRL, m g^-1 DW) decreased for NS fine roots and increased for IF fine roots, indicating a further increase in uptake for NS roots and a decreased uptake for IF roots if calculated on a root length basis. So far, the NS and IF treatments maintain a considerable increase in above-ground biomass with a significantly reduced root biomass and standing crop.     

Effects of elevated soil solution Al concentrations on fine roots in a middle-aged Norway spruce (Picea abies (L.) Karst.) stand

Aluminium (Al), mobilized by acidic deposition, has been claimed to be a major threat to forest vitality. Fine root mortality, decreased root growth and reduced nutrient uptake have been observed in controlled laboratory experiments where roots of tree seedlings were exposed to elevated concentrations of Al. Yet, evidence for Al-induced root damage from forest stands is scarcely reported. Nevertheless, Al dissolved in soil water has received a key role in the critical load concept for forests. Here, we present effects of artificially elevated concentrations of Al in the soil solution on fine roots in a middle-aged stand of Norway spruce (Picea abies (L.) Karst.). Although the inorganic Al concentrations about 200 µM and Ca:Al ratio about 0.7 that were established in the soil solution within this experiment have been associated with reduction of root growth and root mortality for spruce seedlings in hydroponic studies, no acute damage on fine roots was observed. Three years of treatment did not cause visual root damage, nor were effects on fine root necromass observed. Fine root necromass made up about 10% of fine root biomass for all treatments. However, significantly lower molar Ca:Al and Mg:Al ratios in living and dead fine roots were found in the plots where Al concentrations were highest and ratios of Ca to Al in the soil solution were lowest. The lack of response on fine root biomass suggests that forest stands tolerate higher Al levels than results from laboratory experiments indicate. We conclude that effect studies in the laboratory have limited value for field conditions. The key role of Al toxicity, expressed as the Ca/Al ratio, in critical load calculations for forests may have to be reconsidered.     

Influence of needle loss,yellowing and mineral content on monoterpenes in the needles of Picea abies (L.) Karst.

Summary Monoterpenes in needles of Picea abies (L.) Karst. were studied to reveal whether or not the symptoms of forest decline observed in three mountain regions of South Germany (Bavaria) — needle loss and needle yellowing — are related to changes in the amounts of all or individual terpenes. The nutrient supply of the needles was examined in addition to the terpene concentrations, since the disease symptom montane yellowing is often associated with mineral deficiency. At two of the eight locations, trees exhibited Mg-deficiency, at one location K-deficiency was observed, and at another location the S-content of the needles indicated SO₂-pollution. Using various statistical methods of analysis, no correlation was observed between the mineral element content of the needles, the percentage of needle loss and either the absolute or percentile terpene amounts stored within the needles. This finding is independent of needle age, date of sampling and terpene pattern. Concerning needle yellowing, the data show a tendency towards a reduction of the terpene concentrations in needles originating from branches with partly yellow needles compared to needles from exclusively green-needled branches. In spite of the fact that only 2-year-old and older needles show the disease symptom of yellowing, the terpene level is reduced even in still green, juvenile and 1-year-old needles. No terpene was found to be an indicator for early recognition of injury.     

Experiments on the water budget of densely and sparsely needled spruces [Picea abies (L.) Karst.] in a declining stand

Summary The osmotic potentials of needles were compared from numerous trees that had been classified according to needle loss along an altitude profile. With the increasing degree of damage of the trees, the maximum and minimum values deviated more strongly above and below the common mean of all samples. The level of water content of the needles unequivocally reflected the vitality of the trees. Experiments covering a whole vegetation period were performed on a tree pair selected from a natural stand. They were designed to demonstrate differences in water balance between the healthy and damaged state of the trees. For the damaged tree, measurements of the water potentials of single needles showed a greater reduction of potential during the course of the day compared to the healthy tree. Recovery in the evenings was slower and often incomplete. The osmotic potentials of damaged and healthy shoots measured in individual needles also differed both predawn and especially after transpiration stress. Depending on weather and soil desiccation, the differences were more or less pronounced. In the damaged tree, the rises in potential after saturation of the twigs fell far short of the healthy tree. Reduction of water potential, osmotic potential and relative water content under comparable stress conditions suggest a reduced tolerance of drought by damaged trees. In the context of earlier experiments this result was ascribed to a lack of stomatal control, and long-term pollution effects were thus explained as a specific disturbance of hydroregulation.     

Photosynthetic performance,chloroplast pigments,and mineral content of various needle age classes of spruce (Picea abies) with and without the new flush: an experimental approach for analysing forest decline phenomena

Summary Damage in the older needles of Norway spruce [Picea abies (L.) Karst.] in the Fichtelgebirge (NE Bavaria, FRG) appears to result primarily from nutrient imbalances rather than from direct effects of air pollutants on the mesophyll of the needles. Support for this conclusion was obtained by altering the nutrition of older needles through the removal of terminal buds on several branches from a damaged and an undamaged spruce tree in spring. Various photosynthetic parameters, as well as the chloroplast pigment and nutrient concentrations, of 1- to 3-year-old needles on manipulated branches were compared with those of branches on which the new flush was allowed to develop during the course of the growing period. Removal of terminal buds affected only the 1-year-old needles. Elimination of the new flush resulted in a higher Ca and Mn content of the needles of the undamaged tree. This treatment also resulted in an increase of the photosynthetic capacity (under saturating light and CO₂ conditions), carboxylation and light use efficiency, as well as net photosynthesis under natural conditions of the 1-year-old needles on the yellow chlorotic tree. This was accompanied by higher chlorophyll concentrations and an increase in Mg, Ca, Mn, and Zn content, and no visible signs of chlorosis developed in the experiment. By contrast, the needles of twigs in which the new flush was allowed to develop exhibited reductions in mineral content in the middle of the year. This was especially true for the elements Mg and Ca, and was accompanied by needle chlorosis and a depression of the capacity of photosynthesis. Thus it appears that there is a close relationship between the development of needle damage and nutrient imbalances in spruce. The retranslocation of elements from the 1-year-old needles to the new flush seems to play a major role in the development of needle bleaching. This approach thus supports the hypothesis described above and confirms a preliminary test with a similar experimental design, which had been conducted earlier.     

An analysis of needle yellowing in healthy and chlorotic Norway spruce(Picea abies) in a forest decline area of the Fichtelgebirge(N.E. Bavaria)

Summary In a forest decline area (Fichtelgebirge, N.E. Bavaria, FRG), annual time courses of chloroplast pigments in both healthy and chlorotic Norway spruce were studied. The seasonal time courses of green and apparently healthy trees did not generally differ from those reported in the literature for spruce trees of other regions. Chlorophyll content increased from May to October, remained relatively constant or declined slightly during the fall and early winter, and finally decreased markedly from March to early May when pigment is at its minimum before bud break. The annual maximal chlorophyll content increased with needle age from the current year's needles to 4-yearold needles. While carotene content reached its highest concentration in August, the xanthophylls did not peak until February or March. Pigment dynamics of chlorotic trees with lower concentrations, corresponded to those of undamaged trees. Chlorophyll deficits resulted from less pigment formation as well as pigment loss during the growing period. Even when the content of total chlorophyll was low, the ratio of chlorophyll a/chlorophyll b remained almost unchanged. In conjunction with the chlorophyll reductions, a decrease in the chlorophyll/carotenoid ratio and an increase in the xanthophyll/carotene ratio occurred. The periods of needle-chlorophyll reduction did not correlate with those periods of highest concentrations of atmospheric sulphur dioxide, the main air pollutant at the stand. However, chlorophyll formation ceased in the older needle age classes of chlorotic trees when the new flush was sprouting, indicating that nutritional deficiencies affect needle yellowing more than possible direct needle damage by air pollutants.     

Triphenyltetrazolium chloride as an indicator of fine-root vitality and environmental stress in coniferous forest stands: Applications and limitations

The present study is an attempt to investigate whether triphenyltetrazolium chloride (TTC), a chemical compound which measures dehydrogenase activity, could be used to study fine-root vitality from two different points of view: (i) in relation to ageing; (ii) as an indicator of environmental stress, in this case of excess nitrogen. The study was performed with excavated fine-roots from middle-aged Norway spruce and Scots pine stands.The ageing aspect was investigated by applying TTC to fine roots separated into different vitality classes, based on certain morphological characteristics. A significant difference in activity was demonstrated only in the case of roots that could be referred to as living and dead, respectively. The use of TTC on fine roots grown at different nitrogen supply levels indicates a possible increase in dehydrogenase activity with increasing nitrogen supply.     

Analysis of allozyme variability in three Plantago species and a comparison to morphological variability

Summary The level of electrophoretic variability in three Plantago species, P. major, P. coronopus, and P. lanceolata, was analyzed in relation to their breeding systems and compared with their morphological variability. From each species several populations were analyzed. The outcrossing P. lanceolata had the highest level of electrophoretic variability and the lowest population differentiation. The inbreeding P. major showed the opposite: a low level of electrophoretic variability and a high population differentiation. P. coronopus, with an intermediate breeding system, had an intermediate level of variability and differentiation. In comparing the species, it appeared that P. coronopus and P. major showed good concordance in the distribution of both kinds of variability, each having only a slightly higher morphological than electrophoretic differentiation between populations. P. lanceolata showed a higher morphological than electrophoretic differentiation between populations. A comparison of populations, within species, revealed good concordance of electrophoretic and morphological variability only within P. coronopus, while some populations of the other two species had relatively lower morphological variability compared with electrophoretic variability.Grassland Species Research Group Publication No. 182     

The use of neural networks to aid in microorganism identification: a case study ofHaemophilus species identification

The adipamidase of a mutant strainBrevibacterium sp. R312 involved in the degradation of adiponitrile to adipic acid was purified. Its N-terminal amino acid sequence was shown to be identical toBrevibacterium sp. R312 enantio-selective amidase andRhodococcus sp. N-774 amidase.