Drought Response and Genetic Variation in Scots Pine Seedlings' Provenances: Insights From High‐Throughput Phenotyping for Climate‐Resilient Forestry

Large pine weevil (Hylobius abietis L.) damages different organs of Scots pine (Pinus sylvestris L.) seedlings in unclosed plantations, including foliage, buds, stem bark and roots. Simulated damage of respective organs, particularly debudding is one of the ways to evaluate qualitative and quantitative influence of such damage on tree condition.
 The aim of research was to evaluate the dependence of Scots pine seedlings mortality and height increment on artificial and large pine weevil caused bud damage.
 The growth of apical shoots of Scots pine seedlings was registered since the beginning of May to the end of June. Development of new shoots in seedlings with manually bud removal or large pine weevil damage started in beginning of May and continued in July.
 Mortality of Scots pine seedlings with 20% of lateral buds damaged did not differ from that in undamaged plants. The seedlings with apical and lateral buds manually removed as well as with apical and over 70% lateral buds damaged by large pine weevil have died in the year of treatment or damage. The seedlings with apical buds damaged as well as with apical and over 50% lateral buds damaged died in the 4th year after damage.
 Removal or damage up to 50% lateral buds did not affect the growth of Scots pine seedlings. Height increment of seedlings with apical buds manually removed or damaged by large pine weevil did not differ significantly in May and June of the year of treatment or damage. In July the height increment was statistically greater in the variant with manually removal of apical bud.
 Previously suggested scale of Scots pine seedlings bud damage satisfactory reflects the score of large pine weevil injuriousness.
 We can suggest that height increment in the next years after bud damage does not depend on damage way and intensity but mainly on ecological conditions.

Plants possess regulatory mechanisms that enhance nitrogen (N) uptake under conditions of spatial and temporal variation in N availability. Study of regulatory mechanisms has focused almost exclusively on the uptake of inorganic N sources (i.e., ammonium (NH4+), nitrate (NO3-). Several lines of evidence, however, suggest that amino acids may constitute a potential source of N for a number of plant species, including conifers. In the present study, we investigated the uptake of amino acids and inorganic N in Scots pine (Pinus sylvestris L.) seedlings grown at different N concentrations. We compared the uptake rate of the individual N sources using U-[13C2], [15N]-glycine, U-[13C6], [15N4]-arginine, 15NH4, or 15NO3, and tested the short-term effect of N supply on the uptake rate of glycine, arginine and in field-grown Scots pine seedlings. Our data indicate that Scots pine seedlings can absorb substantial amounts of N in the form of intact arginine and glycine molecules. The data also suggest that Scots pine seedlings down-regulate their uptake of NH4+-N and arginine-N, but not of glycine-N in response to increased endogenous N concentrations.

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.

The idea that water deficit strengthening induces concerted changes of plant physiological parameters is rather widespread. However, such changes are often difficult to identify due to challenges in establishments and maintenance of required water stress intensities using solid substrates. Therefore, we exposed Scots pine (Pinus sylvestris L.) seedlings to the range of water potentials from–0.15 to–1.5 MPa in PEG-water culture to identify the series of physiological parameters differently sensitive to water stress. We observed that even mild water stress (–0.15 MPa) inhibited root elongation, which could be one of the main pine seedlings vulnerabilities under drought. Active accumulation of osmolytes was already induced by mild water deficit and further increased with water stress severity. Plant fresh biomass growth sensitivity was more related to changes of relative water content (RWC) than to changes in tissue water content or dry weight accumulation. Plants were able to grow and accumulate dry weight down to–0.5 MPa, but lower medium water potentials (–1.0 and–1.5 MPa) suppressed growth and heavily damaged root cells, as judged from many-fold increase of Ca2+ content in roots. Chlorophyll a content was surprisingly sensitive to water stress, while carotenoids level was increased under severe stress conditions. In conclusion, the experimental system with stepwise water potential values allowed us to analyze the sensitivity scale of a range of P. sylvestris physiological processes to water stress. It was largely similar to those described earlier for other plant species, but its peculiarities were high sensitivity of root elongation, marked resistance of biomass growth to water deficit and well-developed ability to osmotic adjustment.

Deep planting is recommended in Nordic countries only for normal-sized container seedlings planted on mounds. Its effects on smaller-sized seedlings are poorly understood. We studied the effects of planting depth on the early field performance of small-sized silver birch (Betula pendula Roth) and Scots pine (Pinus sylvestris L.) container seedlings. Silver birch seedlings (mean height of 16 cm) were planted to depths of 3, 6 and 8 cm on spot mounds in May 2016. Scots pine seedlings (mean height of 9 cm) were planted to depths of 2, 5 and 8 cm on inverted mounds in September 2018 and May 2019. At the end of the first growing season, the deeper-planted birch seedlings were the tallest, as opposed to the deeper-planted Scots pine seedlings. However, the height differences between the planting depths were not apparent until the end of the second growing season in both tree species. Deeper planting decreased damage in Scots pine seedlings in the first growing season, which was not observed in silver birch. Based on our findings, small-sized Scots pine and silver birch seedlings can be planted safely at 6–8 cm planting depths, if at least 20% and 50% of their shoots, respectively, are above ground.

The effects of Cd and Zn on cross‐colonization by Paxillus involutus of Scots pine seedlings was examined by using pairs of ectomycorrhizal (ECM) and non‐mycorrhizal (NM) seedlings grown in the same vessel. This was done to assess, first, the ability of P. involutus to colonize NM Scots pine seedlings by growth from colonized roots of other Scots pine seedlings in the presence of Cd or Zn, and, second whether ECM colonization of Scots pine by P. involutus provided a competitive advantage over NM seedlings. Ectomycorrhizal colonization of Scots pine was shown to be more sensitive than Scots pine itself to Cd and Zn, but prior colonization did provide a competitive advantage with respect to biomass production. This beneficial effect over NM seedlings was, however, equal in the control, Cd and Zn treatments, and was due simply to growth stimulation in the presence of ECM colonization. Cross‐colonization from an ECM to a NM seedling was reduced but not prevented by Cd and Zn. Cd had a more negative effect on cross‐colonization than on initial colonization of seedlings, whereas Zn had an equally inhibitory effect on both parameters. These results have important implications for plant establishment on metal‐contaminated sites. If cross‐colonization between plants is reduced by toxic metals, plant establishment on contaminated sites might be retarded.

Scots pine (Pinus sylvestris L.) is one of the most widespread and economically important conifer species in the world. Applications like genomic selection and association studies, which could help accelerate breeding cycles, are challenging in Scots pine because of its large and repetitive genome. For this reason, genotyping tools for conifer species, and in particular for Scots pine, are commonly based on transcribed regions of the genome. In this article, we present the Axiom Psyl50K array, the first single nucleotide polymorphism (SNP) genotyping array for Scots pine based on whole-genome resequencing, that represents both genic and intergenic regions. This array was designed following a two-step procedure: first, 192 trees were sequenced, and a 430K SNP screening array was constructed. Then, 480 samples, including haploid megagametophytes, full-sib family trios, breeding population, and range-wide individuals from across Eurasia were genotyped with the screening array. The best 50K SNPs were selected based on quality, replicability, distribution across the draft genome assembly, balance between genic and intergenic regions, and genotype-environment and genotype-phenotype associations. Of the final 49 877 probes tiled in the array, 20 372 (40.84%) occur inside gene models, while the rest lie in intergenic regions. We also show that the Psyl50K array can yield enough high-confidence SNPs for genetic studies in pine species from North America and Eurasia. This new genotyping tool will be a valuable resource for high-throughput fundamental and applied research of Scots pine and other pine species.

Mycorrhiza formation is not needed for early growth induction and growth-related changes in polyamines in Scots pine seedlings in vitro

Economic pressures have driven an ever-widening period during which foresters use machines to plant Scots pine (Pinus sylvestris L.) seedlings. In Fennoscandia, this period has recently stretched to the entire growing season. To evaluate the performance of seedlings planted during this extended period, three experiments were carried out in Central and Northern Finland over 2 years. One-year-old and current-year seedlings were planted in mounds or disc-trenched furrows when soil temperatures were >0°C. When 1-year-old seedlings grown for spring planting and overwintered outdoors were planted after mid-June, more needles browned and growth was reduced, possibly because seedlings were oversized with respect to planting density and the volume of growth media. When current-year seedlings sown in spring were planted from July to November, those planted in late September and October grew less in later years than those planted earlier, but survival was unaffected. No large differences in field performance were found with respect to whether seedlings were planted in mounds or disc-trenched furrows. In conclusion, Scots pine seedlings can be machine planted in mounds or furrows during May and early June (later in the North) and then continued from early August until late September, provided climatic conditions in late spring and early autumn are typical and similar to those experienced in Central Finland.

Phenolic metabolites in forest trees play a key role in the defence against biotic and abiotic stressors, yet we lack information about the effects of combined abiotic factors on phenolic compounds in conifers. We studied the effects of combined abiotic factors (ozone × temperature, ozone × nitrogen, temperature × nitrogen, and ozone × temperature × nitrogen) on phenolic concentrations in stems of Scots pine (Pinus sylvestris L.) seedlings in a 3-year-long field experiment in central Finland. In current-year stems, elevated ozone increased the concentrations of total phenolics, soluble proanthocyanidins, and total proanthocyanidins, while warming reduced the concentrations of piceatannol glucoside, pinosylvin, isorhamnetin + kaempferol-3-rhamnoside, and monocoumaroyl isoquercitrin 1. Complex interaction effects on current-year stems showed that nitrogen addition increased the concentrations of some flavonoids in ambient ozone and temperature levels. In the stems from the previous years, ozone decreased the concentrations of total phenolics, total proanthocyanidins, and several flavonoids in ambient temperature, while warming increased their concentrations in combination with elevated ozone. Our results suggest that phenolic defence responses in Scots pine seedlings are affected by all three factors, but that the level of phenolics in the stems from previous years may increase under the combined exposure to elevated temperature and ozone — an expected climate trend in the Northern Hemisphere.

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.

We studied effects of soil temperature on shoot and root extension growth and biomass and carbohydrate allocation in Scots pine (Pinus sylvestris L.) seedlings at the beginning of the growing season. One-year-old Scots pine seedlings were grown for 9 weeks at soil temperatures of 5, 9, 13 and 17 degrees C and an air temperature of 17 degrees C. Date of bud burst, and the elongation of shoots and roots were monitored. Biomass of current and previous season roots, stem and needles was determined at 3-week intervals. Starch, sucrose, glucose, fructose, sorbitol and inositol concentrations were determined in all plant parts except new roots. The timing of both bud burst and the onset of root elongation were unaffected by soil temperature. At Week 9, height growth was reduced and root extension growth was much less at a soil temperature of 5 degrees C than at higher soil temperatures. Total seedling biomass was lowest in the 5 degrees C soil temperature treatment and highest in the 13 degrees C treatment, but there was no statistically significant difference in total biomass between seedlings grown at 13 and 17 degrees C. In response to increasing soil temperature, below-ground biomass increased markedly, resulting in a slightly higher allocation of biomass to below-ground parts. Among treatments, root length was greatest at a soil temperature of 17 degrees C. The sugar content of old roots was unaffected by soil temperature, but the sugar content of new needles increased with increasing soil temperature. The starch content of all seedling parts was lowest in seedlings grown at 17 degrees C. Otherwise, soil temperature had no effect on seedling starch content.

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.

Six old-growth, late postfire Scots pine (Pinussylvestris L.) dominated forest stands of the Vaccinium–Cladina type were selected along a latitudinal gradient in northern Sweden. In two of the stands, Scots pine seedlings that had naturally regenerated during the last 40 years were surveyed in relation to field- and bottom-layer vegetation. The most abundant forest floor species, viz. Cladina spp., occupied 41% of the ground cover and dominated the microhabitat of Scots pine seedlings <10 years old. The second most common species, Pleuroziumschreberi (Bird.) Mitt., which occupied 10 and 20% of the ground cover at the two sites, was, in contrast, only found to cover less than 3% of the microhabitat of the seedlings < 10 years old. With increasing age of seedlings (>10 years), however, the cover of Cladina spp. significantly decreased and the cover of P. schreberi and various ericaceous species slightly increased. Thus Scots pine seedlings initially establish in lichen-dominated vegetation but as they develop P. schreberi and ericaceous species gradually become more abundant in the ground vegetation. The relationships between natural establishment patterns and microhabitat were further empirically tested by sowing seeds of Scots pine in patches of Cladina spp. and patches of P. schreberi. Seeding was performed in six sites during 3 consecutive years. A significant higher number of established Scots pine seedlings were found in Cladina spp. vegetation than in P. schreberi dominated vegetation, and Scots pine seedlings in P. schreberi experienced a significantly higher mortality. Laboratory studies indicated a negative chemical influence by P. schreberi on Scots pine seed germination but not on radicle growth of pregerminated seeds. The negative influence of P. schreberi on Scots pine seed germination and seedling survival in the field experiments is interpreted as an effect of moisture factors, chemical interference, and barriers of nutrient availability. Age structures of naturally established Scots pine showed evidence of continuous regeneration over the last 40 years. The study emphasizes the importance of biotically mediated regeneration patterns in explaining spatial stand structure and temporal dynamics in northern boreal Scots pine forest ecosystems during prolonged absence of fire. Tree regeneration tends not to be associated with mesoscale characteristics such as gaps and tip-up mounds, but rather with microhabitat conditions. The results have implications for predicting the effect of climatic warming.

In this study, we investigated the impact of elevated atmospheric CO2 (ambient + 350 μmol mol–1) on fine root production and respiration in Scots pine (Pinus sylvestris L.) seedlings. After six months exposure to elevated CO2, root production measured by root in‐growth bags, showed significant increases in mean total root length and biomass, which were more than 100% greater compared to the ambient treatment. This increased root length may have lead to a more intensive soil exploration. Chemical analysis of the roots showed that the roots in the elevated treatment accumulated more starch and had a lower C/N‐ratio. Specific root respiration rates were significantly higher in the elevated treatment and this was probably attributed to increased nitrogen concentrations in the roots. Rhizospheric respiration and soil CO2 efflux were also enhanced in the elevated treatment. These results clearly indicate that under elevated atmospheric CO2 root production and development in Scots pine seedlings is altered and respiratory carbon losses through the root system are increased.