Small RNAs Originating from Symptomless and Damaged Spruces (Picea abies L., Karst.) II.

A nucleic acid of about 450 nucleotides, called RNA₄₅₀, was extracted from needles of symptomless and damaged spruces (Picea abies L., Karst.) and was analysed by two-dimensional gel electrophoresis. The samples originated from 14-years-old spruces of two differently exposed forest sections of the same location in the Hils area. Trees of section 65 were symptomless whereas those of section 103 were retarded in their growth and had partly yellow coloured needles. A new tree was selected for each sampling. Samples were taken at monthly intervals from June 1985 to March 1986, and bimonthly from March to December 1986. The aim of the study is to find out whether the RNA₄₅₀ indicates the damage of trees in the Hils. The RNA₄₅₀ was characterized in the accompanying paper (Beuther et al. 1988) as an endogeneous RNA, which is influenced in its concentration by tree damages. A comparison of the RNA patterns of needle extracts from four different needle year classes of symptomless and damaged trees led to the result that the concentration of RNA₄₅₀ is influenced by season, needle age, and damage. The amplitude of the seasonal changes in the RNA₄₅₀ level was found to be larger in younger than in older needles and is not influenced by the position of the branch. The period of metabolic activity, as observed on the level of RNA₄₅₀, is markedly shorter in damaged than in symptomless trees. The usefulness of the RNA₄₅₀ as a diagnostic test system will be discussed.     

Photosynthetic performance and nutrient status of Norway spruce [Picea abies (L.) Karst.] at forest sites in the Ore Mountains (Erzgebirge)

Summary Photosynthetic performance of several needle age classes of Norway spruce trees [Picea abies (L.) Karst.] in highly SO₂-polluted and heavily damaged forest sites was measured at two different locations in the Ore Mountains (Erzgebirge, Krusne Hory) during early summer. The carboxylation efficiency showed a dramatic drop from current-year's needles to 1-year-old needles with only a slight further decrease with increased needle age. The light use efficiency also revealed these characteristics. For both parameters, no linear decrease with needle age could be found. In contrast, the maximum photosynthetic capacity (A₂₅₀₀) decreased linearly with time and revealed a good correlation with the total sulfur content of the needles. Absolute values measured for A₂₅₀₀ were approximately 50% lower than those of comparable trees in the nearby Fichtelgebirge. Mineral deficiencies or acute nutrient imbalances of the needles were not detected. In contrast to the situation in the forests of the Fichtelgebirge, a direct effect of gaseous SO₂ on the trees in the Ore Mountains seems plausible.This paper is dedicated to our teacher Professor Otto Ludwig Lange on the occasion of his 65th birthday     

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.     

Control of Adventitious Root Production and Hypocotyl Hypertrophy of Sunflower (Helianthus annuus) in Response to Flooding

Needles of 20-year-old Scots pine trees (Pinus silvestris L.) were permitted to photoassimilate ¹⁴CO₂ for 1 h on different dates during the growing season. The loss of radioactivity from current, 1-year-old, and 2-year-old needles was followed, and the translocation of photoassimilated ¹⁴CO₂ from older needle age-classes to the elongating new needles studied. The effects of good mineral and water supply on translocation were also considered. In the spring, 1-year-old and 2-year-old needles accumulated ¹⁴C. These reserves, together with current photosynthate, were utilized when the trees started growing. The 1-year-old needles exported ¹⁴C to the current needles during the first weeks of elongation of the later, while no such translocation occurred from the 2-year-old needles. Removal of the 1-year-old needles resulted in translocation of assimilates from the 2-year-old needles to the current needles. The general pattern of translocation observed in the control trees was not changed when the trees were fertilized and irrigated. The new needles started to export assimilates in the middle of July when the photosynthetic rate per needle was comparable with that of the older age-classes. This occurred about 4 weeks after positive net photosynthesis was first measured for the current shoot. The current needles of trees with good nutrient and water supply seemed to become self-sufficient in photoassimilates earlier than the current needles of the control trees.     

The chlorophyll fluorescence ratio F690/F730 in leaves of different chlorophyll content

The red laser-induced chlorophyll-fluorescence induction kinetics of predarkened leaf samples were registered simultaneously in the 690 and 730 nm regions i.e., in the region of the two chlorophyll fluorescence emission maxima. From the induction kinetics the chlorophyll fluorescence ratio F690/F730 was calculated. The ratio F690/F730 shows to be dependent on the chlorophyll content of leaves. It is significantly higher in needles of damaged spruces (values of 0.45–0.9) than in normal green needles of healthy trees (values of 0.35–0.5). During development and greening of maple leaves the ratio F690/F730 decreases with increasing chlorophyll content. Determination of the ratio F690/F730 can be a suitable method of monitoring changes in chlorophyll content in a non-destructive way in the same leaves during development or the yellowish-green discolouration of needles of damaged spruces in the Black Forest with the typical tree decline symptoms.Abbreviations F690/F730 ratio of the fluorescence yield at the two fluorescence-emission maxima in the 690 and 730 nm regions - F_m maximum fluorescence - F_s steady-state fluorescence     

Time-resolving luminescence techniques for possible detection of forest decline

Needles from spruces at different environmental and physiological conditions were analyzed by picosecond fluorescence spectroscopy using a novel laser diode and single photon counting detection. The decay curves of chlorophyll fluorescence showed a superposition of three exponentially decaying components with time constants of T1 = 100-200 ps, T2 = 300-500 ps and T3 = 2.0-3.5 ns. A high relative intensity of the long-lived component was found in damaged spruces as well as in trees showing first symptoms of yellowing, needle loss or parasite infection, although all measurements were carried out with green needles which appeared visually intact. Therefore, fluorescence spectroscopy with subnanosecond time resolution seems to be a valuable attempt for an early detection of forest decline.     

Phytohormones in needles of healthy and declining silver fir (Abies alba Mill.): I. Indole-3-acetic acid

 Levels of indole-3-acetic acid (IAA) were determined in needles from silver fir (Abies alba Mill.) trees in the northern Black Forest. IAA was quantified by gas chromatography (GC) as 1-heptafluorobutyryl-IAA-methylester (HFB-IAA-ME) using electron capture detection. Prior to GC analysis, extensive purification of needle extracts was performed employing two HPLC steps. Peak identity of HFB-IAA-ME was confirmed by combined gas chromatography-mass spectrometry in selected samples. Levels of IAA in needles belonging to different needle age-classes exhibited a cyclic seasonal pattern with highest concentrations in winter and lowest levels in spring when bud-break occurred. Such a cyclic seasonal pattern of IAA levels was also observed in needles from declining fir trees or fir trees suffering from a strong sulfur impact (S-impact) in the field due to a local SO₂ source. Levels of IAA increased with increasing needle age. This age dependency of IAA concentrations was most pronounced in late autumn when IAA levels were high and nearly disappeared in spring when IAA levels reached their minimum. In needles from declining fir trees or fir trees suffering from a strong S-impact in the field, IAA levels hardly increased with increasing needle age. It is suggested that in healthy trees high levels of IAA protect older needles from abscission and that the considerable losses of older needles of declining fir trees or of fir trees under S-impact are a consequence of the low levels of IAA found in older needles of such trees. Received: 4 May 1995 / Accepted: 29 August 1995     

Short- versus Long-Term Effects of Elevated CO2 on Night-Time Respiration of Needles of Scots Pine (Pinus sylvestris L.)

Dark respiration rate in the night (R _D) was measured in five-year-old Scots pine (Pinus sylvestris L.) trees grown for two years under ambient (AC) and elevated (AC + 400 µmol mol^–1 = EC) CO₂ concentrations in open top chambers. Two needle age classes (i.e., current-year and one-year-old) were measured at AC and EC in both AC- and EC-grown pines. Additionally different chemical characteristics were determined on the needles, such as nitrogen (N), carbon (C), starch, and soluble sugar concentrations as well as specific leaf area. The direct, short-term and indirect, long-term effects of EC on R _D for the two needle age classes were examined. R _D was expressed on a per needle area, needle mass, N, C, and C/N bases. Direct effects were only pronounced in the AC treatment where inhibition of R _D was found at EC in both current- and one-year-old needles. Indirect effects were only significant in one-year-old needles where a decrease was found in the EC grown trees as compared with AC ones when R _D was expressed per unit needle mass, C, or C/N. R _D per unit needle area and needle N were not sensitive to long-term EC, in any needle age class. Long-term EC treatment also influenced the response of the two needle age classes. One-year-old needles from the EC treatment had significantly lower R _D than current-year needles, but no such response was observed in the AC treatment. Our experiment re-emphasised the importance of expressing R _D on different bases for a correct interpretation of the responses to EC. Moreover, we showed that different needle age classes can respond differently to a CO₂ enrichment.     

Effect of pine foliage damage on the incidence of larval diapause in the pine caterpillar Dendrolimus punctatus (Lepidoptera: Lasiocampidae)

The pine caterpillar Dendrolimus punctatus (Walker) with a larval facultative diapause is one of the most destructive insect pests of the pine tree Pinus massoniana in China. The larvae feeding on pine trees with different damage levels were studied to determine the induction of diapause under both laboratory and field conditions. Developmental duration of larvae before the third instar was the longest when fed with 75%–90% damaged needles, followed by 25%–40% damaged needles and intact pine needles, whereas mortalities did not differ among different treatments under the conditions of 25° and critical photoperiod 13.5:10.5 L:D. At 25°, no diapause was induced under 15:9 L:D, whereas 100% diapause occurred under 12:12 L:D regardless of the levels of needle damage. Incidences of larvae entering diapause when they were fed with intact, 25%–40% and 75%–90% damaged pine needles were 51.7%, 70.8% and 81% under 13.5:10.5 L:D, respectively. Similar results were obtained in the field experiment. Incidence of diapause was significantly different among the pine needle damage levels of pine trees when the photoperiod was close to the critical day length, indicating that the effect of host plants on diapause induction was dependent on the range of photoperiod. The content of amino acid and sugar decreased and tannin increased in pine needles after feeding by the pine caterpillars, suggesting that changed levels of nutrients in damaged needles or a particular substance emitted by damaged pine trees was perhaps involved in the diapause induction of the pine caterpillar.     

The effect of nutrition on the seasonal course of needle respiration in Norway spruce stands

 Respiration of 1-year-old needles of 30-year-old Norway spruce trees [Picea abies (L.) Karst.] was studied in a nutrient optimisation experiment in northern Sweden. Respiration rates of detached needles, from ten control (C) and ten irrigated-fertilised (IL) trees, were measured on 16 occasions from June 1992 to June 1993. The aim of the study was to determine the influence of temperature on the seasonal course of needle maintenance respiration, and the effect of nitrogen concentration [N] and carbohydrate content on needle respiration in young Norway spruce trees subjected to long-term fertilisation. The IL treatment significantly affected needle size, in terms of dry mass and length, but not specific needle length (SNL). There was, however, a strong tree-specific effect on SNL (P<10^–9, R ² = 0.75). Needle starch content varied markedly with season (0–25% of total dry mass). This, unless accounted for, would cause erroneous estimates of nutrient concentrations, and of rates of needle respiration, within and between treatments. There was considerable seasonal variation in needle respiration, both in terms of maintenance respiration and temperature dependence (Q₁₀). Q₁₀ had its highest value (2.8) during winter and its lowest (2.0) in the middle of summer. In early autumn (August, September), respiration rate and needle [N] were significantly related (C: P = 0.001, IL: P<0.0005). There was no significant difference in the slope between the two regression lines, but a difference in intercept. At the same needle [N], needles from IL-plots always had a lower respiration rate than needles from control plots. No obvious explanation for the observed difference in intercept was found, but some plausible assumptions are put forward and discussed. Received: 24 January 1997 / Accepted: 1 July 1997     

“Natural” and NH3-induced variation in epicuticular needle wax morphology of Pseudotsuga menziesii (Mirb.) Franco

Summary Changes in epicuticular wax morphology of Pseudotsuga menziesii needles were studied with scanning electron microscopy throughout the growing season in current-year and older needles in 20 trees from two sites, Kootwijk and Garderen (The Netherlands). Fusion of crystalline wax rods leading to a reticulate structure and ultimately to degradation of the regular three-dimensional porous structure started several weeks after bud break and reached a high level at the end of the first growing season. The increase in amorphous (solid) wax showed a similar, but slightly slower development. In 1- and 2-year-old needles the degradation of crystalline wax and increase in amorphous wax had progressed only slightly further. The rates of change in crystalline wax morphology were very similar for sun-exposed and shaded needles, for the two sites, and for the two tree vitality classes included in the samples. The development of epiphyllous fungi also appeared to be largely needle age dependent. The effects of NH₃ on needle wax morphology were studied in young trees used in fumigation experiments. Short-term fumigation (approx. 5 weeks) did not affect wax morphology in current-year needles, but 1-year-old needles which had been exposed to different concentrations from bud break onwards showed a severe degradation of the crystalline wax, regardless of the NH₃ concentration used. In 2-yearold needles the effect of NH₃ could not be traced and was overshadowed by the natural ageing process. Ambient O₃, SO₂ and NO_x levels did not effect epicuticular wax morphology. It is suggested that the variation recorded for the two forest stands does not show effects of local pollution levels.     

Different Responses of Norway Spruce Needles from Shaded and Exposed Crown Layers to the Prolonged Exposure to Elevated CO2 Studied by Various Chlorophyll a Fluorescence Techniques

The acclimation depression of capacity of photon utilisation in photochemical reactions of photosystem 2 (PS2) can develop already after three months of cultivation of the Norway spruces (Picea abies [L.] Karst.) under elevated concentrations of CO₂ (i.e., ambient, AC, + 350 µmol(CO₂) mol^–1 = EC) in glass domes with adjustable windows. To examine the role that duration of EC plays in acclimation response, we determined pigment contents, rate of photosynthesis, and parameters of chlorophyll a fluorescence for sun and shade needles after three seasons of EC exposure. We found responses of shaded and exposed needles to EC. Whereas the shaded needles still profited from the EC and revealed stimulated electron transport, for the exposed needles the stimulation of both electron transport activity and irradiance saturated rate of CO₂ assimilation (P _Nmax) under EC already disappeared. No signs of the PS2 impairment were observed as judged from high values of potential quantum yield of PS2 photochemistry (F_V/F_M) and uniform kinetics of Q_A reoxidation for all variants. Therefore, the long-term acclimation of the sun-exposed needles to EC is not necessarily accompanied with the damage to the PS2 reaction centres. The eco-physiological significance of the reported differentiation between the responses of shaded and sun exposed needles to prolonged EC may be in changed contribution of the upper and lower crown layers to the production activity of the tree. Whereas for the AC spruces, P _Nmax of shaded needles was only less than 25 % compared to exposed ones, for the EC spruces the P _Nmax of shaded needles reached nearly 40 % of that estimated for the exposed ones. Thus, the lower shaded part of the crown may become an effective consumer of CO₂.     

Physiological characteristics of Japanese red pine, Pinus densiflora Sieb. et Zucc., in declined forests at Mt. Gokurakuji in Hiroshima Prefecture, Japan

The decline of Japanese red pine trees (Pinus densiflora Sieb. et Zucc.) at Mt. Gokurakuji (693 m a.s.l.), 30 km west of Hiroshima city, west Japan, was studied. The effects of air pollution and acid deposition on the physiological characteristics of the trees, especially those of the needles, were investigated. Ozone concentration was not correlated with the physiological status of the needles and SO₂ concentration was not high in the declined area. NO₂ concentration correlated negatively with needle longevity while it correlated positively with ethylene emission from 1-year-old needles. Average needle longevity was about 2.8 years in non-declined areas; however the longevity was 1.3 years in the most polluted area. The minimal fluorescence at night (F ₀)of 1-year-old needles decreased with increasing NO₂ concentration. The maximum stomatal conductance (gl), net photosynthesis (P _n)and intercellular CO₂ concentration (C _i) in the declined areas were lower than in the non- declined areas (about 50%, 30% and 20% lower, respectively). The lower C _isuggested that the major part of the decrease in P _ncan be explained by stomatal restriction. The soil pH, N content and C/N ratio showed no significant difference between the declined and non-declined areas. The physiological disorders of needles were due to the damage by air pollutants, and important roles of NO₂ are suggested. Lowering of P _n and the shortening of needle longevity appear to be the main causes of the decline in pines in the forest decline area. Received: 16 December 1998 / Accepted: 7 January 2000     

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.     

Fungi in Living Symptomless Needles of Pinus sylvestris with Respect to Some Observed Disease Processes

The mycoflora in living symptomless needles of Pinus sylvestris was investigated in three 5–8 year-old plantations in Southern Poland from 1987 to 1990. The needles from trees with symptoms of the autumn needle cast and from trees without such symptoms were treated separately. In total 960 of the current year and 720 of the previous year needles were collected. Fungi were isolated from 80.1 % of needles and 3671 cultures belonging to 86 fungal taxa were obtained. Seven species of fungi showed the rate of infection above 5%: Anthostomella formosa, Lophodermium seditiosum, Cydaneusma minus, Cenangium ferruginosum, Lophodermium pinastri, Sclerophoma pythiophila and Anthostomella pedemontana. The frequency of infection of needles by these species depended on needle age, season of the year, calendar year and site. The spread of fungi in needles was not too high. They were found in 35.8 % of needle sections (isolations were made from 10,080 sections). The needle base, middle and top were infected with more or less similar frequency, but the differences were considerable for the individual fungal species. Anthostomella formosa and A. pedemontana generally occurred slightly more frequently on needles of trees without symptoms of the autumn needle cast, while the other four common species ( Cenangium ferruginosum, Cyclaneusma minus, Lophodermium pinastri, Lophodermium seditiosum ) generally occurred more frequently on needles of trees showing such symptoms. There were considerable differences in this respect among plantations investigated.     

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.     

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.     

Photosynthesis and respiration ofPinus pumila needles in relation to needle age and season

Rates of net photosynthesis and dark respiration were measured for detached needles ofPinus pumila trees growing on the Kiso mountain range in central Japan in 1987. Dependency of photosynthesis on light and temperature was examined in relation to needle age and season. The light saturation point of net photosynthesis was lower in 3- and 4-yr-old needles than that in current (flushed in 1987), 1- and 2-yr-old needles.P _nmax, net photosynthetic rates at 1000 μmol m⁻² s⁻¹ and 15°C, of needles from 1- to 4-yr-old generally decreased with needle age.P _nmax of 1- to 4-yr-old needles became higher in August than in other months, andP _nmax of current needles did so in September. Current needles showed high respiration rates (at 15°C) only in August. Optimum air temperatures for net photosynthesis at 1000 μmol m⁻² s⁻¹ were between 10 and 15°C for current and 1-yr-old needles. The temperature coefficient of dark respiration rates was 2.3–3.3 for current needles from August to October, and 2.2 for 1-yr-old needles in mid-July.     

Photosynthetic capacity,chloroplast pigments,and mineral content of the previous year's spruce needles with and without the new flush: analysis of the forest-decline phenomenon of needle bleaching

Summary Spruce (Picea abies) damage in the Fichtelgebirge (FRG) occurs as needle bleaching and a depression of CO₂ assimilation. Such injury may primarily result from the direct, above-ground effects of air pollution or indirect, below-ground changes in mineral uptake.Typically, the new flush of spruce needles is green and exhibits high photosynthetic capacity. Mies and Zöttl concluded that the older foliage is damaged when nutrients are withdrawn to supply the current year's needles. By removing the terminal buds of single branches in the spring, we produced an experimental set of the previous year's needles with greater mineral reserves than the control needles. During the course of the growing period, the performance of the experimental needles, which lacked competition from the new flush, was compared to that of the control needles of the same age-class on intact branches with the new flush.Throughout the experiment, chloroplast pigments of a healthy control tree were not affected by the elimination of the new flush. However, the chlorophyll and carotenoid content as well as the photosynthetic capacity of the previous year's needles on those branches of a heavily damaged tree where the new flush had been eliminated increased substantially. This increase was associated with an increase in minerals, which seemed to be deficient in the control needles with the new flush. Thus, in contrast to needles of the same age-class on intact branches with undisturbed new growth in the same atmospheric environment, the experimental needles escaped bleaching and a decrease in photosynthesis. It would seem that the bleaching and the loss in photosynthetic capacity typical of trees damaged by forest decline indirectly result from nutrient deficiencies through soil environment changes and/or root damage than directly from atmospheric pollutants.