Evaluating methods for storability assessment and determination of vitality status of container grown Norway spruce transplants after frozen storage

ABSTRACT

Autumn sown small seedlings for later transplanting into large containers have been introduced in Swedish forest tree nurseries. Containerized transplants of Norway spruce (Picea abies (L.) Karst.) from three Swedish nurseries were frozen stored during the autumn of 2014 to find out storability and post-storage vitality. Seedling storability was determined by measuring electrolyte leakage after freezing shoots to ?25°C (SE L _diff?25), by measurements of dry matter content (DMC) of seedling shoots and by the commercial molecular test ColdNSure?. Vitality of seedlings after storage was determined by measuring the leakage of electrolytes from shoots (SEL), and seedlings were also tested in regrowth tests. All three methods for storability assessment gave similar predictions, except in one case where DMC showed “not storable” for successfully stored seedlings. Our results indicated that young transplants can be successfully short term stored before reaching the target levels for safe long-term storage of conventional seedlings. Early storage of young transplants resulted in low post-storage survival and vitality expressed as root growth capacity and shoot electrolyte leakage (SEL). A prolonged duration in storage generally resulted in lower survival as well as lower root growth capacity and higher levels of SEL, especially for seedlings stored at earlier dates.     

Development of a molecular test to determine the vitality status of Norway spruce (Picea abies) seedlings during frozen storage

In boreal forest regions, a great portion of forest tree seedlings are stored indoors in late autumn to prevent seedlings from outdoor winter damage. For seedlings to be able to survive in storage it is crucial that they store well and can cope with the dark and cold storage environment. The aim of this study was to search for genes that can determine the vitality status of Norway spruce (Picea abies (L.) Karst.) seedlings during frozen storage. Furthermore, the sensitivity of the ColdNSure? test, a gene activity test that predicts storability was assessed. The storability of seedlings was tested biweekly by evaluating damage with the gene activity test and the electrolyte leakage test after freezing seedlings to ?25?°C (the SEL_diff-25 method). In parallel, seedlings were frozen stored at ?3?°C. According to both methods, seedlings were considered storable from week 41. This also corresponded to the post storage results determined at the end of the storage period. In order to identify vitality indicators, Next Generation Sequencing (NGS) was performed on bud samples collected during storage. Comparing physiological post storage data to gene analysis data revealed numerous vitality related genes. To validate the results, a second trial was performed. In this trial, gene activity was better in predicting seedling storability than the conventional freezing test; this indicates a high sensitivity level of this molecular assay. For multiple indicators a clear switch between damaged and vital seedlings was observed. A collection of indicators will be used in the future development of a commercial vitality test.     

Assessment of seedling storability of Quercus robur and Pinus sylvestris

Pedunculate oak (Quercus robur L.) and Scots pine (Pinus sylvestris L.) seedlings were lifted on several occasions during autumn 1997 to determine the relationships between storability and frost hardiness. On each lifting date their physiological status was determined by assessment of shoot and root electrolyte leakage and frost hardiness, assessed as freeze-induced electrolyte leakage. Additional seedlings were simultaneously cold-stored for field planting and assessment of preplanting root growth potential in April 1998. First year field performance was determined the following winter. Storability and cold acclimation patterns differed between the two species. Both were negatively affected by early lifting, but oak was less sensitive with respect to survival, and pine attained tolerance to cold storage more rapidly and earlier with respect to growth increment. The correlations between shoot frost hardiness and performance suggest that freeze-induced shoot electrolyte leakage (SEL_diff?20) below a threshold of 5% is a good storability predictor for Scots pine in Denmark. A completely reliable criterion for pedunculate oak could not be established.     

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.     

Root growth capacity and field performance of Pinus sylvestris and Picea abies seedlings

Correlations between root growth capacity (RGC), at the time of planting, and field performance were studied for Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings. Before planting a gradient in seedling viability was generated through exposure to low root temperatures and different winter storage regimes. The hypothesis that high RGC values would improve field performance was to some extent verified for pine seedlings while no correlations could be registered for spruce. Reasons for these results are discussed.     

Fast, nondestructive measurement of frost hardiness in conifer seedlings by VIS+NIR spectroscopy

Frost hardiness development from mid-August to mid-November was evaluated in seedlings of three provenances of Norway spruce (Picea abies (L.) Karst.) and three provenances of Scots pine (Pinus sylvestris L.) raised at nurseries in north, central and south Sweden. Measurements of the visible + near infrared (VIS+NIR) spectra of shoots were made simultaneously with estimates of frost hardiness based on electrolyte leakage following artificial freezing. Nine physiological variables known to influence frost hardiness were measured throughout the experiment. Multivariate analysis showed that VIS+NIR spectra explained 69% and 72% of the variation in frost hardiness in Scots pine and Norway spruce, respectively. Stem lignification, dry weight fraction, and starch, glucose, fructose, galactose, sucrose, raffinose and stachyose concentrations together explained 80% and 85% of the variation in frost hardiness in Scots pine and Norway spruce, respectively when used as independent X variables in a partial least squares model. These physiological variables could be related to varying degrees with variation in the VIS+NIR spectra. We conclude that VIS+NIR spectroscopy provides a rapid nondestructive technique for measuring frost hardiness in conifer seedlings based on causal relationships between the spectra and the physiology of seedling frost hardiness.     

Persistence of deltamethrin against Hylobius abietis on Norway spruce seedlings

Abstract

The pine weevil Hylobius abietis L. is major threat to forest regeneration in the Nordic countries. The persistence of the deltamethrin insecticide used against pine weevil on Norway spruce seedlings was studied after the seedlings were dipped or sprayed. Insecticide application was timed to occur either before or after frozen storage. Bioassays with the stems of Norway spruce seedlings were used to determine the effect of the insecticide against feeding by the pine weevil. The measures of the control effect were reduction in area of gnawed bark and the state of health of the pine weevils. The concentration of deltamethrin decreased rapidly in seedlings, especially after spraying treatment, which did not efficiently protect seedlings against the pine weevil 6 weeks after planting. There were no signs of degradation of deltamethrin or of an effect on seedling height after frozen storage. In bioassay, the amount of deltamethrin that efficiently prevented feeding by the pine weevil was 5.5 µg g^?1 fresh weight. After one growing season in the field, about 1.76–2.24 µg g^?1 (13–15% of the initial level) of dipped deltamethrin remained in the seedlings. In seedlings treated by spraying, 0.93–0.98 µg g^?1 (7–8% of the initial level) of the deltamethrin remained. According to bioassays, these amounts were no longer sufficient to protect seedlings from feeding by the pine weevil. Therefore, in the first summer, dipping was a significantly more efficient method of application for control of pine weevils.     

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.     

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.     

Water relations and morphological development of bare-root jack pine and white spruce seedlings: seedling establishment on a boreal cut-over site

Bare-root jack pine (Pinus banksiana Lamb.) and white spruce (Picea glauca (Moench) Voss) seedlings were planted on a boreal cut-over site and subsequent growth and seedling water relation patterns were monitored over the first growing season. Comparison of morphological development between white spruce and jack pine showed jack pine seedlings had greater new root development and a lower new shoot/new root ratio, while white spruce seedlings had greater new shoot development. Seasonal water relation patterns showed white spruce seedlings to have a greater decrease in xylem pressure potential (_x) per unit increase in transpirational flux density in comparison to jack pine seedlings. These results suggest that the greater resistance to water flow through the soil-plant-atmosphere continuum in white spruce seedlings compared to jack pine seedlings may be due to the relative lack of new root development in white spruce. Stomatal response of the seedlings showed that as absolute humidity deficit between the needles and air (AHD) increased, needle conductance (gwv) decreased in both species, but at low AHD levels white spruce had gwv approximately 35% higher than jack pine. For white spruce seedlings, gwv decreased as _x became more negative in a predictable curvilinear manner, while gwv of jack pine seedlings responded to _x with a threshold closure phenomenon at approximately - 1.75 MPa. Tissue water potential components for jack pine and white spruce seedlings at the beginning and end of the growing season showed jack pine to reach turgor loss at 76% relative water content while white spruce reached turgor loss at 88% relative water content. White spruce seedlings showed osmotic adjustment over the growing season, with an osmotic potential at turgor loss of - 1.27 MPa and - 1.92 MPa at the beginning and end of the growing season, respectively. Jack pine did not show any osmotic adjustment over the growing season. The implication of morphological development on water relation patterns are discussed with reference to successful seedling establishment.     

Overwinter Storability of Conifer Planting Stock: Operational Testing of Fall Frost Hardiness

Operational stock-testing facilities that estimate overwinter storability of seedlings (ability to survive and grow after storage) need a reliable method that provides fast results to forest nurseries. We compared three methods using container-grown seedlings of Douglas-fir, interior spruce, lodgepole pine, and western larch from forest nurseries in British Columbia. On three to nine dates in autumn, frost hardiness at −18°C was estimated using visible injury of foliage or stems (VI), electrolyte leakage from needles or stems (EL), and chlorophyll fluorescence of shoots (CF). Seedlings were placed into overwinter cold storage (−2°C). In the spring, stored seedlings were planted in nursery beds; survival and growth were assessed after one growing season. There were close correlations (r ≥ 0.93) between the assessment methods. Seedlings lifted after they reached thresholds of 69% or higher for CF and 25% or lower for EL and VI had over 90% survival at harvest and doubled shoot dry weight compared with seedlings lifted earlier. Measuring CF was the fastest and most easily replicated method to estimate successful storability, and reduced testing time by 6 days relative to VI tests.     

Effects of planting time on pine weevil (Hylobius abietis) damage to Norway spruce seedlings

Feeding by pine weevil (Hylobius abietis L.) causes severe damage to newly planted conifer seedlings in most parts of Scandinavia. We investigated the effect of planting time and insecticide treatment on pine weevil damage and seedling growth. The main objective was to study if planting in early autumn on fresh clear-cuts would promote seedling establishment and reduce the amount of damage caused by pine weevil the following season. The experiment was conducted in southern Sweden and in south-eastern Norway with an identical experimental design at three sites in each country. On each site, Norway spruce seedlings with or without insecticide treatment were planted at four different planting times: August, September, November and May the following year. In Sweden, the proportion of untreated seedlings that were killed by pine weevils was reduced when seedlings were planted at the earliest time (August/September) compared to late planting in November, or May the following year. This pattern was not found in Norway. The average length of leading shoot, diameter growth and biomass were clearly benefited by planting in August in both countries. Insecticide treatment decreased the number of seedlings killed or severely damaged in both Norway and Sweden.     

Silvicultural options to promote seedling establishment on Kalmia–Vaccinium-dominated sites

Seedling growth is often hampered on sites dominated by Kalmia angustifolia. In June 2000, a trial was established on a clear-cut site in Quebec, Canada, with a high cover of Kalmia and Vaccinium species. The objectives were to evaluate how soil scarification and fertilization at the time of planting influence early growth and establishment of black spruce [Picea mariana (Mill.) BSP] and jack pine (Pinus banksiana Lamb.) seedlings. During the first 2 years, scarification reduced Kalmia cover three-fold and doubled the distance from seedlings to the nearest Kalmia stem. Scarification did not increase soil-extractable NH₄-N concentration, and reduced soil potassium, calcium and magnesium. Scarification had no effect on seedling water stress. Seedling growth improved and foliar nutrient concentrations were generally higher in scarified plots than in unscarified control plots. No differences were observed between single- and double-pass scarification for any variables except for ground-level stem diameter of seedlings, which was greater with double-pass scarification (12.1 vs 13.1 mm). Spot fertilization increased seedling growth and foliar nitrogen concentrations. Jack pine growth was greater than black spruce growth, an effect enhanced when seedlings were fertilized.     

Effects of roe deer browsing and site preparation on performance of planted broadleaved and conifer seedlings when using temporary fences

Abstract

The effects of fencing and site preparation on performance of birch (Betula pendula Roth), oak (Quercus robur L.), pine (Pinus sylvestris L.) and spruce [Picea abies (L.) Karst.] seedlings were studied over four growing seasons in southern Sweden. The experiment was composed of four browsing treatments, from which roe deer (Capreolus capreolus L.) were excluded for 0, 12, 24 or 42 months, and four soil treatments. Natural browsing was combined with artificial browsing (clipping). It was demonstrated that browsing by roe deer could be isolated from browsing by moose (Alces alces L.) by selective fencing. Except for birch, browsing had little effect on seedling survival. Browsing reduced height growth by more than 100% for oak and pine, and more than 60% of pine seedling developed multiple stems. Except for oak, site preparation increased seedling survival. Inverting site preparation, in combination with or without fertilization, produced positive growth responses in seedlings, whereas patch scarification did not. There was no interaction between browsing and soil treatments on seedling performance. It was concluded that long-term protection against browsing by roe deer is needed for oak and pine, whereas short-term fences might be used for birch, and that spruce is relatively unaffected by browsing.     

Shoot and root sensitivity of containerized black spruce,white spruce and jack pine seedlings to late fall freezing

Damage to containerized forest seedlings due to freezing can occur in the fall or early winter in Canadian forest nurseries. The following spring, damage to shoots and impairment of growth is observed. The objectives of this experiment were to measure the impact of late fall low temperatures (0° to --30°C) on whole seedlings of the three most common species used for reforestation in Quebec: black spruce (BS), white spruce (WS) and jack pine (JP). Impacts of freezing temperatures on (i) whole seedling and apical bud mortality, (ii) shoot growth and root mortality, (iii) stem electrical resistance, (iv) shoot and root water relations, (v) concentrations of N, P, K, Ca, Mg, and total sugars in shoots were assessed. JP showed the highest rate of whole seedling mortality while WS showed the highest rate of apical bud mortality. JP was the most severely affected: destruction of the root system at low temperatures as well as a reduction of shoot growth and stem diameter and a decrease (more negative) in shoot and root water potential. WS showed a reduction of shoot growth despite no apparent damage to the root system at low temperatures. BS was not affected by temperatures as low as --30°C. Nutrient and sugar concentrations were not affected by low temperature treatments.     

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.     

Outdoor winter storage of container stock on raised pallets—effects on root zone temperatures and seedling growth

Containerized seedlings of Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) were overwintered on the ground and above ground on pallets. Soil temperatures in elevated containers were lower and showed greater fluctuation than containers on the ground. The lowest temperatures (‐15 to ‐16°C) were observed in containers stored on pallets with little or no snow cover during the winter. Temperatures in the edge rows of containers were lower than interior bed soil temperatures. Lower temperatures were also observed in the top than in the bottom of the container. The storage on pallets resulted in reduced shoot and root growth. Although insulation preventing air movements beneath the container units improved soil temperature conditions and subsequent seedling growth, the best result was obtained when seedlings were stored directly on the ground surface.     

Influence of nutrient supply on spring frost hardiness and time of bud break in Norway spruce (Picea abies (L.) Karst.) seedlings

When spring frosts occur on recently planted forest sites, severe damage may occur to the seedlings. The aim of the present study was to test how different low levels of nutrient concentrations in Norway spruce (Picea abies (L.) Karst.) seedlings affected spring frost hardiness and time of bud break. Seedlings were grown in a greenhouse for one season and supplied with fertiliser containing 22, 43 and 72 mg N l^–1, respectively. The treatments resulted in needle nitrogen concentrations ranging from 0.9 to 1.8% in autumn. After winter storage at 0 °C, bud break was recorded on seedlings growing in the greenhouse, outdoors and in growth chambers at 12 °C and at 17 °C. Freezing tests were performed on seedlings directly removed from winter storage and following one week growth in the greenhouse. Seedlings receiving fertiliser with 43 mg N l^–1 had less freezing injury than the two other fertilisation treatments in the present study. The earliest bud break occurred in seedlings receiving 72 mg N l^–1.     

Infection of Norway spruce container seedlings by Gremmeniella abietina

First‐ and second‐year containerized Norway spruce seedlings were inoculated with conidia of type A (large tree type) and type B (small tree type) of Gremmeniella abietina var. abietina at different times during the summer. The appearance of symptoms after artificial inoculation and natural infection on spruce seedlings were recorded the following spring and compared with the disease symptoms on Scots pine seedlings. The proportion of diseased seedlings after inoculation reached as high as 80%. The susceptible period during the summer began later on the first‐year seedlings than on the second‐year seedlings, and was similar for the pine seedlings. Susceptibility of first‐year seedlings was highest in August and on second‐year seedlings in July. The accumulated temperature sum, relative humidity and height growth for first‐ and second‐year seedlings was assessed. Natural infection in 2002 caused more disease on pine than on spruce seedlings. Experimental thinning of seedlings had no effect on disease incidence. In a preliminary comparison between the ability of A and B types to cause disease in Norway spruce seedlings, type B caused more damage than type A after inoculation. However, type A caused a high disease frequency in other experiments in this study. Symptoms on Norway spruce seedlings often first occurred in the mid‐section of the shoot, and were similar to those observed on pine seedlings: needles turned brown, starting at the needle base, in the spring following inoculation. On first‐year spruce, diseased needles were shed rapidly, in contrast to a slower rate of shedding on first‐year pine seedlings. Pycnidia developed about 2 years after inoculation (on pine 1 year after inoculation). On Norway spruce seedlings the lower part of the shoot, including the lateral shoots, often remained alive. The experiments show that G. abietina can cause disease on containerized Norway spruce seedlings under nursery conditions in Finland. The coincidence of spore dispersal, seedling susceptibility and predisposing factors are important in disease development.     

Root preparation technique and storage affect results of seedling quality evaluation in Norway spruce

Evaluation of tree seedling quality is necessary for improving technology for forest nursery production and seedling handling. Nutrient status can be measured to determine seedling quality, but it can be affected by seedling handling techniques. In this study effects of root preparation technique and storage regime on content of macro nutrients (N, P, K, S, Ca, Mg) in fine roots of Norway spruce (Picea abies (L.) Karst.) plants were investigated. The root preparation techniques were: (a) rinse in tap or (b) deionised water, and (c) dry preparation. These techniques were tested on seedlings subjected to four storage regimes: (1) no storage, (2) deep freezing below −20°C, and long-term (3) cold (+1 to +3°C) and (4) frozen (−3 to −4°C) storage. From the results it was concluded that the nutrient status in needles is not sufficient to describe the whole plant nutrient status in stored dormant plants, fine roots should also be included. The results also showed that deep freezing of fine roots before nutrient analyses should be avoided. Losses of K, P, S, and Mg were substantial with this method. Deionised water or dry preparation is preferred since tap water contains substantial amounts of ions that may affect the analyses.