Changes in cellular structures and enzymatic activities during browning of Scots pine callus derived from mature buds

Visible browning is a typical feature of callus cultures derived from shoot tips of mature Scots pine (Pinus sylvestris L.). Because the ability of callus to regenerate is low, we determined the effect of browning on growth and changes in cellular structure during culture. Striking alterations in cellular structure were detected by LM (light microscopy), EM (electron microscopy) and SEM (scanning electron microscopy). Accumulation of phenolic substances was shown by histochemical staining. Staining for beta-glucosidase activity of soluble proteins that had been subjected to polyacrylamide gel electrophoresis indicated lignification of cells. The measured growth rate of callus was low compared with a hypothetical growth curve. Peroxidase activity increased rapidly soon after the start of the culture period, but especially between the second and third weeks of culture. At this time, the degradation of cell membranes and browning began coincident with the loss of chlorophyll. We conclude that browning is associated with cell disorganization and eventual cell death, making tissue culture of mature pine especially difficult.     

Interference of bracken (Pteridium aquilinum L. Kuhn) with Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies L. Karst.) seedling establishment

The nature of interference of bracken with Scots pine and Norway spruce seedling establishment was considered in three field experiments. In a seeding experiment, it was found that Scots pine germination was highest on exposed mineral soil and lowest when intact bracken litter and humus were present, suggesting adverse effects of litter and humus on pine regeneration probably due to phytotoxicity. In a second experiment, smothering by bracken caused high mortality of Scots pine seedlings while Norway spruce seedlings were relatively unaffected. Mortality for both Scots pine and Norway spruce seedlings was low when planted in a adjacent Scots pine-bilberry stand with no bracken. Annual shoot growth of Norway spruce was higher in bracken than in Scots pine-bilberry vegetation while no differences in shoot growth between these two vegetation types occurred for Scots pine. In a third experiment, activated carbon was added to the ground under Norway spruce seedlings planted in bracken to adsorb possible phytotoxic compounds released by bracken. The addition of carbon had no effect on seedling mortality or growth rate, indicating that the seedlings were not susceptible to allelochemicals released by bracken. Since large Norway spruce seedlings were relatively unaffected by bracken interference in this study, artificial regeneration with containerized Norway spruce seedlings is suggested to achieve an acceptable conifer tree establishment on clear-cuts invaded by bracken.     

Damage by pine weevil Hylobius abietis to conifer seedlings after shelterwood removal

Abstract

Pine weevil (Hylobius abietis L.) damage to seedlings after overstorey removal was investigated in a survey study in six shelterwoods in the south–central part of Sweden. The shelterwoods predominantly consisted of Scots pine, except at one site where the shelter trees mainly consisted of Norway spruce. Before final cutting, 10 plots were laid out at each site and measurements of shelter trees and marked seedlings were taken. The seedlings were examined during the 2 years after final cutting. The study showed that removal of shelter trees increases the risk of severe damage by pine weevil and the variable that was most strongly correlated with the risk was the seedling root collar diameter. Both Scots pine and Norway spruce seedlings were severely damaged by pine weevil, and most of the feeding occurred during the first year after cutting. The amount of debarked area was significantly larger for Scots pine than for Norway spruce seedlings. Vitality (growth of the leading shoot before final cutting) of the seedlings also affected the probability of damage. Seedlings with high vitality were less damaged by pine weevil than seedlings with low vitality. For Scots pine the shelterwood density before final cutting was correlated to the intensity of pine weevil feeding after cutting. In conclusion, after the final cutting of a pine or spruce shelterwood, pine weevils will probably invade the area. To avoid serious damage, Norway spruce and Scots pine seedlings should have reached a diameter of at least 10–12 mm.     

Height growth of planted conifer seedlings in relation to solar radiation and position in Scots pine shelterwood

Seedlings of different provenances of Scots pine (Pinus sylvestris L.), lodgepole pine (Pinus contorta Dougl., var. latifolia Engelm.) and Norway spruce (Picea abies (L.) Karst.) were planted in three Scots pine shelterwoods (125, 65 and 43 stems ha⁻¹) and a clear-cut, all in northern Sweden. The sites were mounded and planting took place during 2 consecutive years (1988 and 1989). The solar radiation experienced by the individual seedlings was determined using a simulation model. Height development of the seedlings was examined during their first 6 years after planting. During the final 3 years of the study, height growth of Norway spruce was relatively poor, both in the shelterwoods and the clear-cut area. Height growth of lodgepole pine was significantly greater than that of Scots pine, both in the shelterwoods and the clear-cut. In contrast to Norway spruce, Scots pine and lodgepole pine displayed significantly greater height growth in the clear-cut than in the shelterwoods. For all three species in the shelterwoods, regression analyses showed that height growth was more strongly correlated with the distance to the nearest tree than with the amount of radiation reaching the ground, i.e. growth was reduced in the vicinity of shelter trees. Therefore, we conclude that the significant reduction in height growth of seedlings of Scots pine and lodgepole pine in Scots pine shelterwoods was partially caused by factors associated with the distance to the nearest shelter tree. Because the substrate was a nitrogen-poor sandy soil, we suggest that root competition for mineral nutrients, especially nitrogen, accounts for the reduction in height growth.     

Effect of uninucleate Rhizoctonia on Scots pine and Norway spruce seedlings

One‐year‐old container‐grown seedlings were planted in spring on clear cut areas: the Norway spruce (Picea abies) on a moist upland site (Myrtillus‐type) and Scots pine (Pinus sylvestris) on a dryish upland site (Vaccinium‐type). While still in the nursery, half of the seedlings of each species had been inoculated during the previous summer, with a uninucleate Rhizoctonia sp., a root dieback fungus. At outplanting all the seedlings appeared healthy and had a normal apical bud, although the height of the inoculated seedlings was less than that of the uninoculated control seedlings. At the end of the first growing season after planting, the mortality of inoculated Scots pine and Norway spruce seedlings was 25 and 69%, respectively. After two growing seasons the mortality of inoculated seedlings had increased to 38% for Scots pine and 93% for Norway spruce. The mortality of control seedlings after two growing seasons in the forest was 2% for Scots pine and 13% for Norway spruce. After outplanting the annual growth of inoculated seedlings was poor compared with the growth of control seedlings. These results show that, although Rhizoctonia‐affected seedlings are alive and green in the nursery, the disease subsequently affects both their survival and growth in the forest.     

Fungi colonizing Scots-pine cone scales and seeds and their pathogenicity

Fungi were isolated from the cone scales and seeds of Scots pine using plating on malt-extract agar and/or the standard blotter method in a Jacobsen's apparatus. Alternaria alternata, Epicoccum purpurascens and Ulocladium atrum were isolated from damping-off seedlings germinating on agar or filter paper, but, in pathogenicity tests with peat-sand (1:3) growth substrate, they were not pathogenic. All the Fusarium species isolated were pathogenic in growth substrate. Some F. avenaceum colonies formed no aerial hyphae and they proved to be a mixed culture of the fungus, a fluorescent Pseudomonas sp., and a gram-negative bacterium. The bacterial associates appeared to increase the pathogenicity of F. avenaceum. All the micro-organisms tested were more pathogenic in sterilized than in unsterilized peat-sand substrate.     

Responses of in vitro grown pine seedlings to infection by four strains of Heterobasidion annosum1

Scots pine seedlings growing in pure cultures were inoculated with 4 isolates of Heterobasidion annosum. Significant variation in resistance to this pathogen was observed within and among progenies of pine trees selected at two different locations. Mycorrhizal synthesis occurred more frequently in cases where seedlings were inoculated with less pathogenic isolates of the pathogen.     

无菌松材线虫对10年生黑松致病性的研究

2001年9月,用无菌的松材线虫对10年生黑松进行接种.2002年8月,检查黑松发病情况,并对接种的松树进行线虫和细菌的再分离.试验发现:接种的8株黑松11个月后仍有5株表现正常,没有出现任何症状,而从其木质部却能分离到数量较多的无菌松材线虫.结果说明:无菌的松材线虫虽然能侵入松树木质部,但在不携带细菌的情况下对松树是没有致病力的.     

Boron retranslocation in Scots pine and Norway spruce

We previously traced 10B-enriched boric acid from shoots to roots to demonstrate the translocation of boron (B) in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings. To gain a more detailed understanding of B translocation, we sought: (1) to demonstrate B retranslocation directly, by showing that foliar-applied 10B is located in the new growth after dormancy; and (2) to assess whether shoot-applied B affects growth in the long term. We applied 10B-enriched boric acid to needles of Scots pine and Norway spruce seedlings. After a dormancy period and 9 weeks of growth, small but significant increases in the 10B isotope were found in the new stem and needles of both species. In Scots pine, the total B concentration of the new stem was also increased. Both species contained polyols, particularly pinitol and inositol. Boron-polyol complexes may provide a mechanism for mobilizing B in these species. To determine the long-term effects of applied B, seedlings were grown for two growing seasons after the application of 10B to shoots. In Norway spruce, the proportion of 10B in the root systems and current needles of the harvest year was slightly higher than in the controls, and in Scots pine root systems, marginally so. The B treatment had no effect on growth of Norway spruce seedlings. In Scots pine seedlings, the B treatment caused a 33% increase in total dry mass and significantly increased the number of side branches.     

Infection experiments with Gremmeniella abietina on seedlings of Norway spruce and Scots pine

Conidia of Gremmeniella abietina infected and caused disease symptoms in annual shoots of both Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) seedlings. In Norway spruce shoots the infection remained largely latent, with only a few seedlings showing symptoms. Mycelial growth inside the shoots was faster in Scots pine than in Norway spruce and was favoured by low temperature in both hosts. The shoots of Norway spruce seedlings had higher endophyte populations than those of Scots pine, and the populations were decreased by low temperatures. Reductions in the normal epiphytic or endophytic flora by acid mist treatments seemed to favour the development of G. abietina.     

Responses of Scots pine (Pinus sylvestris) seedlings grown in different nutrient regimes to changing root zone temperature in spring

We examined effects of nutrient availability and changing root zone temperature (RZT) on growth, gas exchange and plasma membrane H(+)-ATPase (PM-ATPase) activity of roots of 1-year-old Scots pine (Pinus sylvestris L.) seedlings during spring flushing. The 6-week growth-chamber experiment was carried out in hydroponic cultures that supplied the seedlings with low (0.5 mM N) or high (3 mM N) nutrient concentration and two rates of increase in RZT were simulated: slow warming (SW-treatment) and fast warming (FW-treatment). Air temperature, humidity, and light conditions were similar in all treatments. Growth of roots and shoots was retarded at low RZT, and fresh mass increment of roots was closely correlated with RZT sum. High nutrient availability increased nitrogen concentrations of needles and stems, but only at RZTs >13 degrees C. Low RZT and low availability of nutrients suppressed gas exchange of the seedlings. Real PM-ATPase activity was highly dependent on RZT. At high RZTs, real PM-ATPase activity was affected by nutrient availability but this effect was related to root growth. We conclude that, under conditions of high nutrient availability, Scots pine seedlings can compensate for the suppressive effects of long-term exposure to low RZT by rapidly accelerating growth, gas exchange and root metabolism, but only when RZT has increased above a threshold value, which was 13 degrees C in this study.     

Seedling shoot,needle and bud development in three provenances of Pinus sylvestris and Pinus contorta cultivated in northern Sweden

The patterns of current‐year shoot, needle and terminal bud elongation in seedlings of three Scots pine (Pinus sylvestris L.) and three lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) provenances were compared during the third and fourth growing seasons after planting. Lodgepole pine produced longer shoots and buds than did Scots pine, mainly because lodgepole pine formed more stem units and elongated at a faster rate. Stem unit length and the duration of shoot and bud elongation differed relatively little between species and provenances. Lammas or polycyclic growth occurred in some lodgepole pine provenances, but not in any Scots pine provenance, and was associated with enhanced shoot elongation. Needle elongation commenced earlier, proceeded at a faster rate, and was greater in lodgepole pine than in Scots pine, but ceased about the same time in all species and provenances. The heat sum required to attain 50% of final length was lower for shoots and needles in lodgepole pine than in Scots pine, and for shoots in northern provenances than in southern ones. Mitotic activity in the apical meristem of the terminal bud, which occurred less than one week after the seedlings were free from snow, started and ceased about the same time in each species, but was higher in lodgepole pine than in Scots pine early in the shoot elongation period.     

Effects of Fencing and Ground Preparation on Natural Regeneration of Native Pinewood over 12 years in Glen Tanar, Aberdeenshire

An enumeration was carried out in 1989 to assess the effectsof fencing and ground treatment 11 years previously on survivaland growth of naturally disseminated Scots pine seedlings inthe native pinewood at Glen Tanar, Aberdeenshire. Results showeda significant difference between unfenced and fenced plots onpine seedling density and height in 1989. Initial effects ofseedbed treatment on Scots pine seedling density, 2 years afterthe experiment began had largely disappeared by 1989. If removalof ground cover is used to enhance pine seed germination, theexposed seedlings may need to be protected from browsing byfencing. Received 19 March 1990.     

Browsing by large herbivores on Scots pine (Pinus sylvestris) seedlings in mixture with ash (Fraxinus excelsior) or silver birch (Betula pendula)

Abstract

Food selection by large herbivores occurs at a hierarchy of scales, for example landscape, patch or plant. Several hypotheses regarding food plant selection on patch or plant level have been developed. In this cafeteria-type design field experiment, conducted during one winter immediately after planting, we tested the effect of species mixture on browsing by large herbivores (mainly roe deer) on Scots pine seedlings in mixture with seedlings of ash (highly preferred) or silver birch (less preferred). Browsing on Scots pine was not affected by species mixture, neither in terms of the number of browsed pines nor browsing intensity. Instead, browsed biomass was positively and significantly correlated to the total biomass available for browsing. Also, there were differences due to species, with ash being most browsed (44.6%), followed by Scots pine (18.9%) and silver birch (11.6%). Browsed biomass per browsed seedling, however, was largest for Scots pine. In addition, browsed seedlings were initially taller compared to unbrowsed seedlings for all species. The main management implication in this study is that the species mixture did not influence large herbivore browsing on Scots pine seedlings. Hence, removing or discouraging more (or less) attractive browse species in early stages of pine regeneration activities seems unnecessary from the point of large herbivore browsing.     

Growth reactions of a Gremmeniella abietina isolate and Scots pine embryogenic tissue cultures differ in a host–parasite in vitro system

Summary The aim of this study was to screen Scots pine embryogenic tissue culture material to Gremmeniella abietina. According to the results Scots pine (Pinus sylvestris) embryogenic tissue lines (27 lines) could be ranked based on significant differences in G. abietina effects estimated by changes in glucosamine concentrations, growth, and dry matter contents of inoculated tissue pieces. The ranking order of the extreme tissue lines stayed basically similar, regardless of the variable measured. These findings imply a possibility to use the ranked embryogenic tissue lines in more detailed investigations of interactions between G. abietina and its host. This is the first report of using embryogenic tissue cultures in studying host–parasite interaction between Scots pine and G. abietina.     

Freezing temperatures in the root zone—effects on growth of containerized Pinus sylvestris and Picea abies seedlings

Roots of 1‐year‐old containerized seedlings of Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) were experimentally frozen in December. The seedlings were then grown for 3 weeks in a growth chamber and evaluated with regard to root growth capacity (RGC) and shoot elongation. The subsequent RGC of Scots pine declined as root zone temperatures were lowered from ‐6°C to ‐11°C and from ‐11°C to ‐16°C. Almost no root growth was observed after exposure to ‐20°C. Shoot growth was also negatively affected by low root temperatures but less than root growth. Low root temperatures did not affect Norway spruce as much as Scots pine, although root and shoot growth of Norway spruce were reduced after exposure to the lowest test temperatures (‐16°C and ‐20°C). The length of exposure, ranging between 1 and 8 hours had no effect on subsequent growth.     

The effect of acid rain treatments on the susceptibility of Pinus sylvestris to Gremmeniella abietina

Scots pine (Pinus sylvestris) seedlings were planted in a nursery bed 20 cm thick. The substrate had been removed from an illuvial horizon in the soil underlying a stand of Scots pine in Finland, which had suffered from severe infection by Gremmeniella abietina. The seedlings were irrigated for 3 years, applying three pH levels (2.5, 3.5, and 4.5) using sulphuric acid, nitric acid, and a 3:1 mixture of sulphuric and nitric acid. The pH of the pure water used in the control treatment was 6.1–6.4. The seedlings were inoculated with the conidiospores of G. abietina to find out their susceptibility to infection with the Scleroderris canker disease. Because to the adequacy of its buffering capacity, the soil was observed to become more acidic only in the treatments at pH 2.5. The concentrations of extractable Ca and Mg cations decreased while the concentration of Al and Fe increased with soil acidification during the 3-year experiment. Acidification did not increase the susceptibility of Scots pine to infection by the Scleroderris canker. Seedling growth was at its maximum in the acidification treatments where most nitrogen was added.     

Growth and visible responses of Scots pine (Pinus sylvestris L.) seedlings to simulated summer frost

Scots pine seedlings, 20–30 days old, were exposed to simulated summer frost in controlled environment growth chambers. The responses observed showed extreme variation between individual seedlings; seedlings suffering from needle-necrosis, healthy-looking, and dead seedlings were found in the same treatment. Four days exposure to temperatures below –4.5°C caused multiple-leaders maximally in 7.7 % of the pine seedlings and resulted in decreased shoot and root dry weight and shoot length.     

Control of bilberry vegetation by steam treatment – effects on seeded Scots pine and associated mycorrhizal fungi

In boreal forest, ground vegetation of bilberry (Vaccinium myrtillus L.) and feather mosses strongly suppress seed regeneration of Scots pine (Pinus sylvestris L.). We investigated a site preparation technique by steam treatment to control bilberry and moss vegetation after clear cutting. Sowing experiments were performed between 1993 and 1995 with Scots pine in four treatments: (1) untreated vegetation, (2) activated carbon added to the soil surface of otherwise intact ground vegetation, (3) steam treated vegetation, and (4) activated carbon added to the soil surface of steam treated vegetation. Activated carbon was added to adsorb possible phenolic compounds released from bilberry leaves and litter. Steam killed ground vegetation effectively and recolonisation of vascular plants and mosses was slow. Scots pine seedling establishment was improved by steam treatment especially when activated carbon was added to the soil surface. We interpret that activated carbon adsorbed and reduced the levels of phenolics from bilberry litter and humus, which otherwise may inhibit seed germination. Dry weight of four year old seedlings was three times higher in steam treated plots than in untreated plots. Seedling nutrient content was also strongly improved by steam treatment. We interpret the increased growth of seedlings in steam treated plots mainly as an effect of reduced resource competition from ground vegetation. Irrespective of treatment, almost all fine roots were colonised by ectomycorrhizal fungi. Using PCR-based molecular methods we were able to identify 28 taxa forming mycorrhiza on the seedlings. Steam treatment did not affect species richness and abundance of ectomycorrhizal inocculum. We conclude that steam treatment has the potential to be an efficient and environmentally acceptable method to reduce negative influence of bilberry vegetation on Scots pine seedling establishment and early growth.