The Loss of Structural Integrity in Damaged Spruce Needles from Locations Exposed to Air Pollution I. Mesophyll and Central Cylinder

Abstract In connection with the new type of forest damage, the individual disease situation of two-year-old spruce ( Picea abies ) needles was analyzed histopathologically in forest areas exposed to different levels of O₃-, SO₂- and NO₃- pollution.
Early damage results from losses of chlorophyll in the mesophyll cells. The bleaching is more intensive towards the apex in severely damaged needles. The cytoplasm is aggregated at the cell wall and the chloroplasts show definite structural damage as well.
The mesophyll cells below the epidermis, or the cells adjacent to the vascular bundle sheath, appear to be particularly susceptible. Collapsed cells (bone cells), which increase in number with damage, can lead to tissue death in certain needle areas, (brown tips, transverse bands).
Necrotic spots are manifested as groups of dissociated cells in which hypertrophic and collapsed cells as well as abnormal proliferations can be observed.
Hypertrophy and cell collapse appear in the central cylinder in addition to severe phenol deposits.
Bone cells and chlorophyll losses can already be detected in the green needles of damaged trees, indicating latent damage, which becomes macroscopically visible only after more extensive damage.
Our results indicate that no biotic stress factors take part in the damage of the spruce needles investigated here. Anthropogenic air pollutants in addition to abiotic stress factors must be regarded as a main cause of damage.     

SEASONAL CHANGES IN LEAF SURFACE WAXES OF PICEA PUNGENS

The morphological and chronological development of epicuticular waxes and their effects on the boundary layer of blue spruce needles were studied with a scanning electron microscope. Structural or crystalline-like waxes develop first in the epistomatal chambers of needles in an expanding bud. However, as the needles emerge from the bud the structural waxes develop over the entire needle surface on glaucous foliage but are mainly restricted to the stomatal areas on non-glaucous foliage. The epistomatal chambers are occluded with surface waxes as the needles emerge from the bud and they remained occluded. Structural waxes occlude the epistomatal chambers but as these waxes degrade the occlusion becomes amorphous in appearance. Structural waxes are degraded to an amorphous layer over the entire needle surface by weathering. Calculations reveal that the surface waxes of blue spruce are too small to have a significant effect on the boundary layer of the needle.     

Differences in the probing behaviour of Elatobium abietinum feeding on Sitka and Norway spruces

The probing behaviour of Elatobium abietinum (Walk.) on the needles of Norway spruce and Sitka spruce was directly related to the stomatal frequency accompanied by heavy deposition of saliva, and multiple branching and divergence of the salivary sheath, all of which were associated with the impenetrability of the endodermis. On Norway spruce these features were very greatly reduced where there was a higher rate of probing coupled with a higher rate of penetration into the phloem, which was not accompanied by early uptake of phloem sap when compared with aphids on Sitka spruce. These differences in probing behaviour are related to damage symptoms exhibited by the host trees.     

Damage caused by the Green Spruce Aphid to Norway and Sitka spruce needles

Three-month-old needles of Sitka spruce were less susceptible to Elatobium abietinutn than 15-month-old needles. Symptoms appeared after longer aphid feeding times but only a proportion of damaged needles fell. After short feeding periods symptoms appeared in more Norway spruce needles than in Sitka spruce, whereas longer feeding periods resulted in more needles producing symptoms in the Sitka spruce. The symptoms took 4—6 days longer to appear in Norway spruce, and needle fall followed a longer feeding period than on Sitka spruce. Following 72 h feeding, needle fall occurred more quickly on Sitka spruce than on Norway spruce. The time taken for needle fall to occur was inversely related to the feeding time in Sitka spruce but such a response was not evident in Norway spruce. The results are discussed in relation to the differences exhibited in the probing behaviour of the aphid on the two spruces.     

Changes in stomatal frequency and size during elongation of Tsuga heterophylla needles

BACKGROUND AND AIMS: The inverse relationship between the number of stomata and atmospheric CO2 levels observed in different plant species is increasingly used for reconstructions of past CO2 concentrations. To validate this relationship, the potential influence of other environmental conditions and ontogenetical development stage on stomatal densities must be investigated as well. Quantitative data on the changes in stomatal density of conifers in relation to leaf development is reported. METHODS: Stomatal frequency and epidermal cells of Tsuga heterophylla needles during different stages of budburst were measured using computerized image analysis systems on light microscope slides. KEY RESULTS: Stomata first appear in the apical region and subsequently spread basipetally towards the needle base during development. The number of stomatal rows on a needle does not change during ontogeny, but stomatal density decreases nonlinearly with increasing needle area, until about 50 % of the final needle area. The total number of stomata on the needle increases during the entire developmental period, indicating that stomatal and epidermal cell formation continues until the needle has matured completely. CONCLUSIONS: Epidermal characteristics in developing conifer needles appear to be fundamentally different from angiosperm dicot leaves, where in general leaf expansion in the final stages is due to cell expansion rather than cell formation. The lack of further change in either stomatal density or stomatal density per millimetre needle length (the stomatal characteristic most sensitive to CO2 in conifers) in the final stages of leaf growth indicates that in conifers the stage of leaf maturation would not influence CO2 reconstructions based on stomatal density.     

Variations of stomatal density and carbon isotope values of<Emphasis Type="Italic"> Picea crassifolia</Emphasis> at different altitudes in the Qilian Mountains

The stomatal characteristics, length and dry weight as well as stable carbon isotope composition (i¹³C) of needles and tree rings of Qinghai spruce (Picea crassifolia) at different altitudes in the Qilian mountains were investigated. The results showed that stomatal density, distribution pattern on epidermis, and length and dry weight of needles all increased with altitude below 3,000 m. In contrast, these parameters all decreased with increasing altitude above 3,000 m. Furthermore an assay of tree rings showed that near 3,000 m in altitude was the optimum zone for growth and development of Qinghai spruce. Although atmospheric CO₂ concentration influences stomatal density, the effects of many environmental factors, such as temperature, light and rainfall, could obscure the effects of changes in atmospheric CO₂ concentration on stomatal density at altitudes higher than the optimum for growth. The correlation of stomatal density with atmospheric CO₂ concentration and i¹³C of Qinghai spruce needles was significant below 3,000 m, but was insignificant above 3,000 m altitude. The i¹³C value of needles, however, was negatively correlated with atmospheric CO₂ concentrations. Therefore, the influence of altitude should be considered in research on plant physiological ecology and the relationship of stomatal density with i¹³C value or atmospheric CO₂ concentration.     

Fluorescence Microscopy of Fresh Tissue as a Rapid Technique for Assessing Early Injury to Mesophyll

A technique was developed for sectioning fresh red spruce foliage (Picea rubens Sarg.) for use in fluorescence microscopy. This allowed rapid examination of mesophyll in 3-5 mm needle sections. Healthy, ozone treated and cold stressed needles were examined to assess the utility of this technique for early detection of damage. Healthy mesophyll cells fluoresced bright red, while injured cells fluoresced yellow-green in ozone treated needles, and yellow-orange in frozen needles. Shifts in fluorescence wavelengths may be useful for early detection of injury to mesophyll before it is evident by standard light or electron microscopy.     

Image analysis of the stomatal cell walls of Picea abies (L.) Karst. in pure and ozone-enriched air

 The method of image analysis was used to re-investigate previously described structural changes in spruce needles following their exposure to ozone. The changes observed in fumigation experiments were compared with the modifications observed at a site with a similar ozone load. Use was made in each case of needles for which the varying behavioural patterns were known from physiological experiments. In samples with reduced ability to regulate stomatal aperture, the cell walls of the stomatal apparatus showed a reduction in absorptive power at 280 nm. This was interpreted to be a result of delignification of the cell walls in question due to the direct impact of ozone on the surface of the needle. Received: 21 May 1997 / Accepted: 30 June 1997     

Growth characteristics of Sakhalin spruce (Picea glehnii) planted on the northern Japanese hillsides exposed to strong winds

Sakhalin spruce (Picea glehnii), a native species typically found in northern Japan, has been used in reforestation on hillsides exposed to strong winds. In the reforestation areas, there are south-facing (S-slope) and northwest-facing slopes (NW-slope). Climatic conditions are contrasting between the two slopes, with shallower snow cover on the S-slopes. We compared growth responses of the spruce to micro-environment between the S- and NW-slopes through soil nutrients, needle longevity, water status, photosynthesis, and nutrients in the needles. These parameters were measured in needles exposed above the snow in winter and in lower needles protected by snow cover. High-position needles suffered from drought stress, especially in winter, and needles were shed early in the year on both slopes. Low-position needles did not suffer from drought stress, and maintained a high photosynthetic rate on both slopes. However, needle longevity was reduced on the S-slope, and concentrations of nitrogen, phosphorus, and potassium in the needles decreased with needle age. Soil nutrient concentrations were low on the S-slope, which suggests that the needles on the S-slope may remobilize nutrients from aged needles to younger needles prior to shedding. This characteristic is probably an adaptation in Sakhalin spruce to poor soil conditions.     

Morphological and stomatal responses of Norway spruce foliage to irradiance within a canopy depending on shoot age

Morphological and stomatal responses of Norway spruce (Picea abies) foliage to light availability were studied in respect to shoot age. Needle minor diameter (D(1), anatomical width), major diameter (D(2), anatomical thickness), dry weight (M), and tissue density index (I(D)) increased, and needle flatness (Fl) and specific leaf area (SLA) decreased with foliage age, while shade foliage demonstrated higher morphological plasticity as compared to sun foliage. Needle minor diameter, dry weight, and the ratio of total to projected leaf area increased, and needle flatness and specific leaf area decreased with daily average photosynthetic photon flux density (Q(D)). The current-year foliage exhibited the highest variation with irradiance, while the morphological plasticity decreased with needle ageing. The morphological characteristics of needles were independent of irradiance if Q(D) was above 300 μmol m(-2) s(-1). D(1) was the only linear needle characteristic which significantly changed with light availability within a canopy, and thus determined needle flatness, SLA, as well as the ratio of total to projected leaf area (TLA/PLA). Needle flatness was a characteristic responding most sensitively to the photosynthetic photon flux density, R(2) was 0.68, 0.44, and 0.49 for the current-year, 1-year-old, and 2-year-old foliage, respectively. TLA/PLA ranged from 2.2 to 4.0 depending on D(1). Variation in SLA in response to light availability can be attributed to changes both in needle shape and tissue density. Stomatal responses to photosynthetic photon flux density (Q(P)) depended on foliage type (sun or shade) and age. Sun needles demonstrated higher daily maximum leaf conductances to water vapour compared to shade needles. The shade needles responded more sensitively to changes in Q(P) at dawn and sunset than the sun needles, while older needles of both foliage types exhibited faster stomatal responses. The light-saturation of leaf conductance (g(L)) was achieved by 20 μmol m(-2) s(-1) for shade foliage, and approximately by 50 μmol m(-2) s(-1) for sun foliage. As a rule, g(L) changed in response to irradiance faster in the evening, i.e. at decreasing irradiance. Stomata were not usually completely closed in the dark before sunrise and after sunset, the phenomenon being more pronounced in older shoots and sun needles. Nightly water losses from spruce foliage are attributable primarily to older shoots, and are related to age-dependent changes in stomatal responsiveness.     

Seasonal variation in the monoterpenes in needles of Picea abies (L.) Karst.

Summary This investigation was conducted to obtain information about the fluctuations in composition and amount of needle monoterpenes during the development of spruce needles. Studies conducted with two Norway spruce clones clearly revealed the existence of fluctuations. In juvenile needles, the amounts of the oxygenated terpenes increase constantly with age during the first 2 months of needle growth. The hydrocarbon terpenes dominate within the first weeks, some of them even showing a very distinct first maximum within the first 3 weeks after bud burst. All terpenes, including the oxygenated ones, have a maximum in June/July, which favours the hypothesis of a substitution of the hydrocarbons later on. There are significant changes even in mature needles of Norway spruce. The terpene level of 1-year-old needles of the clonal trees increased from spring to early summer and then dropped again towards winter. In addition, fluctuations in mature needles were shown for a set of ten wild trees. Needles of the same age class, which emerged in 1986, were sampled 4 times from 1986 to 1988. The needle terpene concentrations of the 1 -year-old needles were considerably lower in spring at the time of bud burst than in autumn. The terpene level of older needles thus seems to be influenced by biosynthetic and catabolic activities.     

Developmental disorders in buds and needles of mature Norway spruce, Picea abies (L.) Karst., in relation to needle boron concentrations

Dieback of the terminal shoot and consequently bushy growth induced by boron deficiency have been reported widely throughout the world in several tree species. Recently, similar growth damage was documented in half of the young spruce stands in eastern Finland. To clarify the role of B deficiency, the light microscopic structure of emerging buds and of developing and previous-year needles of mature Norway spruce (Picea abies L. Karst.) from damaged (D stand), partly damaged (PD stand) and healthy (H stand) stands were analysed. The samples, on which needle nutrient concentrations were also determined, were taken seven times between early spring (April) and early winter (November). Cell death characterized by precipitation of the cell content, possibly due to the release of tannins after membrane rupture, was seen in the apex of emerging buds, and this led to fatal damage in about half of the buds in the trees from the D stand, where the needle B concentration was well below the deficiency level of 4–5 mg kg⁻¹. Furthermore, an increase in living cells that accumulated tannins in the vacuoles, which is a common stress and/or defense reaction, was found in the primordial shoots of buds and in the differentiating needles in the PD and D stands. The increase in the areas of the central cylinder and of the xylem found in the needles indicate structural plasticity during needle differentiation to drought. The time frame for bud emergence from late May up to mid-September means that an adequate B supply is necessary throughout the summer in order to avoid fatal bud damage and thus bushy growth of the trees.     

Investigations on the Starch Content and Ultrastructure of Spruce Needles Relative to the Occurrence of Novel Forest Decline

The starch content and ultrastructure of needles of Norway spruce trees [Picea abies (L.) Karst.] taken from three natural habitats, undamaged or with symptoms of novel forest decline, were investigated during the course of three years. The starch content was clearly dependent on the seasons, with a maximum in spring and a decline during summer and autumn, leading to a minimum in winter. Needles of damaged trees from one habitat exhibited in all three years from August to October a significantly higher starch content than their undamaged counterparts. Microscopic investigations of these needle samples exhibited severe damage symptoms to the phloem in macroscopically green needles, though more intensely in the needles with yellowing symptoms. The phenomenon of starch accumulation is interpreted as a delay in starch mobilization, caused by a reduction in assimilate transport capacity of the needle phloem. The lower degree of starch accumulation in needles of damaged trees from a second natural habitat corresponded well to the lesser extent of phloem damage. The difference in damage patterns are discussed in terms of the differing air pollution situations acting on both habitats.     

Does timing of boron application affect needle and bud structure in Scots pine and Norway spruce seedlings?

Loss of apical dominance is a well-known boron (B) deficiency symptom in trees. Recent field studies indicate that B deficiency may cause irreversible damage in emerging leader buds leading to bushy growth, and changes in developing needles in mature Norway spruce trees. We experimentally studied if timing of B application affects needles and buds of Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings with low initial B levels. The treatments were: no B (B0); B supply from the beginning of the simulated summer (B1); starting soon after bud burst (B2) and starting at the occurrence of first needle primordia in new spruce buds (B3). At the end of the experiment, B concentration in B1 was 23 mg kg⁻¹ (pine) or 17 mg kg⁻¹ (spruce) and lower in the later applications. In B0 it was at deficiency limit. In B0, B2 and B3, there were fewer sclerenchyma cells, and cavities occurred in vascular cylinders in pine needles, and in spruce buds there were more tanniferous cells in the primordial shoots compared to B1. Furthermore, in all but B1 there was cell collapse in the bud apex of some spruce seedlings. The experimentally induced changes were the same as earlier reported in B deficient conifers in the field, and indicate, similarly as in the field that adequate B is necessary throughout the growing season for healthy growth, particularly for spruce. The differences between spruce and pines are due, at least partly, to the differences in time frame of needle development and in the differences in development of conducting tissues in the buds.     

叉蕨科30种植物叶表皮形态特征研究

利用光镜对叉蕨科7属30种植物叶表皮形态特征进行详细观察研究。结果显示：（1）叉蕨科30种植物的叶上表皮和下表皮细胞形状均为不规则型,垂周壁式样为深波状或浅波状,具单晶或针晶;上表皮细胞的长宽比为1.62~4.0,下表皮细胞的长宽比为1.63~3.06。（2）在30种植物中共观察到7种气孔器类型,分别为：极细胞型、腋下细胞型、聚合极细胞型、聚腋下细胞型、不等细胞型、无规则四细胞型和不规则型,每种植物分别具有4~7种气孔器类型,均为下生型气孔;气孔长宽比为1.22~1.91,气孔密度为8~76个/mm²,气孔指数为3.9%~25.7%。（3）基于气孔器类型组成进行聚类分析,可将30种植物分成3个类群。（4）对叶表皮形态特征分析认为,轴脉蕨属应介于叉蕨属和肋毛蕨属之间,且与叉蕨属关系更近;叉蕨属的范畴还有待进一步研究;支持将肋毛蕨属从叉蕨科中分离出来置于鳞毛蕨科,但不支持黄腺羽蕨属归入鳞毛蕨科。     

Effects of ozone on organic acids in needles of Norway spruce and Scots pine

Summary The effect of ozone, needle age, and season on the pH of homogenate and acid contents of Scots pine and Norway spruce needles is presented. In addition enzyme activities of cytochrome C-oxidase (cyt. C-ox), phosphoenolpyruvate-carboxylase (PEPC), shikimic acid-dehydrogenase (SHDH) and malate-dehydrogenase (MDH) were measured in Scots pine needles. In freshly sprouted spruce needles the level of quinic acid is high and the pH of the needle homogenate is low. Shikimic acid starts at low levels, increases with increasing needle age and becomes dominant, whereas the quinic acid content decreases. Malic acid has a marked seasonal trend; no trend was found in citric acid. Ozone (200 g/m³) decreased shikimic acid and quinic acid, whereas pH, malic acid and citric acid increased. Ozone (100 g/m³) had a similar effect, except in the current-year spruce needles. In Scots pine needles ozone led to increased enzymatic activities of cyt. C-ox, PEPC and SHDH, and a decrease in the activity of MDH. This effect was more pronounced in summer than in autumn, but the visible damage was greater in autumn. These effects can be found with other stresses and are not specific for ozone.     

Is long-lived foliage in Picea mariana an adaptation to nutrient-poor conditions?

Summary This study evaluated the contribution of different ages of foliage to the nutrient and carbon balance of black spruce (Picea mariana (Mill.) B.S.P.) from a nutrient-poor peatland in Alberta. Seasonal patterns of foliar nitrogen and phosphorus concentration and content were examined in six needle cohorts up to 10 years old. Trees were treated to simulate excess nutrient deficiency (removal of all one-year-old foliage), nutrient excess (fertilized with 250, 50, 100 kg ha^–1 NPK split application in June and July), or left as controls. Gas exchange (net assimilation-Na, stomatal conductance-g_s, mesophyll conductance-g_m, water-use efficiency-WUE, dark respiration-RS) was measured on six different needle cohorts in several control trees in 1989 and 1990. Nitrogen and phosphorus concentration decreased with needle age. Foliar nutrient concentration fell from April to June and then was stable until September except for the fertilized trees where it increased. There was no evidence of greater than normal retranslocation of nutrients from older needles for defoliated trees or greater than normal nutrient loading in older needles of fertilized trees. NA, g_s, g_m, WUE, and RS were similar for all needles up to six or eight years old, these older needles having NA of 65% of current needles and similar RS. The results do not support to conclusion that older needles of black spruce are retained as an adaptation to nutrient stress. It does not appear that older needles serve as a nutrient storage site in conditions of excess nutrient availability or a greater than normal nutrient source during times of excess nutrient deficiency. It appears that the maintenance of long-livedfoliage in black spruce does not provide for greater flexibility in tree nutrient allocation. Their contribution to the carbon balance of the tree seems to be sufficient to explain their retention.     

Poor stomatal control of water balance and the abscission of green needles from a declining stand of spruce trees [Picea abies (L.) Karst.] from the northern Alps

On a site on the west slope of the Wank in the northern Alps changes in water potential, osmotic potential and transpiration rate were measured in spruce trees during the dry summer months of 1991. The pattern of decrease in water potential and osmotic potential on days of varying evaporative demand from trees of widely different decline conditions was used to describe the relative ability of the trees to withstand drought stress. Stress diagrams served as a tool for interpreting the state of health of each tree. The criterion is independent of the water situation of the tree and the other external conditions of the respective experiment. These diagrams clearly show that the foliage of spruces with high needle losses reaches the limit of endurance relatively early. For equal evaporative demand much lower turgor levels were observed in spruces with high needle loss compared to undamaged trees. Associated with the occurrence of low turgor values was the shedding of green needles. The abscission zone was shown in sections. The accumulation of highly fluorescent substances in the bundle sheath cells of the same material was described. Incomplete to non-existent stomatal control over water loss was attributed to modifications in the cell walls of the stomata which appear to alter the ability of the guard cells to sense changes in either atmospheric or cellular hydration. Our studies point to the following situation: air pollution directly affects stomatal control, the loss of stomatal control changes the drought avoidance abilities of the foliage and, as a consequence, low turgor levels occur and premature needle abscission is induced. As site water balance decreases, either due to a dry year or to poor moisture holding abilities of the soil, these conditions become apparent.     

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.     

Diversity of endophytic fungi of single Norway spruce needles and their role as pioneer decomposers

The diversity of endophytic fungi within single symptomless Norway spruce needles is described and their possible role as pioneer decomposers after needle detachment is investigated. The majority (90%) of all 182 isolates from green intact needles were identified as Lophodermium piceae. Up to 34 isolates were obtained from single needles. Generally, all isolates within single needles had distinct randomly amplified microsatellite (RAMS) patterns. Single trees may thus contain a higher number of L. piceae individuals than the number of their needles. To investigate the ability of needle endophytes to act as pioneer decomposers, surface-sterilized needles were incubated on sterile sand inoculated with autoclaved or live spruce forest humus layer. The dry weight loss of 13-17% found in needles after a 20-week incubation did not significantly differ between the sterilized and live treatments. Hence, fungi surviving the surface sterilization of needles can act as pioneer decomposers. A considerable portion of the needles remained green during the incubation. Brown and black needles, in which the weight loss had presumably taken place, were invaded throughout by single haplotypes different from L. piceae. Instead, Tiarasporella parca, a less common needle endophyte, occurred among these invaders of brown needles. Needle endophytes of Norway spruce seem thus to have different abilities to decompose host tissues after needle cast. L. piceae is obviously not an important pioneer decomposer of Norway spruce needles. The diversity of fungal individuals drops sharply when needles start to decompose. Thus, in single needles the decomposing mycota is considerably less diverse than the endophytic mycota.