effects of root freezing on the physiology and growth of picea glauca , picea mariana and pinus...

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Scand. J. For. Res. 17: 206–217, 2002 Effects of Root Freezing on the Physiology and Growth of Picea glauca, Picea mariana and Pinus banksiana Seedlings Under Different Soil Moisture Regimes CAROLE COURSOLLE 1 , FRANCINE J. BIGRAS 2 and HANK A. MARGOLIS 1 1 Centre de recherche en biologie forestie `re, Faculte ´ de foresterie et de ge ´omatique, Universite ´ Laval, Que ´bec, QC, Canada G1K 7P4, and 2 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 3800, Sainte -Foy, QC, Canada G1V 4C7 Coursolle, C. 1 , Bigras, F. J. 2 and Margolis, H. A. 1 ( 1 Centre de recherche en biologie forestie `re, Faculte ´ de foresterie et de ge ´omatique, Universite ´ Laval, Que ´bec, QC, Canada G1K 7P4, and 2 Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O. Box 3800, Sainte-Foy, QC, Canada G1V 4C7). Effects of root freezing on the physiology and growth of Picea glauca, Picea mariana and Pinus banksiana seedlings under different soil moisture regimes. Received June 5 , 2001. Accepted November 12, 2001. Scand. J. For. Res. 17: 206–217, 2002. The root systems of 2-yr-old Picea glauca, Picea mariana and Pinus banksiana seedlings were submitted to various frost temperatures during an arti cial frost to induce different levels of root damage. Frost-damaged and control seedlings were placed in a greenhouse under high and low soil moisture regimes. Seedling growth and physiology were evaluated periodically. Seedling survival was reduced when root damage reached levels of 60–80%. Root systems of all three species showed partial to total recovery by the end of the experiment. In general, root freezing damage caused reductions in seedling growth, with these reductions becoming less signi cant over time. Root damage had little to no effect on black spruce and jack pine seedling physiology, while white spruce CO 2 uptake decreased with increasing root damage. Shoot nitrogen content of all three species decreased slightly with increasing root damage. Key words : biomass, black spruce, containerized seedlings, jack pine , photosynthesis , root :shoot ratio, water potential, white spruce. Correspondence to: F. J. Bigras, e-mail: [email protected] INTRODUCTION The production of forest tree seedlings in containers overwintered outside exposes root systems to freezing temperatures that can cause damage (Lindstro ¨m & Mattsson 1994). Root damage can reduce seedling survival, growth and outplanting performance (Lind- stro ¨m 1986, Bigras 1997, 1998). Past studies have shown that survival and growth of root-damaged seedlings varied according to the degree of damage (Bigras 1997, 1998) and:or the temperature to which root systems were exposed (Lindstro ¨m 1986, Lind- stro ¨m & Stattin 1994). Furthermore, root damage induced by frost or boiling has been reported to affect negatively photosynthesis and transpiration of hardened Pinus sylvestris L. and Picea abies (L.) Karst. seedlings (Langerud et al. 1991, Troeng 1991). Root damage also seems to have varying effects on subsequent root growth. Langerud et al. (1991) re- ported that the root growth capacity of hardened Picea abies seedlings was negatively affected when 5 0% of the root system was plunged into boiling water, and Blake (1983) found that 75 % of the root system of hardened Picea glauca (Moench) Voss seedlings had to be pruned before subsequent root growth was affected. Lindstro ¨m (1986) and Lind- stro ¨m & Stattin (1994) found that the root growth capacity of hardened P. sylvestris and P. abies seedlings began to decrease when roots were exposed to temperatures between ¼ 10 and ¼ 12°C. Given the high cost of seedling production, it would be advantageous to determine the level of root damage a seedling can sustain without exhibiting major reductions in survival and growth. Unfortu- nately, few intensive studies concerning the effects of different levels of frost-induced damage on the physi- ology and growth of seedlings ready for outplanting (2 yrs old) exist. This is particularly true for studies that have isolated the effects of root damage by protecting the aerial portions of the plant. Studies separating the effects of root and shoot frost damage are important since both can occur separately in the nursery depending on the timing of the frost and the amount of snow cover. Furthermore, since root dam- age can affect seedling water relations (Stupendick & © 2002 Taylor & Francis. ISSN 0282-75 81

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Page 1: Effects of Root Freezing on the Physiology and Growth of Picea glauca , Picea mariana and Pinus banksiana Seedlings Under Different Soil Moisture Regimes

Scand. J. For. Res. 17: 206–217, 2002

Effects of Root Freezing on the Physiology and Growth ofPicea glauca, Picea mariana and Pinus banksiana SeedlingsUnder Different Soil Moisture Regimes

CAROLE COURSOLLE1, FRANCINE J. BIGRAS2 and HANK A. MARGOLIS1

1Centre de recherche en biologie forestiere, Faculte de foresterie et de geomatique, Universite Laval, Quebec, QC, CanadaG1K 7P4, and 2Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du P.E.P.S., P.O.Box 3800, Sainte -Foy, QC, Canada G1V 4C7

Coursolle, C.1, Bigras, F. J.2 and Margolis, H. A.1 (1Centre de recherche en biologie forestiere,Faculte de foresterie et de geomatique, Universite Laval, Quebec, QC, Canada G1K 7P4, and2Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 duP.E.P.S., P.O. Box 3800, Sainte-Foy, QC, Canada G1V 4C7). Effects of root freezing on thephysiology and growth of Picea glauca, Picea mariana and Pinus banksiana seedlings underdifferent soil moisture regimes. Received June 5, 2001. Accepted November 12, 2001. Scand. J.For. Res. 17: 206–217, 2002.

The root systems of 2-yr-old Picea glauca, Picea mariana and Pinus banksiana seedlings weresubmitted to various frost temperatures during an arti� cial frost to induce different levels ofroot damage. Frost-damaged and control seedlings were placed in a greenhouse under high andlow soil moisture regimes. Seedling growth and physiology were evaluated periodically. Seedlingsurvival was reduced when root damage reached levels of 60–80%. Root systems of all threespecies showed partial to total recovery by the end of the experiment. In general, root freezingdamage caused reductions in seedling growth, with these reductions becoming less signi� cantover time. Root damage had little to no effect on black spruce and jack pine seedling physiology,while white spruce CO2 uptake decreased with increasing root damage. Shoot nitrogen contentof all three species decreased slightly with increasing root damage. Key words: biomass, blackspruce, containerized seedlings, jack pine, photosynthesis, root:shoot ratio, water potential, whitespruce.

Correspondence to: F. J. Bigras, e-mail: [email protected]

INTRODUCTION

The production of forest tree seedlings in containersoverwintered outside exposes root systems to freezingtemperatures that can cause damage (Lindstrom &Mattsson 1994). Root damage can reduce seedlingsurvival, growth and outplanting performance (Lind-strom 1986, Bigras 1997, 1998). Past studies haveshown that survival and growth of root-damagedseedlings varied according to the degree of damage(Bigras 1997, 1998) and:or the temperature to whichroot systems were exposed (Lindstrom 1986, Lind-strom & Stattin 1994). Furthermore, root damageinduced by frost or boiling has been reported toaffect negatively photosynthesis and transpiration ofhardened Pinus sylvestris L. and Picea abies (L.)Karst. seedlings (Langerud et al. 1991, Troeng 1991).

Root damage also seems to have varying effects onsubsequent root growth. Langerud et al. (1991) re-ported that the root growth capacity of hardenedPicea abies seedlings was negatively affected when50% of the root system was plunged into boilingwater, and Blake (1983) found that 75% of the root

system of hardened Picea glauca (Moench) Vossseedlings had to be pruned before subsequent rootgrowth was affected. Lindstrom (1986) and Lind-strom & Stattin (1994) found that the root growthcapacity of hardened P. sylvestris and P. abiesseedlings began to decrease when roots were exposedto temperatures between ¼10 and ¼12°C.

Given the high cost of seedling production, itwould be advantageous to determine the level of rootdamage a seedling can sustain without exhibitingmajor reductions in survival and growth. Unfortu-nately, few intensive studies concerning the effects ofdifferent levels of frost-induced damage on the physi-ology and growth of seedlings ready for outplanting(2 yrs old) exist. This is particularly true for studiesthat have isolated the effects of root damage byprotecting the aerial portions of the plant. Studiesseparating the effects of root and shoot frost damageare important since both can occur separately in thenursery depending on the timing of the frost and theamount of snow cover. Furthermore, since root dam-age can affect seedling water relations (Stupendick &

© 2002 Taylor & Francis. ISSN 0282-7581

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Root freezing effects on conifer seedlings 207Scand. J. For. Res. 17 (2002)

Shepherd 1980, Langerud et al. 1991, Langerud &Sandvik 1991) and soil moisture content has beenreported to affect root regeneration (Day &MacGillivray 1975), it is important to determine theeffects of soil moisture content on the development ofroot-damaged seedlings.

The objective of this experiment was to determinethe physiological and morphological effects of � vedifferent levels of frost-induced root damage on whitespruce (Picea glauca), black spruce [Picea mariana(Mill.) BSP] and jack pine (Pinus banksiana Lamb.)seedlings grown under two different moisture regimes(dry and wet) and in a controlled environment, thelatter serving to eliminate the effects of other environ-mental variables.

MATERIALS AND METHODS

Plant material

Two-year-old containerized (IPL 45-110, IPL®; Saint-Damien, QC, Canada) white spruce, black spruce andjack pine seedlings were used during the experiment.Seedlings were grown on a peat moss:vermiculitesubstrate at a forest tree nursery (ReboisementMauricie, Saint-Etienne-des-Gres, QC, Canada) ac-cording to standard conditions for seedling productionin Quebec (Ministere de l’Energie et des Ressources,Anon. 1990). Seedlings were transferred to the Lauren-tian Forestry Centre (47°00Æ N, 71°00Æ W) in Sainte-Foy, Quebec, on 1 October 1997, removed from theircontainers, inserted into individual Ray Leach Cone-tainer cells (low-density RLC Super ‘‘Stubby’’ model,115 cm3, Stuewe and Sons, Corvallis, OR, USA) andplaced outside until 27 October 1997. Seedlings werethen placed in a cold room in the dark at 2°C for 14days while a preliminary test was performed to deter-mine which freezing temperatures should be used. Thisensured that any further hardening was minimizedbetween the preliminary test and the time the arti� cialfrost was applied.

Arti� cial frost

Seedling root systems were submitted to an arti� cialfrost using a modi� ed programmable cold room on 11November 1997. Aerial portions of seedlings were putin plastic bags with moistened paper towels and theninserted head � rst up to the root collar into polystyreneinsulation boxes (Coursolle et al. 2000) to protect themfrom frost. The initial temperature in the cold roomwas kept at 2.5°C for approximately 6 h and then

lowered at a rate of 2.5°C h¼1 with 1 h plateauxbetween each 2.5°C drop in temperature. Six temper-atures (one control, T1, and � ve freezing, T2–T6) wereused for the experiment. The � ve freezing temperatureswere determined using the results from the preliminarytest and were selected to damage approximately 20, 40,60, 80 and nearly 100% of the root system. Testtemperatures used (air temperature) were ¼10,¼12.5, ¼15, ¼20 and ¼22.5°C for white spruce,¼12.5, ¼15, ¼17.5, ¼20 and ¼22.5°C for blackspruce, and ¼10, ¼12.5, ¼15, ¼17.5 and ¼20°Cfor jack pine. Seedlings were removed at the end of the1 h plateau corresponding to the test temperaturesought. Sampled seedlings were placed in a cold roomin the dark at 2°C until they had thawed. Controlseedlings were kept in the cold room at 2°C for theduration of the arti� cial frost. Temperatures in plasticbags and root plugs were monitored using copperconstantan thermocouples attached to a datalogger(CR21X, Campbell Scienti� c, Logan, UT, USA). Tem-peratures in plastic bags did not fall below ¼5°C.Actual root plug temperatures (T2–T6) attained dur-ing the frost were: ¼5.6, ¼9.7, ¼12.2, ¼19.2 and¼22.8°C for white spruce, ¼9.7, ¼12.2, ¼15.6,¼19.2 and ¼22.8°C for black spruce, and ¼5.6,¼9.7, ¼12.2, ¼15.6 and ¼19.2°C for jack pineseedlings. Once thawed, seedlings were placed in card-board boxes and stored in a cold room in the dark at2°C until 15 January 1998 to allow dormancy require-ments to be completed. Seedlings showed no indicationof bud � ushing or mould upon removal from storage.

Greenhouse growth

Following cold storage, seedlings were repotted in IPL15-320 containers (15 cavities per container, 320 cm3;IPL®, Saint-Damien, QC, Canada) using a peat moss:vermiculite substrate (4:1, v:v). Seedlings were fertil-ized using a controlled release fertilizer (Nutricote18-6-8, type 180, with microelements; Plant ProductsCo., Brampton, ON, Canada) which was added to thesubstrate at the time of repotting at a rate of 1.44 gcavity¼1 (0.259 g N, 0.038 g P, 0.095 g K). Repottedseedlings were placed in a greenhouse on 19 January1998 at 20°:15°C day:night temperatures under a 16 hphotoperiod supplemented with high-pressure sodiumlamps (Sylvania Lumalux®; Osram Sylvania, Man-chester, NH, USA) to give a minimum of 60 mmol m¼2

s¼1 until the end of the experiment on 13 May 1998.Seedlings were placed under either a wet or dry soilmoisture regime from 19 January to 13 May. Soilmoisture was monitored daily using an MP-917 (time

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C. Coursolle et al. Scand. J. For. Res. 17 (2002)208

domain re� ectometry; E.S.I. Environmental Sensors,Victoria, BC, Canada) with a single diode probe(SDP, 407 mm length) inserted (at the midway pointbetween the top and bottom of plugs) through holesdrilled through the � ve middle cavities of containersrepotted with control seedlings. Software parameterswere corrected to factor in the total length of theprobe that was actually in contact with the substrateversus the length in contact with air (Lambany et al.1996). A second diode was attached to the end of theprobe to enhance signal reception (Lambany et al.1997). Containers were watered individually when thesoil moisture content of at least half the containersmeasured dropped below either º40% v:v for thewet regime or º15% for the dry regime. Enoughwater was added to bring moisture levels back up tosaturation (º45–60%) for the wet regime and up toº30% for the dry regime. This resulted in containersbeing watered every 2–3 days for the wet regime andabout every 5–6 days for the dry regime.

Morphological measurements

Initial root damage (damaged root mass:total rootmass½100) and live root dry mass (LRDM) wereassessed by separating damaged roots from un-damaged roots using a stereomicroscope. Damagedroots were identi� ed by a mushy cortex and:or abrown discoloration of the cambium. Percentage ofroot damage was assessed at the beginning of theexperiment, while LRDM was assessed four times(D16, D44, D72, D100), every 28 days, beginning 16days after placement in the greenhouse.

Aerial growth was assessed using shoot dry mass(SDM) and new terminal shoot length (TSL). SDMmeasurements were made at the same time asLRDM, while new TSL was assessed weekly, begin-ning 24 days after placement in the greenhouse. Theroot-to-shoot ratio (R:S ratio) was calculated foreach sampling date and the percentage of survivingseedlings was evaluated at the end of the experiment.Seedlings with 100% desiccated shoots were judged tobe dead.

Physiological measurements

CO2 uptake (An), stomatal conductance to CO2 (gs)and intercellular CO2 concentration (Ci) were mea-sured in the morning and on the same days as forSDM and LRDM using a portable photosynthesissystem (LI-6200; LI-COR, Lincoln, NE, USA) with a0.25 l chamber. Measurements were made at º1000mmol m¼2 s¼1 using a halogen lamp (type 13186,

14.5 V, 90 W; Philips, Germany). Measurements weremade on the last 4 cm of the 2nd year terminal shoot(1-yr-old needles), and no current-year needles wereplaced in the chamber.

Shoot water potential (Cs) was measured at thebase of the main stem using a pressure chamber(PMS Instruments Co., Corvallis, OR, USA). Mea-surements were made on the same day and immedi-ately after the gas-exchange measurements.

The maximal PSII photochemical ef� ciency (Fv:Fm) was measured on � ve 1-yr-old needles sampledfrom the � rst 5 cm below the terminal bud scar usinga � uorometer (PAM-2000; Heinz Walz, Effeltrich,Germany). Measurements were made on the samedays as for gas exchange. Needles were placed inPetri dishes on moistened � lter paper and placed inthe dark for 30 min before measurement. The � breoptic cable was placed at a 60° angle to the needlesduring measurement.

Shoot nitrogen (N) content on a dry mass basiswas evaluated using the Kjeldahl method (Bremner &Mulvaney 1982).

Experimental design and statistical analysis

Experimental units consisted of groups of tenseedlings, one seedling per sampling date (four dates)for An, gs, Ci, cs, Fv:Fm, LRDM, SDM and R:Sratio combined, and six seedlings for TSL andseedling survival. The experiment was designed asthree complete replicates of the 36 treatment combi-nations (2 moisture regimes½6 temperatures½3 spe-cies). Each replicate was organized into twoincomplete blocks, each corresponding to an insula-tion box containing 18 experimental units. Each com-bination of a species with a freezing temperatureappeared once in each insulation box with either ofthe two moisture levels in such a way that a differentcomponent of the three-way interaction between spe-cies, temperature and moisture level was confoundedwith the blocks in each replicate. In total, 1080seedlings were used, 648 for new terminal shootgrowth measurements and 432 for other variables.

The appropriate statistical models were � tted withthe MIXED procedure of SAS (Littell et al. 1996).All variables were analysed in the same way exceptfor new TSL, which had a repeated date effect, andinitial root damage, which had only species andfreezing temperature effects and was analysed using acompletely random design. With the exception ofinitial root damage, the � xed part of the initial mod-els contained effects for moisture regime, species,

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Root freezing effects on conifer seedlings 209Scand. J. For. Res. 17 (2002)

date, freezing temperatures and their interactions.Since freezing temperatures were not the same for allspecies, temperature was introduced as a covariate inthe initial models, along with its quadratic (T2) andcubic (T3) effects.

The random part of the initial models containedeffects for blocks within replicates, replicates andexperimental units (residuals). The model for therepeated new TSL measurements contained a randomeffect for experimental unit, the six seedlings withinany such unit being the subjects. The random part ofthe initial models (except initial root damage) wasreduced in a series of likelihood-ratio tests on thevariance components in each model (Littell et al.1996, p. 44, Bernier-Cardou & Bigras 2001). A ran-dom effect was removed from the model if its vari-ance component did not reach signi� cance at the 0.3level (Milliken & Johnson 1984, p. 262). The � xedpart of the model was also reduced in a hierarchicalfashion, starting with interaction terms involving T3.Higher order terms (T4 and T5) were not included inthe models since these effects were judged to be oflittle biological interest. Tests were conducted at thea¾0.05 level. If an interaction involving an effectwas signi� cant, this effect was kept in the model, andthe form of the polynomial in T was investigated foreach moisture level and each species, reducing themodel to its most parsimonious form whenever possi-ble. When there was no interaction, the model wasreduced for all levels of the non-interacting factor(s)simultaneously. Reduction was implemented in accor-dance with Rule 1 of Draper & Smith (1998, p. 268)for polynomial regression: terms of higher order wereremoved as long as they were non-signi� cant, andreduction was stopped when a signi� cant term was

found in the decreasing order of powers. Results arepresented as polynomial regressions of the responsevariable on frost temperature. The degree of thecurve presented depends on the statistical results. The� xed part of the model for the repeated new terminalshoot measurements involved time and its interac-tions with other factors or covariates in addition tothe � xed effects already included in the model forother variables. Dead seedlings were excluded fromthe analyses except for survival and initial root dam-age. Several variables required transformation (Box-Cox) to stabilize their residual variance: An ( x), cs

(x0.4), Fv:Fm (x19), TSL (x0.15), SDM ( x), LRDM(x0.01), R:S ratio (x0.01) and survival (arcsine( x)).

RESULTS

Initial root damage and seedling survival

Preplanting root damage varied with species (p50.0001) and increased quadratically (p50.0001) withdecreasing temperatures (T1–T6, Table 1). Jack pineseedlings exhibited the highest levels of root damage,while white spruce seedlings had the lowest levels.

The analysis of variance (ANOVA) for seedling sur-vival showed a signi� cant quadratic temperature½moisture regime interaction (p¾0.005, Table 1).White spruce survival showed moderate decreasesbeginning at T5 (91 and 82% survival for the dry andwet regime, respectively), with seedlings from the dryregime showing a larger decrease at T6 (69% sur-vival). Black spruce seedling survival began decreas-ing at T4 (87%) for the dry regime and at T5 (92%)for the wet regime with larger decreases occurring atT6 (64 and 50%). Jack pine survival started decreas-ing at T4 for the dry regime (81%) and at T5 for the

Table 1. Root plug frost temperatures, preplanting root damage levels and seedling survival (n¾18) for whitespruce, black spruce and jack pine seedlings

White spruce Black spruce Jack pine

Survival SurvivalSurvivalRoot Root (%)(%)Root (%)

Frost TemperatureTemperature damage Temperature damage damage(%)WetDry DryWet(%) Wet(°C)treatment (°C)(%)(°C) Dry

2 5 100 1002 3T1 100 100 2 1 99 10010010040¼5.61009738¼9.710010029¼5.6T2

¼9.7 55 100 100¼9.7 33 99 100T3 ¼12.2 51 97 100T4 ¼12.2 44 98 100 ¼15.6 59 87 100 ¼12.2 66 81 97

915278¼15.6929379T5 ¼19.2829161¼19.2T6 ¼22.8 69 69 84 ¼22.8 72 64 50 ¼19.2 84 2 40

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C. Coursolle et al. Scand. J. For. Res. 17 (2002)210

Fig. 1. Dry and wet regime: (A,B) white spruce (WS), (C, D)black spruce (BS), and (E, F)jack pine (JP) terminal shootlength with respect to frost tem-perature and sampling week.Treatment temperatures (T1–T6)are, respectively, 2, ¼10,¼12.5, ¼15, ¼20 and¼22.5°C for WS, 2, ¼12.5,¼15, ¼17.5, ¼20 and¼22.5°C for BS and 2, ¼10,¼12.5, ¼15, ¼17.5 and¼20°C for JP.

wet regime (91%), with T6 seedlings from bothregimes being severely affected (2 and 40%).

Morphological measurements

The analysis of TSL showed a signi� cant cubic tem-perature ½ species½moisture regime½ sampling dateinteraction (p¾0.003). Seedlings grown in the drymoisture regime (Fig. 1A, C, E) showed little differ-ence among frost temperatures (1, 1.7 and 3 cmdecreases for T6 white spruce, black spruce and jackpine seedlings, respectively, at D100 compared withT1) and exhibited less terminal shoot growth com-pared with seedlings grown in the wet regime (Fig.1B, D, F). Final TSL of white spruce and jack pineseedlings in the wet regime decreased with decreasingtemperature (4.2 and 11.5 cm decreases, respectively,for T6 seedlings compared with T1) but little differ-ence was observed for black spruce seedlings.

SDM of seedlings from the wet regime was greaterthan that of seedlings from the dry regime (p50.0001, Figs 2A, B, 3A, B, 4A, B) on D44, D72 andD100 for all three species. White spruce and jack pineSDM decreased with decreasing temperatures onD44, D72 and D100 (p50.045) (Figs 2A, B, 4A, B).Black spruce SDM decreased signi� cantly with de-creasing temperatures (p50.0001) at D100 only (Fig.3A, B). All three species showed higher rates ofdecrease with respect to frost temperature in the wetregime.

The results indicate the presence of a signi� cantcubic temperature½species½moisture regime½sam-pling date interaction (p¾0.029) for LRDM. In gen-eral, white spruce and jack pine LRDM from bothmoisture regimes, as well as black spruce LRDMfrom the dry regime decreased with increasingroot damage (Figs 2C, D, 3C, 4C, D). However, theamplitude of this decrease diminished over time,

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Root freezing effects on conifer seedlings 211Scand. J. For. Res. 17 (2002)

Fig. 2. Dry and wet regime white spruce: (A, B)shoot dry mass (SDM), (C, D) live root drymass (LRDM), (E, F) root-to-shoot (R:S) ratio,(G, H) shoot water potential (Cs), (I, J) CO2

uptake (An), (K, L) internal CO2 concentration(Ci), and (M, N) whole seedling nitrogen (N)concentration for each sampling date and withrespect to frost temperature. D16, D44, D72 andD100 refer to the number of days after place-ment in the greenhouse. Arrows indicate treat-ment temperatures.

with LRDM of white spruce, black spruce and jackpine seedlings decreasing (from T1 to T6) by 37–39%, 50% and 21–34% respectively, on D100, com-pared with decreases of 65–67%, 75% and 74–93%on D16. Finally, LRDM of black spruce seedlings

from the wet regime decreased (from T1 to T6) by 69and 48% on D16 and D44, but increased by 28 and40% on D72 and D100 (Fig. 3D).

The ANOVA for R:S ratio showed signi� cantquadratic temperature½species½date (p¾0.047),

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C. Coursolle et al. Scand. J. For. Res. 17 (2002)212

Fig. 3. Dry and wet regime black spruce: (A, B)shoot dry mass (SDM), (C, D) live root dry mass(LRDM), (E, F) root-to-shoot (R:S) ratio, (G, H)shoot water potential (Cs), (I, J) CO2 uptake (An),(K, L) internal CO2 concentration (Ci), and (M,N) whole seedling nitrogen (N) concentration foreach sampling date and with respect to frosttemperature. D16, D44, D72 and D100 refer tothe number of days after placement in the green-house. Arrows indicate treatment temperatures.

and signi� cant cubic temperature½moistureregime½date (p¾0.005) interactions. In general, theR:S ratio decreased from T1 to T6 on D16, andeither showed little change or increased with increas-ing root damage by the last sampling date on D100(Figs 2E, F, 3E, F, 4E, F).

Physiological measurements

Analyses of shoot water potential (cs) showed signi� -cant species (p50.0001) and moisture½date (p50.0001) effects, but no frost treatment effects(p\0.05). Black spruce seedlings exhibited the lowestcs, and jack pine the highest (Figs 2G, H, 3G, H, 4G,

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Root freezing effects on conifer seedlings 213Scand. J. For. Res. 17 (2002)

Fig. 4. Dry and wet regime jack pine: (A, B) shootdry mass (SDM), (C, D) live root dry mass(LRDM), (E, F) root-to-shoot (R:S) ratio, (G, H)shoot water potential (Cs), (I, J) CO2 uptake (An),(K, L) internal CO2 concentration (Ci), and (M, N)whole seedling nitrogen (N) concentration for eachsampling date and with respect to frost tempera-ture. D16, D44, D72 and D100 refer to the numberof days after placement in the greenhouse. Arrowsindicate treatment temperatures.

H). Shoot water potentials in the dry regime com-pared with the wet regime were higher on D16 andD100, similar on D44 and slightly lower on D72.

All three species exhibited signi� cantly higher ratesof net CO2 uptake (An) in the wet regime comparedwith the dry regime (p50.0001, Figs 2I, J, 3I, J, 4I,

J). White spruce An (Fig. 2I, J) exhibited a 37 and31% linear decrease (from T1 to T6, p¾0.005) withdecreasing temperature for the dry and wet regime,respectively. Black spruce An was not affected byfrost temperature (p\0.05, Fig. 3I, J), while jackpine An varied cubically (p¾0.002, Fig. 4I, J) with

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C. Coursolle et al. Scand. J. For. Res. 17 (2002)214

temperature. The effects of sampling date on An

varied according to species (p50.0001). Rates wereat their highest on D16 and on D44 for jack pine andblack spruce, respectively, and at their lowest forboth species on D100. White spruce An showed littlevariation with time.

Leaf stomatal conductance to CO2 (gs) varied sig-ni� cantly with moisture regime (p50.0001) and sam-pling date (p50.0001), but not with temperature(p\0.05). Leaf gs was reduced by approximately 49%in the dry regime compared with the wet regime anddecreased throughout the course of the experiment(results not shown).

Intercellular CO2 concentration (Ci) was greater inthe wet regime than in the dry regime (p50.0001,Figs 2K, L, 3K, L, 4K, L). White spruce Ci on D72decreased linearly (p¾0.003) with decreasing temper-ature but did not change with temperature for theother three sampling dates (p\0.05, Fig. 2I, J).Black spruce Ci did not vary with temperature (p\0.05, Fig. 3I, J). Jack pine Ci decreased from T1 toT2, then increased to T6 on D16 (p¾0.0006) anddecreased linearly (p¾0.025) on D100 (Fig. 4K, L).

The effects of moisture regime on needle chloro-phyll � uorescence (Fv:Fm) varied with respect tospecies (p¾0.0002, results not shown) and samplingdate (p50.0001, results not shown), while root frostdamage had no effect (p\0.05). Fv:Fm values variedfrom approximately 0.81 to 0.85, irrespective of spe-cies or date.

The effect of both moisture regime and species onshoot nitrogen concentration (N) varied with sam-pling date (p50.0001, Figs 2M, N, 3M, N, 4M, N).In general, whole plant N increased over the courseof the experiment and differences between dates weregreater in the dry regime. N content of white andblack spruce seedlings was higher in the wet regimethan in the dry regime for the � rst two samplingdates, and lower for the � nal two dates (Figs 2M, N,3M, N). Jack pine seedlings exhibited greater N inthe dry regime than in the wet regime (Fig. 4M, N).Shoot N of all species decreased slightly (p¾0.009)with decreasing temperature.

DISCUSSION

The results indicate that high levels of root frostdamage were required before seedling survival wasaffected. Moderate to signi� cant reductions in sur-vival began appearing when initial root damage hadreached levels of 61–69%, 72% and 66–78% for white

spruce, black spruce and jack pine seedlings, respec-tively. Furthermore, these damage levels had littleeffect on TSL, and slight to moderate effects on SDMand LRDM of surviving seedlings in the dry regime.Similar � ndings were reported by Lindstrom (1986),Bigras & Calme (1994), and Lindstrom & Stattin(1994), who found that survival and growth of hard-ened Pinus sylvestris, Picea mariana and Picea abiesseedlings only started to be affected by root damagewhen freezing temperatures had reached levels of¼15 to ¼25°C. Bigras (1997, 1998) also found that40–60% of hardened Picea mariana seedling rootsystems needed to be damaged before growth andsurvival were signi� cantly affected. Seedlings fromthe wet regime showed greater growth than seedlingsfrom the dry regime in the present study. However,white spruce and jack pine seedlings from the wetregime experienced greater reductions, compared withthe dry regime, in TSL, SDM and LRDM owing toextensive root damage. This would indicate thatwhite spruce and jack pine seedlings with extensiveroot damage could not fully take advantage of theincreased water supply in the wet regime and that soilmoisture stress may have had a more important effecton seedling growth than did root damage.

LRDM and R:S ratio measurements indicate thatroot systems from all three species were able torecover either partially or totally (black spruceseedlings from the wet regime) after severe frostdamage, since differences between control and dam-aged seedlings decreased over the course of the exper-iment. Coursolle et al. (2000) reported near totalrecovery of black spruce root systems with 52% dam-age after 60 days of growth in a controlled environ-ment, and partial recovery of white spruce and jackpine root systems with 59 and 86% damage, respec-tively. Furthermore, Bigras & Calme (1994) reportedthat black spruce root systems frozen at ¼20°C hadpartially recovered after 239 days of growth in acontrolled environment, while Bigras (1997) foundthat frozen root systems exhibited a lower dry massthan unfrozen controls after 6 months of greenhousegrowth. Blake (1983) reported that after 6 weeks ofgrowth in a controlled environment, 75% of whitespruce root systems needed to be pruned before rootdry mass was negatively affected. Root growth capac-ity has also been found either to stay the same(Langerud & Sandvik 1991) or to decrease (Lind-strom 1986, Lindstrom & Mattsson 1994, Lindstrom& Stattin 1994) with increasing root damage. Finally,in some instances (white spruce LRDM and R:S

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Root freezing effects on conifer seedlings 215Scand. J. For. Res. 17 (2002)

ratio, black spruce LRDM and jack pine SDM,LRDM and R:S ratio), shoot and root growth mea-surements seem to suggest that moderate root dam-age may stimulate seedling growth. Lindstrom (1986)and Langerud & Sandvik (1991) also reported in-creased growth, compared with controls, for P.sylvestris and P. abies seedlings with moderate rootdamage.

Root freezing damage had little or no effect onblack spruce and jack pine seedling physiology. Jackpine An and Ci (D16 and D100 only) varied with frosttemperature, but these variations at lower tempera-tures may be an aberration caused by decreasedsurvival which led to a reduced number of seedlingsavailable for measurement at these temperatures. Aslight decrease (º 0.1%) with increasing root dam-age was also observed for shoot N content. Langerudet al. (1991) and Langerud & Sandvik (1991) reporteddecreased levels of photosynthesis (CO2 uptake) andtranspiration for P. abies seedlings with 50% of theirroot systems damaged by boiling water. Troeng(1991) also reported a decrease in photosynthesi s withdecreasing root zone temperatures for P. abies and P.sylvestris seedlings. Stupendick & Shepherd (1980)reported a decrease in photosynthesis and an increasein stomatal resistance with a concurrent decrease inleaf water potential of Pinus radiata D. Don seedlingsduring the � rst 8 days after root pruning, and agradual recovery thereafter which intensi� ed onceroots had started regenerating. Blake (1983) reportedlittle long-term effect of root pruning on white sprucestomatal resistance, transpiration and needle waterpotential. The fact that cs, An, gs and Fv:Fm wereunaffected by root freezing damage seems to indicateeither that black spruce and jack pine seedlings hadadapted to root damage induced by water stressbefore the � rst sampling date, or that stress levelswere not severe enough to cause changes in thesevariables. In fact, Stewart & Bernier (1995) reportedthat black spruce photosynthesis and conductancewere signi� cantly affected by moisture stress onlywhen root plug water content had decreased to 10%(v:v) under high evaporative demand and that shootwater potential was relatively unaffected at theselevels.

White spruce An decreased with decreasing temper-ature while all the other physiological variables re-mained relatively unaffected. Since neither Ci nor gs

was affected by root damage, it is proposed that An

decreased as a result of either a decrease in thecarboxylation capacity, an increase in respiration, or

a combination of these two. A decrease in the car-boxylation capacity would be required to maintainsteady Ci when the uptake of CO2 (An) is decreased.There are several possibilities as to why the carboxy-lation capacity may have decreased. Rubisco is sensi-tive to water stress and water absorption may havebeen limited at lower temperatures owing to reduceduptake by the damaged roots or the resultant smallerroot systems. N content, which decreased slightlywith increasing root damage, is an essential compo-nent of carboxylation enzymes, so a reduction in Nmay lead to a reduction in carboxylation. Reductionsin An have been correlated with reductions in leaf N(Reich et al. 1995, Dang et al. 1997). Increases inrespiration at lower temperatures would also result inlower measurements of net CO2 uptake by the leafand these increases may have been triggered by rootdamage. van den Driessche (1987) reported thatconifer root growth was primarily achieved usingcurrent photosynthates . The extensive root regenera-tion required by damaged root systems would haverequired larger quantities of photosynthates , whichmay have led to increases in respiration.

Soil moisture regime signi� cantly affected seedlingphysiology. Seedlings from the wet regime hadgreater An, Ci and cs. White and black spruceseedling N content from the wet regime was higher,compared with the dry regime, on the � rst two sam-pling dates and lower on the last two dates. Jack pineseedling N was higher in the dry regime than in thewet regime. White and black spruce seedling cs fromthe dry regime indicates a slight to moderate waterstress which is re� ected in lower rates of An and gs.Grossnickle & Blake (1986) found that gs of whiteand black spruce seedlings decreased steadily withdecreasing cs, while Beadle et al. (1979) found that gs

of Picea sitchensis Bong. (Carr.) was affected when cs

fell below ¼1.4 MPa. Jack pine cs values (below¼0.4 MPa) indicate, at � rst glance, an absence ofwater stress in the dry regime. However, these valueswere accompanied by slightly lower An and lower gs

measurements, which would indicate a slight effect.Jack pine seedlings may have responded to waterstress by partially closing their stomata to prevent cs

from being affected. Stewart & Bernier (1995) alsofound that black spruce cs varied little with increas-ing water stress while stomatal conductance de-creased, which indicated stomatal regulation as ameans to avoid water stress.

Black spruce and jack pine seedlings showed mod-erate decreases in aerial growth (SDM in particular)

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C. Coursolle et al. Scand. J. For. Res. 17 (2002)216

in response to root frost damage, although seedlingAn was little affected. This probably resulted from achange in photosynthate allocation with increasingroot damage. Changes in R:S ratio and LRDM overthe course of the experiment probably indicate that alarger proportion of photosynthates was allocated toroot systems to facilitate root regeneration. Rootfrost damage to white spruce seedlings caused de-creases in An rates, which may have contributed tothe decrease in aerial growth but did not seem toaffect root regeneration.

In conclusion, seedling survival began to be af-fected when approximately 60–80% of root systemswere damaged by freezing temperatures. Aerialgrowth was affected by root damage to a greaterdegree in the wet regime, but root systems both beganand completed regeneration earlier in the wet regime.The reduced aerial growth of root damaged seedlingswas probably a result of a greater allocation ofphotosynthates to the regenerating root systems.Black spruce and jack pine physiology was not de-tectably affected by root damage, while white spruceAn decreased with increasing root damage. It is pro-posed that this decrease was the result of either areduction in � xation and:or carboxylation and:or anincrease in respiration. With the exception of wholeseedling N and cs, growth and physiological mea-surements were greater in the wet regime.

ACKNOWLEDGEMENTS

We thank Yves Dubuc, Jacques Prevost, JenniferWright and David Gaumont-Guay for technical as-sistance, Jasmin Fredette and Lajmi Lakhal Chaiebfor the statistical analysis, and Michele Bernier-Car-dou, senior statistician, for statistical advice. We alsothank Annick Bertrand for reviewing the manuscript,and Reboisement Mauricie Inc. and the ministere desRessources naturelles du Quebec for providing theseedlings. This research was funded by the ministeredes Ressources naturelles du Quebec, the CanadianForest Service and the Natural Sciences and Engi-neering Research Council of Canada.

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