New Forests 13: 29–49, 1996. c 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Shoot and root sensitivity of containerized black spruce, white spruce and jack pine seedlings to late fall freezing F.J. BIGRAS1; and H.A. MARGOLIS2

1 Natural Resources Canada, Canadian Forest Service – Quebec Region, P.O. Box 3800, Sainte-Foy, Qu´ebec G1V 4C7, Canada; 2 Centre de recherche en biologie foresti`ere, Universit´e Laval, Sainte-Foy, Qu´ebec G1K 7P4, Canada ( Author for correspondence)

Received 15 December 1994; accepted 1 September 1995

Key words: frost hardiness, Picea glauca, Picea mariana, Pinus banksiana, root damage Application. It is demonstrated that shoots and roots of white spruce, black spruce and jack pine seedlings differ in their sensitivity to frost. Thus, overwintering conditions in nurseries should be adapted to suit each individual species. Jack pine needs greater protection for its roots while white spruce requires greater protection for its apical buds. Black spruce was the most frost tolerant species. Abstract. 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.

Introduction Damage to containerized forest seedlings due to extreme cold temperatures can occur each fall or early winter in Canadian forest nurseries (Colombo et al. 1982, Lindstr¨om 1987, Forˆets Qu´ebec 1992). Furthermore, this damage is usually greater in years with minimal snow cover (Lindstr¨om 1986a). Damage may appear on both shoots and roots even if the shoots have a higher hardening capacity than roots (Smit-Spinks et al. 1985, Bigras and D’Aoust 1992, 1993, Colombo 1994, Colombo et al. 1995). Damage arises if hardening has been impaired by inappropriate cultural practices, for example, or if the intensity

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30 of frost exceeds the genetic capacity of hardening of either shoots or roots (Sakai and Larcher 1987). Damage to the aerial parts of seedlings is easily identified the following spring. However, damage to roots is not easily recognized and measured (Ritchie 1990, Bigras and Calm´e 1994). As reforestation success is based on survival and growth of the seedlings, root damage to seedlings has to be detected before planting. Stem electrical resistance has been used to evaluate cold hardiness and frost injury of the aerial parts of conifer seedlings (van den Driessche 1973, Glerum 1980) and could possibly be useful in determining root damage as well. To our knowledge, it has never been tested to detect root freezing damage (Bigras and Calm´e 1994). The influence of root damage on subsequent growth and physiology of the seedlings is difficult to evaluate (Ritchie 1990). Loss of roots may be detrimental to water and nutrient uptake, available reserves, and hormone synthesis (Kramer and Kozlowski 1979). Measurements of nutrient and sugar contents and water potentials after a regrowth period following an episode of frost would possibly tell us the extent of root damage. The objectives of this experiment were to measure the impact of simulated late fall low temperatures on entire containerized seedlings of the three most common species used for reforestation in Quebec: black spruce, white spruce and jack pine. Impacts on (i) whole seedling and apical bud mortality, (ii) shoot growth and root mortality, (iii) stem electrical resistance, (iv) water relations of shoots and roots, and (v) nutrient and sugar concentrations in shoots were evaluated.

Materials and methods Plant material Five- to six-month-old black spruce (Picea mariana [Mill.] B.S.P.) (BS), white spruce (Picea glauca [Moench] Voss) (WS) and jack pine (Pinus banksiana Lamb.) (JP) seedlings were grown at a commercial forest tree nursery (Centre de production de plants forestiers de Qu´ebec inc., SainteAnne-de-Beaupr´e, QC) in multicellular containers, IPL –45–110 (45 cells per container, 110 cm3 per cell; IPL, Saint-Damien, Bellechasse, QC) with a peat moss-vermiculite substrate (4:1, v/v). The seedlings were grown under normal nursery conditions (unheated polyhouses) for containerized seedling production in Quebec. Three to four seeds were sown per cavity on May 3 for WS, May 24 for BS, and June 13, 1991 for JP and seedlings were thinned 30 d later to one per cavity. Seedlings were placed outside in mid-October. On November 27, seedlings were shipped to the Laurentian Forestry Centre

r

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31 Table 1. Seedling characteristics measured on November 4 (n = 72). Total nutrients applied since Dry mass sowing Species

Provenance

Age Shoot height Diameter Shoot Root N (d)

Black spruce 47 450 N 70 050 W 165 8.0 White spruce 46 450 N 71 450 W 186 6.6 Jack pine 50 050 N 66 460 W 145 6.1

(cm)

(mm) 0.99 1.29 1.30

(mg) 155 242 226

P

K

(mg/seedling)

79 7.3 5.7 6.5 111 15.1 4.0 7.5 182 10.2 9.2 11.6

(Canadian Forest Service – Quebec Region), Sainte-Foy (47  N 71  W) and kept in a cold room at –2  C. The characteristics of the seedlings (Nov. 4), measured at the nursery, are given in Table 1. Meteorological data show that the minimum air temperature observed from the beginning of October to late November ranged from 10.9 to –10.8  C; it is possible that freezing damage to the seedlings occurred at that time. Freezing tests On December 6, entire seedlings were removed from their containers along with their substrate. Seedlings with damage to the shoots or roots (surface of root plugs) were excluded. Seedlings were then placed in plastic bags in groups of five and maintained at –2  C until the freezing tests. The seedlings were submitted to temperatures of 0 (control), –4.3 , –9.8 , –15.6 , –20.0 , –24.5 , or –30.7  C; both air and root plugs reached these temperatures. Each temperature tested corresponded to an individual freezing test (i.e. one temperature tested at a time in one freezer). The order of the freezing temperatures was randomly selected. Simulated frosts were always tested in the same modified freezer (model E271, W.C. Woods Co. Ltd., Guelph, ON) programmed with a controller (model 2010, LFE Corp., Clinton, MA). The temperature in the freezer was monitored inside the bags (air and inside root plugs) using thermocouples connected to a datalogger (model 21X, Campbell Scientific, Logan, UT). During these tests, seedlings were maintained in the freezer for 12 h at 0  C, followed by a decrease of 2.5  C per hour for 2 h and a 2-h plateau following each 5 drop in temperature until the test temperature was reached. Seedlings were removed from the freezer at the end of the 2-h plateau. Control seedlings were kept in the freezer for a 12-h period at 0  C and then removed. After the freezing test, bags were placed for 5 to 13 d in a cold room at 2  C for slow thawing, and repotted in the same type of multicellular container as that in which they were grown. As the sequence

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32 of temperatures for the freezing tests was randomly selected, the time the seedlings spent in the cold room was dependent on the order in which they were frozen. We proceeded rapidly during the experiment to minimize the time spent at –2  C before the freezing tests or the time spent at +2  C after the freezing tests, but it is still possible that temperature effects were confounded with those of elapsed time at –2  C before freezing or elapsed time at +2  C after freezing. Growing period Regrowth was used as the viability test. Qualified as the ultimate viability test (Larsen 1978), regrowth allows time for full expression of freezing damage. After the period at +2  C, seedlings were placed in growth chambers with a day:night temperature of 20 :15  C under a 16-h photoperiod having a photosynthetic photon flux of 350 mol
m 2
s 1 . Air humidity was maintained at about 60%. Every fourth day, seedlings were irrigated and fertilized by soaking the multicellular container in a solution of commercial fertilizer of 20–8–20 (Plant Products, Brampton, ON) at a concentration of 0.62 g
L 1 until the mass of the container reached between 2500 and 3000 g. This procedure had been used previously to maintain a sufficient water supply under similar conditions (Gonzalez 1985). Morphological and physiological measurements Seedlings were examined daily for bud break after being placed in the growth chambers. Time of spruce bud break was noted when terminal buds showed green needles emerging from parted scales. For jack pine, time of bud break was noted as soon as the emerging shoot swelled the terminal bud. After 90 days in the growth chambers, whole seedling mortality, apical bud mortality and morphological and physiological characteristics were measured. Some seedlings measured for morphological and physiological characteristics showed foliar browning; however, the extent of brown foliage on an individual seedling never exceeded 40%. A seedling was considered dead if all the needles were yellow. Dead apical buds were recognized by lack of growth; since nearly 95% of the control seedlings flushed, the lack of growth could not have resulted from dormant buds. First-year shoot height and shoot growth observed during the growing period were measured with a ruler. Stem diameter was measured 1 cm above the substrate level with a digital caliper (0.01–200 mm, Digimatic, Mitutoyo Corp., Japan). Stem electrical resistance was recorded 0.5 cm above the substrate with a shigometer using fine electrodes (1 mm in diameter) 1 cm apart (model 7950, NEC, Concord, NH). Afterwards, the root systems were gently washed for 30 s under

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33 running tap water at 22  C and root and shoot water potential were immediately measured with a pressure bomb (model 600, PMS Instruments Co., Corvallis, OR). Root systems were then examined under a stereomicroscope to eliminate dead roots; live roots were dried at about 83  C for 22 h and weighed. First-year shoots, new terminal shoots and new lateral shoots were separated, dried as described previously, and weighed. Nutrient analyses were performed on first- and second-year foliage. Nitrogen content was determined on a dry matter basis by the Kjeldahl method (Bremner and Mulvaney 1982) and phosphorus, by colorimetry of the phospho-molybdic complex (Olsen and Sommers 1982). Potassium, calcium, and magnesium concentrations were determined by atomic spectrophotometry (Perkin-Elmer, model 2380) (Chapman and Pratt 1961). Total sugar concentrations were determined using the colorimetric method developed by Blakeney and Mutton (1980). Experimental design and statistical analysis The experimental design was a split-plot in three randomized complete blocks (three growth chambers). Nine containers (three containers per species, three species) were completely randomized within each growth chamber. At the main factor level, the experimental units were the containers. Thirty-five seedlings from each container were randomly divided into seven groups of five seedlings, and each group was randomly assigned to one of the seven freezing temperatures. At the temperature factor level, the experimental units were the bags of five seedlings each. There was a total of 945 seedlings (3 chambers  3 species  3 containers  7 temperatures  5 seedlings). At the time the seedlings were evaluated (after 90 days in the growth chambers), whole seedling mortality was first evaluated and dead seedlings were discarded. On the remaining seedlings, apical bud mortality was examined and seedlings with dead apical buds were also discarded. Morphological and physiological characteristics were analyzed on the remaining seedlings; consequently, means per species per temperature were based on a number of seedlings varying from 1 to 44 seedlings rather than the anticipated 45 (5 seedlings  3 containers  3 growth chambers). No statistical analysis was done for whole seedling mortality and apical bud mortality because of the lack of variation in the results. The appropriate model for the dependent variables for the growth and physiological characteristics (Table 3) was: Dependent variable = constant + chamber + species + error a + error b + temperature + species * temperature + error c + error d + error e

nefo046.tex; 14/03/1997; 2:27; v.5; p.5

34 where error a = chamber * species error b = container (species chamber) error c = block * temperature + chamber * species * temperature error d = container * temperature (species chamber) error e = seedlings (temperature container species chamber). The asterisk indicates an interaction, and parentheses enclose imbedding factors. Species effects, temperature effects, and interactions thereof were considered fixed. Interest lies in the fixed effects, but it is advantageous to first reduce the random part of the model if some of the random effects can confidently be assumed negligible; this provides simpler models, more powerful tests of significance, and avoids zero or negative variance components (Milliken and Johnson 1984, p. 262). Main species effects and associated orthogonal contrasts were tested against error a, which has 4 degrees of freedom (D.F.). Main effects of temperatures, associated orthogonal polynomial contrasts, the interaction between temperature and species and its components were tested against error c, which has 36 D.F. The MIXED procedure of SAS was used to perform the analyses (SAS Institute Inc. 1992). This procedure uses maximum likelihood to estimate variance components for the various error terms enumerated above and parameters for the fixed effects; it does not provide degrees of freedom for the mean squares for random effects. The model for the nutrient and sugar concentrations (Table 4) is basically similar to that for growth characteristics except that there are additional effects for age and interactions between age and other fixed effect factors. Shoot growth, stem diameter, electrical resistance, as well as shoot and root water potential were measured on five seedlings per temperature treatment per container. Shoot dry mass (first year, new terminal shoots, new lateral shoots) was measured on three seedlings per temperature treatment per container. Live root dry mass was also measured on three seedlings per temperature treatment per container but only in one chamber. Nutrient and sugar concentrations were analyzed on the shoots of two seedlings bulked together for each temperature treatment in each container. Hence, there was no residual error between seedlings in the models for these variables. The height of the first-year shoot was used as a covariate for the analysis of terminal shoot increment while the dry mass of the first-year shoot was used as a covariate for the dry mass of the terminal, lateral, and combined terminal and lateral shoots. A logarithmic transformation was used for the analysis of root water potential and live root dry mass to achieve homogenity of the residual variance.

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35 Results Whole seedling and apical bud mortality JP showed the highest rate of whole seedling mortality (Table 2). The whole seedling mortality of JP was 11.1% at –24.5  C and 60.0% at –30.7  C while only 2.2% of WS was affected at –30.7  C. BS did not show any mortality at either –24.5 or –30.7  C. On the other hand, WS showed the highest rate of apical bud mortality as compared with BS or JP (Table 2). On WS, 11.1% of apical bud mortality was observed at –20.0  C while none was observed at that temperature on the other two species. However, there was some minor mortality at lower temperatures. At –30.7  C, 93.3% of WS apical buds were damaged as compared with 62.2% for BS and JP. Shoot growth, stem diameter, and electrical resistance Bud break occurred first for JP (day 12), second for WS (day 23), and last for BS seedlings (day 27). Shoot growth decreased with decreasing test temperature for JP (JP * Tlin., p  0.001) with a growth reduction of 68% between 0 and –30.7  C (Figure 1A and Table 3). The reduction of growth on WS was mainly observed at –30.7  C with a growth reduction of 54% at that temperature as compared with 0  C (WS * Tlin., p < 0.001). Shoot growth was highest for BS seedlings and was not affected by decreasing temperature (BS * T, p > 0.407 for all components of temperature effect for this species). Stem diameter did not change with decreasing temperature for black spruce (BS * T, p > 0.628 for all components of temperature effect for this species) and white spruce (WS * T, p > 0.187 for all components of temperature effect for this species) (Figure 1B and Table 3). However, JP had a reduction of stem diameter of 18% between 0 and –30.7  C (JP * Tlin., p < 0.001). The rate of decrease increased with decreasing temperature (JP * Tqua., p = 0.017). The electrical resistance of the basal part of the stem of JP seedlings decreased between 0 and –15  C and then increased for temperatures below –15  C (JP * Tqua., p = 0.014) while it stayed unchanged for BS (BS * T, p > 0.694 for all components of temperature effect for this species) and WS (WS * T, p > 0.083 for all components of temperature effect for this species) (Figure 2 and Table 3). Shoot and root water potential Shoot water potential decreased slightly (more negative) with decreasing temperature for JP only (JP * Tlin., p = 0.048) (Figure 3A and Table 3); this decrease was 4% between 0 and –30.7  C. For root water potential,

nefo046.tex; 14/03/1997; 2:27; v.5; p.7

0 6.7 0 6.7

Species

Black spruce White spruce Jack pine

0 0 2.2

4.3 0 0 4.4

9.8 0 4.4 0

15.6 2.2 0 2.2

20.0

Whole seedling mortality (%) Temperature ( C)

0 0 11.1

24.5 0 2.2 60.0

30.7 0 4.4 0

0 2.2 0 0

4.3 2.2 0 2.2

9.8 2.2 0 0

15.6 0 11.1 0

20.0

Apical bud mortality (%) Temperature ( C)

Table 2. Whole seedling (n = 45) and apical bud mortality (n = 18 to 45)(%) after exposure to low temperatures.

24.4 62.2 11.5

24.5

62.2 93.3 62.2

30.7

36

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37

Figure 1. New shoot growth (A) and stem diameter (B) of hardened black spruce, white spruce and jack pine seedlings 90 days after exposure to freezing temperatures. Each symbol is the mean of 37 to 44 observations between 0 and –20.0  C, of 17 to 39 at –24.5  C and of 3 to 17 at –30.7  C.

Figure 2. Stem electrical resistance of hardened black spruce, white spruce and jack pine seedlings 90 days after exposure to freezing temperatures. Each symbol is the mean of 37 to 44 observations between 0 and –20.0  C, of 17 to 39 at –24.5  C and of 3 to 17 at –30.7  C.

nefo046.tex; 14/03/1997; 2:27; v.5; p.9

Species (S) Temperature (T) Tlinear (lin.) Tquadratic (qua). Lack-of-fit S*T BS * Tlin. BS * Tqua. Lack-of-fit WS * Tlin. WS * Tqua. Lack-of-fit JP * Tlin. JP * Tqua. Lack-of-fit Covariate

Fixed effects

Source of variation

2 6 (1) (1) (4) 12 (1) (1) (4) (1) (1) (4) (1) (1) (4) (1)

a c c c c c c c c c c c c c c d

Error1

< <

<

<

0.325 0.001 0.756 0.407 0.491 0.001 0.002 0.160 0.001 0.001 0.305

0.003

<0.001 <0.001 <0.001

Shoot growth

<

0.015 0.029 0.001 0.021 0.647 0.107 0.628 0.636 0.999 0.210 0.187 0.254 0.001 0.017 0.927

Stem diameter

0.970 0.188 0.095 0.016 0.489 0.436 0.736 0.927 0.694 0.178 0.083 0.322 0.090 0.014 0.602

Electrical resistance

p 0.107 0.691 0.226 0.076 0.345 0.874 0.083 0.732 0.528 0.640 0.707 0.048 0.877 0.024

>F <0.001

Shoot

0.120 0.543 0.274 0.684 0.712 0.010 0.021 0.529 0.582 0.145 0.899 0.909 0.020 0.093 0.060

Root

Water potential

0.021 0.346 0.133 0.578 0.421 0.286 0.674 0.213 0.723 0.611 0.184 0.341 0.035 0.193 0.665

Live root

0.103 0.158 0.061 0.049 0.170 0.360 0.919 0.122 0.705 0.167 0.093 0.287 0.086 0.938 0.131

<

<

0.057 0.001 0.001 0.010 0.156 0.015 0.230 0.587 0.714 0.024 0.335 0.719 0.001 0.001 0.096 0.001

Shoot – lateral

Dry mass Shoot – terminal

> F) associated with the analysis of variance of growth and physiological characteristics.

D.F.

Table 3. Observed significance (p

<

<

0.005 0.463 0.081 0.655 0.337 0.541 0.008 0.060 0.246 0.001 0.017 0.589 0.001

0.425

<0.001 <0.001

Shoot – terminal + lateral

38

nefo046.tex; 14/03/1997; 2:27; v.5; p.10

>j j

<

0.710 0.228 0.201 0.901 0.005 0.001

< <

0.365 0.311 0.283 —3 0.001 0.001

<

<

0.416 0.226 0.052 0.628 0.022 0.001

0.425 — 0.042 — 0.001 0.001

> jZj

< <

p

<

0.353 0.797 0.116 — 0.021 0.001

2

<

0.481 0.381 0.111 — 0.225 0.001

<

0.447 0.352 0.358 — 0.038 0.001

<

0.368 0.410 0.524 — 0.024 0.001

2

Error term used in the F tests. For live root dry mass, the data from only one chamber was available and –30.7  C was deleted from the analysis because of an empty cell for JP Z for C(S) = 0.187, C * T (S) = 0.090, residual 0.001; species was tested against random effect C(S), and T, and S * T, at this temperature: p against C * T(S). 3 Dashes appear when a random effect was removed from the model in the reduction process.

1

Chamber (B) B * S (error a) Container (C) (S B) (error b) B * T + B * S * T (error c) C * T (S B) (error d) Seedlings (T C S B) (error e)

Random effects

39

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40

Figure 3. Shoot (A) and root (B) water potential of hardened black spruce, white spruce and jack pine seedlings 90 days after exposure to freezing temperatures. Each symbol is the mean of 38 to 44 observations between 0 and –20.0  C, of 38 to 44 at –24.5  C and of 3 to 17 at –30.7  C.

a different pattern was observed for each species (Figure 3B and Table 3). Between 0 and –30.7  C, the root water potential of BS increased 17% (BS * Tlin., p = 0.021), decreased 16% for JP (JP * Tlin., p = 0.020), and stayed unchanged for WS (WS * T > 0.145 for all components of temperature effect for this species). Root and shoot dry mass Live root dry mass showed a 46% decrease between 0 and –24.5  C for JP (JP * Tlin., p = 0.035) (Figure 4A and Table 3) while it remained unchanged with test temperatures for BS (BS * T, p > 0.213 for all components of temperature effect for this species) and WS (WS * T, p > 0.184 for all components of temperature effect for this species). The total shoot growth dry mass (terminal and lateral growth) stayed unchanged at all temperatures for BS (BS * T, p > 0.337 for all components of temperature effect for this species) while it decreased with decreasing

nefo046.tex; 14/03/1997; 2:27; v.5; p.12

41

Figure 4. Live root biomass (A), total (terminal + lateral) shoot growth (B), and lateral shoot growth (C) of hardened black spruce, white spruce and jack pine seedlings 90 days after exposure to freezing temperatures. For roots, each symbol is the mean of 3 to 9 observations; at –30.7  C, means for black and white spruce are shown but were not included in the ANOVA due to an empty cell for jack pine. For shoots, each symbol is the mean of 17 to 26 observations between 0 and –20.0  C, of 8 to 21 at –24.5  C and of 1 to 12 at –30.7  C.

temperature for WS (WS * Tlin., p = 0.008) and JP (JP * Tlin., p < 0.001) (Figure 4B and Table 3). There was some indication that the rate of decrease increased with decreasing temperature (p = 0.060 and p = 0.017 for Tqua. for WS and JP respectively). This decrease was mainly due to the decrease in the dry mass of the lateral shoot growth (Figure 4C and Table 3). For JP, lateral shoot growth generally decreased with decreasing temperature (JP * Tlin., p <

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42 0.001), but the exact trend showed an initial increase between 0 and –10  C followed by a decrease from –10 down to –30.7  C (JP * Tqua., p = 0.001). For WS, the lateral shoot growth decreased with decreasing temperature (WS * Tlin., p = 0.024) while it stayed unchanged for black spruce (BS * T, p > 0.230 for all components of temperature effect for this species). Temperature had no apparent effect on the dry mass of terminal shoot growth (T, p = 0.158) for the three species (S * T, p = 0.360); there was no evidence that terminal shoot growth dry mass differed among species either (S, p = 0.103) (Table 3). Mineral and sugar content We observed no effect of temperature on the concentration of nitrogen, phosphorus, potassium, calcium, and magnesium in the foliage (T, p > 0.111) (Table 4). However, the concentrations of these elements differed with the age of foliage for each species (A * S, p = 0.003, 0.001, 0.013, < 0.001, 0.031 for the variables N, P, K, Ca, and Mg respectively). Total sugar concentrations also differed with foliage age according to species (A * S, p = 0.025) (Table 4). However, their concentrations were not affected by the frost treatments (T, p > 0.157). Discussion In late fall, the root systems of jack pine were more frost sensitive than those of the two spruce species even after 90 d of growth. To our knowledge, no information has been published on seasonal patterns or degree of frost tolerance of jack pine roots. We observed that a temperature of –15  C killed half of the roots of jack pine. Therefore, special care should be taken in nurseries to avoid the exposure of roots to low temperatures. Our results were different for the two spruce species where exposure to low temperatures in late fall did not damage the root systems. Spruce roots are generally recognized to be more frost tolerant than pine roots (Lindstr¨om 1987). Lindstr¨om (1986b) exposed root systems of Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) to freezing temperatures between –6 and –20  C and showed that the root growth capacity after the freezing tests was reduced more on the Scots pine than on the Norway spruce seedlings. Lindstr¨om and Nystr¨om (1987) reported that Scots pine and lodgepole pine roots (Pinus contorta Dougl.) were more sensitive to low temperatures than Norway spruce roots. Also, McKay (1994) showed that root systems of Sitka spruce (Picea sitchensis (Bong.) Carr.) were more frost tolerant that those of Scots pine. However, Jian (1992) showed that roots of red pine (Pinus resinosa Ait.) and black spruce were both damaged by temperatures lower than –10  C.

nefo046.tex; 14/03/1997; 2:27; v.5; p.14

Species (S) Temperature (T)2 S*T Age (A) A*S A*T A*T*S

Fixed effects

Source of variation

Jack pine

2 5 10 1 2 5 10

D.F.

1 2 1 2 1 2

Black spruce White spruce

Foliage (year)

Species

a c c e e e e

Error1

<

0.418 0.871 0.518 0.001 0.003 0.917 0.864

1.61 2.17 1.45 1.72 1.61 2.07

N (%)

0.210 0.996 0.825 0.531 0.001 0.904 0.967

0.33 0.27 0.20 0.23 0.23 0.25

P (%)

<

0.785 0.111 0.230 0.001 0.013 0.895 0.510

0.60 1.00 0.62 0.87 0.66 1.00

K (%)

p

< <

0.350 0.178 0.988 0.001 0.001 0.960 0.999

>F

0.61 0.27 0.38 0.23 0.44 0.23

Ca (%)

<

0.357 0.563 0.382 0.001 0.031 0.674 0.846

0.20 0.18 0.17 0.16 0.23 0.19

Mg (%)

>

<

47.6 28.2 51.2 29.0 38.2 22.2

0.018 0.157 0.538 0.001 0.025 0.514 0.589




Total sugars (mg g 1 )

Table 4. Nutrient concentrations (N, P, K, Ca, and Mg) and total sugars (n = 63) in first- and second-year foliage of hardened black spruce, white spruce, and jack pine seedlings 90 days after exposure to freezing temperatures and observed significance (p F) associated with the analysis of variance.

43

nefo046.tex; 14/03/1997; 2:27; v.5; p.15

3

2

1

< < <

0.107 0.521 —3 0.001 0.001 0.001

< <

— 0.113 0.809 — 0.013 0.001 0.001

<

0.108 0.292 — 0.731 0.022 0.001

p 0.099 — — — 0.001 0.001

< <

> jZ j

Error term used in the F tests. Temperature of –30.7  C has been excluded from the analyses because the samples were too small. Dashes appear when a random effect was removed from the model in the reduction process.

Chamber (B) B * S (error a) Container (C) (S B) (error b) B * T + B * S * T (error c) C * T (S B) (error d) B * S * T * A (error e) T (C S B) (error f)

Random effects

<

0.109 0.209 — 0.006 0.004 0.001

<

— 0.012 — 0.426 0.353 0.001

44

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45 Lindstr¨om (1987) proposed that the higher frost tolerance of spruce species compared with that of pines may be an adaptation to the shallower root development of spruce species. Our results showed that this observation could be extended to nursery studies since in containers, the roots of jack pine, the most sensitive species, showed a well-developed taproot and were twice the dry mass of the tolerant black spruce. In our experiment, the root systems of the spruce species from 45  N (BS) and 46  N (WS) were more frost tolerant than those from jack pine originating from a more northern latitude (50  050 N). This observation corroborates the study by McKay (1994), who reported that the latitude of seed origin alone is a poor guide to the differences in root frost hardiness observed between species. In her study, the roots of Sitka spruce from 46  N were more frost hardy than the roots of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) from 48  N. Coleman et al. (1992) observed that the distributions of sub-alpine species were not influenced by root cold hardiness. However, intraspecific root frost hardiness has been related to the latitude of seed origin for Scots pine, Norway spruce and Sitka spruce (Lindstr¨om and Nystr¨om 1987, McKay 1994). The frost sensitivity of the root systems of jack pine could have caused the high level of whole seedling mortality observed in this species. The damage to the root systems of jack pine, after exposure to low temperatures, might also explain the reduction in shoot growth and stem diameter as well as the reduction of shoot dry mass. Lindstr¨om (1987) reported that shoot growth of Scots pine and Norway spruce was reduced by exposing the root systems to freezing temperatures. While the shoot growth of Scots pine was reduced by 74% at –20  C, the reduction of shoot growth was 50% for Norway spruce at the same temperature. In our experiment, jack pine showed a shoot growth reduction of 68% as compared with 55% for white spruce at –30  C. The reduction of shoot growth may be a response to root destruction for many tree species. Langerud et al. (1991) destroyed half of the root systems of Picea abies by boiling, and showed that transpiration, photosynthesis, and shoot growth of treated seedlings were lower than for control plants after 20 d in a growth chamber. For the same species, Langerud and Sandvik (1991) also reported a reduction in shoot growth capacity when the basal half of the root system was submerged in boiling water. In our experiment, the destruction of the root system of jack pine resulted in a lower water potential of shoots and roots. This result indicates that the seedlings suffered a mild water stress that might be associated with the reduction of shoot growth through an effect on shoot turgor. With black spruce seedlings, Bigras and Calm´e (1994) observed a decrease in root water potential in response to the destruction of live roots by frost that also resulted in a reduction in shoot growth. Our frost treatments did not change the concen-

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46 tration of nutrients or of total sugars in the foliage of the three species. Coker (1984) observed a marked reduction in shoot N concentration immediately after root pruning of Pinus radiata D. Don but after 56 d, N concentration in pruned seedlings approached that before pruning. It seems that in response to our treatments, the reduction of shoot growth in jack pine is better explained by modifications in water relations than by impaired mineral absorption or mobilization. White spruce showed, at –25 and –30  C, a reduction in shoot growth and total shoot growth dry mass despite no apparent reduction of root system. It is possible that in our experiment, damage could have occurred at the root level of white spruce and that a rapid reconstruction of the root systems followed. Another possible explanation of the reduction of shoot growth for white spruce could be that photosynthesis was reduced after the low temperature exposure. For cold-hardened ponderosa pine (Pinus ponderosa Laws.) and Douglas-fir, Pharis et al. (1970) found that full recovery of photosynthesis after exposure to low temperatures (–4 to –15  C) could require several weeks. White spruce is the species that showed the greatest frost sensitivity for the apical bud. Our results confirm those reported by Clements et al. (1972) on the frost sensitivity of white spruce buds before the beginning of bud break. Stiell (1959) has also reported significant damage to apical buds of white spruce in plantations. Recently, Simpson (1994) followed the low temperature exotherms (LTE) of apical buds of white spruce from August until April and showed that LTE ranged from –20 to –34  C in November. In our experiment, exposure to –20.0 , –24.5 , and –30.7  C at the same time of the year also resulted in high apical bud mortality. Since root vitality seems to be essential to the growth of jack pine and other species (Ritchie 1990) but is difficult to evaluate, a reliable indicator of root damage would be valuable. The destruction of 46% of the root dry mass of jack pine resulted in a 4% decrease in shoot water potential from 0 to –30  C and a 16% decrease in root water potential. However, these small differences could hardly be used in predicting root damage. Larger differences were observed with black spruce seedlings by Bigras and Calm´e (1994) who reported a decrease of 70% in root water potential after a destruction of 87.5% of the root system. These authors showed a high negative correlation (r = 0.77) between the two variables and root water potential was a good indicator of freezing damage to roots even after 90 d of growth following the freezing treatments. Electrical impedance (or resistance) has been used to determine the extent of frost injury to the shoots of conifer seedlings after subjecting tissues to freezing temperatures (Glerum 1970, van den Driessche 1973). The electrical

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47 impedance is also used to determine the frost hardiness of bareroot seedlings; however, this method cannot be used to determine frost hardiness of container seedlings because of their small diameter (Glerum 1980). We wanted to determine if this method could be used to measure root damage to the root systems. Our results showed that the electrical resistance for jack pine was not correlated with damage to the root systems. The absence of a correlation between stem electrical resistance and root damage could be due to the small diameters (0.99–1.30 mm) of the seedlings used in this experiment. Viability tests used after a freezing test fall into two categories, depending on the presence or absence of an incubation time after the freezing events (Bigras and Calm´e 1994). Viability tests without incubation time are rapid and well suited for operational purposes. However, these tests do not provide information on the possibility of recovery or reconstruction of damaged tissues. Consequently, damage may be overestimated during operational testing. On the other hand, viability tests measured after an incubation period (usually refers to regrowth tests for whole plants) are long and tedious. However, these tests allow plenty of time for recovery and reconstruction of affected tissues. They are currently used for research. However, these tests do not provide information on the damage observed immediately after the freezing test. In our experiment, it is possible that the absence of correlation between electrical resistance and root damage, for example, was the consequence of tissue recovery after the freezing test, as was also hypothesized previously in the case of the reduction in shoot growth and total shoot growth dry mass without any apparent reduction of the root system. Our results showed a great frost sensitivity of the root systems of jack pine and of the apical buds of white spruce. These results could lead to different overwintering strategies to protect the most sensitive parts of the seedlings. As mentioned by Lindstr¨om (1986b), the negative effect of low root temperature on subsequent growth emphasizes the importance for nurseries to supply sufficient protection against low root temperatures. Acknowledgments The authors express their gratitude to Eric Leclerc, technician, for his collaboration during the project and to Carole H´ebert and Edith Poirier for statistical analyses. The authors also thank Richard Gohier and Rosaire Tremblay from the “Centre de protection de plants forestiers de Qu´ebec” for supplying the seedlings and for their continuing collaboration. They also thank Dr Annick Bertrand, post-doctoral fellow, Dr Andr´e D’Aoust, Mich`ele Bernier-Cardou, from the Canadian Forest Service – Quebec Region, and the anonymous reviewers for their constructive criticism of the first draft of the manuscript.

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