The effect of nursery substrate and fertilization on the growth and ...

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The effect of nursery substrate and fertilization on the growth and ectomycorrhizal status of containerized and outplanted seedlings of Picea abies Lu-Min Vaario, Arja Tervonen, Kati Haukioja, Markku Haukioja, Taina Pennanen, and Sari Timonen

Abstract: Over a 5 year period, we examined the influence of substrate and fertilization on nursery growth and outplanting performance of Norway spruce (Picea abies (L.) Karst.). We focused on the relative growth and development of roots and shoots and the colonization intensity and diversity of ectomycorrhizal (ECM) fungi. In the nursery, a conventional substrate (low-humified Sphagnum peat) supplemented with woody material (wood fibre and pine bark) and either mineral or organic fertilizers yielded shorter seedlings than those grown on the unmodified substrate. However, after outplanting, the growth rate of seedlings cultivated on modified substrates was higher than that of seedlings grown on the unmodified substrate. Seedlings cultivated in modified substrates had significantly higher root/shoot ratios and ECM diversity; the latter remained significant after ‡3 years of outplanting. Seedlings grown on a substrate containing 50% woody material and supplemented with organic fertilizer had the highest growth rate among all seedlings during the 3 year period of outplanting. Colonization intensity of ECM fungi was high in all seedlings except for those grown in heavily fertilized substrate. This study suggests that nursery techniques that produce seedlings with higher root/shoot ratios and ECM diversities could improve plantation success and growth rate for at least the first 3 years of outplanting. Re´sume´ : Sur une pe´riode de 5 ans, les auteurs ont e´tudie´ l’influence du substrat et de la fertilisation sur la croissance en pe´pinie`re et la performance au champ de l’e´piceá commun (Picea abies (L.) Karst.). Ils ont plus particulie`rement e´tudie´ la croissance relative et le de´veloppement des racines et des pousses ainsi que l’intensite´ et la diversite´ de la colonisation par les champignons ectomycorhiziens (ECM). En pe´pinie`re, un substrat conventionnel (mousse de sphaigne peu humifieé) modifie´ par l’ajout de matie`re ligneuse (fibre de bois et ećorce de pin) et de fertilisants mine´ral ou organique a produit des semis plus courts que ceux qui ont e´te´ cultive´s dans le substrat non modifie´. Apre`s la plantation au champ cependant, le taux de croissance des semis cultive´s dans les substrats modifie´s e´tait supe´rieur a` celui des semis cultive´s dans le substrat non modifie´. Les semis cultive´s dans les substrats modifie´s avaient un rapport racine/tige et une diversite´ de champignons ECM significativement plus e´leve´s et la diversite´ est demeureé significativement plus e´leveé apre`s au moins 3 ans au champ. Les semis cultive´s dans les substrats contenant 50 % de matie`re ligneuse et enrichi avec un fertilisant organique avaient le taux de croissance le plus e´leve´ de tous les semis durant la pe´riode de 3 ans au champ. L’intensite´ de la colonisation par les champignons ECM e´tait e´leveé chez tous les semis a` l’exception de ceux qui avaient e´te´ cultive´s dans un substrat fortement fertilise´. Cette e´tude indique que les techniques de pe´pinie`re qui produisent des semis avec un rapport racines/tige plus e´leve´ et une plus grande diversite´ de champignons ECM pourraient ame´liorer le taux de succe`s lors de la plantation et le taux de croissance pendant au moins les 3 premie`res anneés au champ. [Traduit par la Re´daction]

Introduction Received 16 June 2008. Accepted 8 October 2008. Published on the NRC Research Press Web site at cjfr.nrc.ca on 10 December 2008. L. Vaario1 and T. Pennanen. Finnish Forest Research Institute, Vantaa Research Unit, PL 18, FI-01301 Vantaa, Finland. A. Tervonen. Forelia Oy, Nurmija¨rven taimitarha, Kiljavantie 664, 05100 Roÿkka¨, Finland. K. Haukioja and M. Haukioja. Biolan Oy, PL 2, 27501, Kauttua, Finland. S. Timonen. Department of Applied Biology, P.O. Box 27, 00014 University of Helsinki, Finland, and Department of Applied Chemistry and Microbiology, P.O. Box 56, 00014 University of Helsinki, Finland. 1Corresponding

author (e-mail: [email protected]).

Can. J. For. Res. 39: 64–75 (2009)

Norway spruce (Picea abies (L.) Karst.) is widely planted for reforestation in the boreal zone (Grossnickle 2000). Approximately two-thirds of ca. 160 106 seedlings planted by Finnish foresters in 2006 were Norway spruce (Peltola 2007). Unfortunately, nursery-grown seedlings of Norway spruce and other conifers often exhibit poor growth after outplanting in forest sites (Burdett et al. 1984; Grossnickle 2000). Regeneration of Norway spruce with container-grown stock is a common practice in many countries (van den Driessche 1991). As in many other parts of the world, Finnish nurseries rely on low-humified Sphagnum peat as a substrate for containerized tree seedlings (Juntunen and Rikala 2001). Although conventional nursery substrates and

doi:10.1139/X08-156

Published by NRC Research Press

Light Sphagnum peat (50%), wood fibre (25%), crushed pine bark (25%)

Light Sphagnum peat (75%), wood fibre (25%) CW

CWB

Light Sphagnum peat C

*Substrates in C+ and C were Novagro; in CW were Forelia C; and in CWB were Forelia D. { Values for conventional and slow-release fertilizers are kilograms per cubic metre and values for chicken manure are litres per cubic metre. { M, using Kekkila¨ fertilization program, which is mineral fertilizer 4:1:5 to 3:1:6 to 0:4:5 (N:P:K; Kekkila¨ Oy, www.kekkila.fi). Slow-release fertilizer was Nutricote 70; O, using Biolan liquid organic fertilizer, 5:0:0 to 3:0:4 (N:P:K; Biolan Oy, www.biolan.fi); the same amount of nitrogen (about 18 mg N/seedling) was added in both fertilization programs during the first growing season. During the second growing season, fertilization was given according to the growth of the seedlings.

M, O 300 60 80 80

M, O 300 60 80 80

M, O 150 160 180 70 1.5 0.8

78 40

K (mg/L) 135 P (mg/L) 63 N (mg/L) 135 Amount{ 0.9

Fertilization of substrate Conventional mineral fertilization (16:4:17, N:P:K) Slow-release fertilizer Conventional mineral fertilization (12:6:22, N:P:K) Organic fertilization (composted chicken manure) Organic fertilization (composted chicken manure) Substrate contents* Light Sphagnum peat

Preparation of substrates and seedling materials We used commercial preparations (Biolan Oy) of conventional peat substrate containing different quantities of woody material (Table 1, Forelia C and Forelia D). The conventional substrate was limed (2 kg/m3) and fertilized as described in Table 1. Press water conductivity was 23 mS/m3. Hard plastic seedling trays (La¨nnen PL 64 tray, 40 cm 40 cm, with 64 cells each 5 cm 5 cm 5 cm, capacity

Code C+

Materials and methods

Table 1. Characteristics of the different substrates with two types of fertilization.

fertilization techniques produce stock of satisfactory height (Timmer and Munson 1991) the aboveground characteristics of plants can be an unreliable indicator of performance under more natural conditions (Thompson and Schultz 1995; Jacobs et al. 2005). For example, Wakeley reported in 1949 that there was often only a weak relationship between seedling height in the nursery and survival after outplanting for southern pines (see review by Davis and Jacobs 2005). The success of a newly planted seedling is dependent on access to a sufficient supply of soil water and its uptake. The ability of a seedling to draw water is determined by the size and distribution of its root system, the extent of root– soil contact and root hydraulic conductivity (Grossnickle 2005). Several studies have reported that the root morphology of container seedlings can be influenced by nursery fertilization (Jacobs et al. 2004), irrigation (Jacobs et al. 2004), growth stimulants, and container treatments (Arnold and Struve 1993). Heiskanen (1995) studied the conventional substrate (low-humified Sphagnum peat) and found that it can shrink on drying, has a low unsaturated hydraulic conductivity, and has a large air-filled porosity when dry. Furthermore, moist peat-based substrate is known to lose a large amount of water into the drier surrounding soil soon after outplanting (Day and Skoupy 1971; Nelms and Spomer 1983). Even without the extra problems with the peat substrate, water availability in the soil may be a limiting factor during dry years, and competition with field vegetation for water may cause mortality among newly outplanted seedlings (Nilsson ¨ rlander 1995). Seedlings are usually planted in spring; and O thus, a major impediment to the extension of the spring planting period is the risk of drought (Helenius et al. 2005). The slow growth of seedlings after outplanting could be related to water stress, low uptake of nutrients, or to both (Heiskanen 2005). These deficiencies may be a consequence of poor root development. In addition to root system quality, ectomycorrhizal (ECM) fungi are an important factor in determining seedling vigor (Smith and Read 1997), and inadequate colonization in the nursery may also play a role in poor seedling performance. The aim of this study was to test whether different peatbased substrates and fertilizers routinely used in the nursery could affect the root development and ECM fungal colonization of Norway spruce seedlings and consequently improve growth after outplanting. This was carried out by assessing (i) shoot and root development in the nursery under a combination of modified substrates and fertilization programs, (ii) seedling performance after outplanting, and (iii) colonization intensity and diversity of ECM fungi in the nursery and 3 years after outplanting.

65

Fertilization program in nursery{ M, O

Vaario et al.


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Can. J. For. Res. Vol. 39, 2009

Fig. 1. Mean shoot heights of Norway spruce seedlings: (a) first year in nursery; (b) second year in nursery; (c) first year after outplanting; (d) second year after outplanting; (e) third year after outplanting. C+, conventional substrate with slow releasing fertilizer; C, conventional substrate; CW, conventional substrate containing 25% wood fibre; CWB, conventional substrate containing 25% wood fibre and 25% pine bark. Details of the mineral and organic fertilization programs used in the nursery are given in Table 1. Bars are means, and error bars are SEs. Bars with different letters are significantly different (LSD test, p < 0.05).

80 cm3) were machine-filled with test substrate. Treatments (including three replicate trays per treatment) were repeated 24 times to yield a total of 36 864 seedlings. Norway spruce seeds from a regional seed orchard (SY73 Tammela) were sown in each cell in mid-June 2002. All seedlings were grown in an unheated greenhouse without artificial lighting for one growing season (June–November 2002) and fertilized with ca. 18 mg N/seedling. Seedling trays were transferred outside for the second growing season and fertilized according to the programs described in Table 1.

Study sites and treatment The outplanting field site was located in Jokioinen, Finland, and had been clear-cut during the winter of 2003. The site was prepared with an excavator in May 2004 and seedlings were planted manually 1 month later in four randomized blocks each containing the eight treatments (four substrates, two fertilization programs). Seventy seedlings of each treatment were planted in each block, each spaced ca. 2.5 m 2.5 m apart. In total, 2240 seedlings were planted in 2004. Published by NRC Research Press

Note: For each column, values with the same letters are not significantly different according to an LSD test (P < 0.05). *The mineral fertilization used Kekkila¨ fertilization program, which is mineral fertilizer, 4:1:5 to 3:1:6 to 0:4:5 (N:P:K; Kekkila¨ Oy: www.kekkila.fi). Slow release fertilizer was Nutricote 70; The organic fertilization used Biolan liquid organic fertilizer, 5:0:0 to 3:0:4 (N:P:K; Biolan Oy: www.biolan.fi); same amount of nitrogen (about 18 mg N per seedling) was added in both fertilization programs during the first growing season. During the second growing season fertilization was given according to the growth of the seedlings. { Values are means ± SEs. na, not available.

248 423 474 533 99.7±0.2a 99.4±0.6a 100±0a 99.8±0.2a 8.43±0.38a 8.45±0.37a 9.66±0.51acd 10.59±0.48bcd 6.68±0.56ab 8.44±0.53bc 10.23±0.40cd 11.32±1.31d na 0.62±0.06b 0.77±0.08b 0.99±0.08c

250 206 230 210

220 225 222 240

67.9±2.7b 98.0±0.8c 99.5±0.4c 98.9±0.6c

167 224 340 271 99.2±0.5a 99.6±0.3a 99.1±0.6a 99.8±0.1a 46.2±3.3a 98.1±1.1c 97.7±0.8c 94.3±2.4c 218 224 224 230 8.55±0.33a 9.40±0.44ab 9.62±0.38ab 11.11±1.02d 220 220 212 208 5.63±0.49a 9.08±0.30bc 8.97±0.19bc 10.18±0.86cd na 0.35±0.02a 0.41±0.04a 0.41±0.03a

Third year after outplanting{ Second year in nursery{

Fertilization program and sustrate* Mineral C+ C CW CWB Organic C+ C CW CWB

Root/shoot ratio in second year in nursery{

No. of sampled tips

No. of sampled tips

Second year in nursery{

Third year after outplanting{

67 Growth rate (third year after outplanting/ second year in nursery)

ECM fungal colonization intensity No. of root tips/cm

Table 2. Selected root, mycorrhizal, and shoot characteristics of seedlings grown on different substrates and fertilization programs in the second year in the nursery and the third year after outplanting.

Vaario et al.

Sampling and seedling measurements Seedling height was measured during the autumn of 2002 and 2003 in the nursery and in the autumn of 2004, 2005, and 2006 in the outplanting field. Eighteen to 20 seedlings/ treatment were measured in 2002 and 2003, 4–8 were measured in 2004 and 2005, and 100 were measured in 2006. Prior to outplanting in 2003, 20 containerized seedlings per treatment were placed in –20 8C for subsequent dry mass measurement and ECM morphotyping. In 2006, ten seedlings per treatment were carefully removed from the outplanting field along with soil (15 cm 15 cm 20 cm depth) and placed in plastic bags prior to storage at –20 8C for subsequent dry mass measurement and ECM morphotyping. Frozen seedlings were processed in sets of eight to minimize the impact of thawing on fine root observations. Observation and morphotyping of ECM Roots were washed gently in cold tap water to remove substrate and subsequently cut into approximately 2 cm long sections and placed in water. Only roots with diameter of £2 mm were examined. With the aid of a stereomicroscope, randomly selected 2 cm fragments (25 cm/seedling, 206–250 root tips were sampled in each seedling) were examined to determine the number of root tips and the percentage of ECM colonization (= ECM fungal colonization intensity). When fresh roots were swollen and had lost root hair or were covered by fungal mantles they were considered ECM-colonized roots. Fresh ECM roots were sorted into morphotypes according to the criteria and terminology of Agerer (1987–2002). Color, shape, branching, surface texture, hyphal structure and abundance, and mantle pattern were recorded (see Table 3). ECM root tips of each morphotype were counted. A representative samples of one to five root tips for each morphotype in each treatment were placed in separate, sterile centrifuge tubes containing 70% ethanol prepared with sterile DNA free water and stored at –20 8C for subsequent molecular analysis. Following the ECM examinations, dry masses of the shoots and roots were obtained after 3 days at 105 8C. Molecular analyses DNA was extracted from ECM root tips similar to the procedure described by Vainio et al. (1998). Our protocol included cell disruption using quartz sand and a FastPrep cell disrupter (Qbiogene, Inc., Illkirch CEDEX, France) for three 20 s pulses at 4 m/s, a phenol – chloroform – isoamyl alcohol (25:24:1) and chloroform – isoamyl alcohol (24:1) extraction, precipitation with polyethylene glycol, washing with 70% ethanol, drying, and resuspending in 50 mL TE buffer (10 mmol/L tris-HCl, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 8.0). The internal transcribed spacer (ITS) region of the rDNA was amplified with GC-clamped ITS1F (Gardes and Bruns 1993) and ITS2 or ITS4 primers (White et al. 1990) and analyzed using the D-GENE denaturing gradient gel system (Bio-Rad, Hercules, California). Polymerase chain reaction (PCR) amplification of samples for denaturing gradient gel electrophoresis (DGGE) analysis was performed with Biotools polymerase (B & M Laboratories, Madrid, Spain) with the following thermal profile: initial denaturation for 8 min at 95 8C; 35 cycles of denaturation for 1 min at 95 8C, anPublished by NRC Research Press

Morphytype Straight or tortuous; yellowish brown, densely stringy mantle with emanating hyphae Straight; light mantle with roundish, yellow rhizomorphs Straight or tortuous; dense or loose woolly light mantle Straight or bent; dense, loose spiny or smooth mantle Straight or bent; dark mantle with dense or loose spiny hyphae Straight or bent; dark brown or black mantle with shiny, curved emanating hyphae Straight or bent; light or dark brown smooth and shiny mantle Straight or bent, irregularly monopodial-pyramidal; yellowish brown or darkly coloured and smooth mantle with white tip Straight; hairy, bright white mantle with emanating hyphae Straight or bent; dark brown mantle with white tip Straight; smooth, thick, light coloured and semi-transparent mantle with white tip Straight or bent, monopodial, pinnate; dark with brown tip; shiny mantle with grainy rhizomorphs Straight or bent; typically constricted and darker base; white or yellowish brown smooth mantle

*Denaturing gradient gel electrophoresis.

MT19

MT16

MT15

MT13

MT12

MT9b

MT9a

MT7

MT6

MT5

MT4

MT3

Code MT2

Unknown

Unknown

Inocybe sp.

Piceirhiza bicolorata

Piloderma sp.

Tylospora sp.

Tylospora sp.

Cenococcum geophilum

Thelephora terrestris

Thelephora terrestris

Amphinema byssoides

Tomentellopsis sp.

Identity Paxillus involutus

DGGE, sequencing

DGGE, sequencing

Morphotype match with collection strain ectomycorrhiza Identical DGGE with collection strain DGGE, sequencing

Morphotype match with collection strain ectomycorrhiza

Identical DGGE with collection strain Morphotype match with collection strain ectomycorrhiza DGGE, sequencing

DGGE, sequencing

Analysis method Morphotype match with collection strain ectomycorrhiza Morphotype match with collection strain ectomycorrhiza DGGE,* sequencing

bankit1101948 EU810199 Inocybe

bankit1101956 EU810200 Tylospora

bankit1101933 EU810197 Amphinema bankit1101945 EU810198 Thelephora

Accession No.

—

—

Inocybe aurea

—

—

Tylospora sterophora, AF052557 —

—

Amphinema byssoides, AY838271 Thelephora errestris, DQ068970 —

—

Closest GenBank match —

—

—

261/279

—

—

—

263/270

—

—

272/273

244/245

—

Similarity —

Table 3. Morphological characteristics and identification of 13 different ectomycorrhizal types observed on Norway spruce seedling grown in nursery or study site (Jokionen).

—

—

e–111

—

—

—

e–128

—

—

e–151

e–134

—

E —

68 Can. J. For. Res. Vol. 39, 2009


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Fig. 2. Mean distribution of ECM morphotypes among samples: (a) second year in nursery and (b) third year after outplanting. See Fig. 1 for abbreviations. Further explanation of fertilization programs is provided in Table 1. Bars with the same letters are not significantly different (LSD test, p < 0.05). The values in the bar are the percentages of Thelephora terrestris in all ECM morphotypes. Error bars are SEs of the total percent colonization.

nealing for 1 min at 58 8C, and extension for 1 min at 72 8C; and final extension for 7 min at 72 8C (Korkama et al. 2006). The 18%–58% denaturing gradients were produced with 100% denaturing solution containing 40% deionized formamide and 7 mol/L urea. Sample gels were

electrophoresed in TAE buffer (40 mmol/L Tris-acetate, pH 8, and 1 mmol/L EDTA) for 16 h at 75 V and 60 8C (Korkama et al. 2006). The DGGE gels on the GelBond PAG (polyacrylamide gel) film (Cambrex Bio Science Rockland, Inc., Rockland, Published by NRC Research Press

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Can. J. For. Res. Vol. 39, 2009

Fig. 3. Mean number of morphotypes per sample: (a) second year in nursery and (b) third year after outplanting. See Fig. 1 for abbreviations. Further explanation of fertilization programs is provided in Table 1. Bars with the same letters are not significantly different (LSD test, p < 0.05) are marked with different letters. Error bars are SEs.

Maine) were stained with SYBR Gold, visualized under low power ultraviolet light (Dark Reader Transluminator; Clare Chemical Research, Dolores, Colorado), and photographed digitally. The following pure culture controls from the collection of the Finnish Forest Research Institute were used: Thelephora terrestris Ehrh. (R-NC10), Tylospora asterophora (Bonord.) Donk (R-MF02), Piloderma fallax (Libert) Stalpers (RSP02), Cenococcum geophilum Fr. (R-CY01), Paxillus involutus (Batsch. ex Fr.) Fr. (F-CY02), and Amphinema byssoides (Pers.) J. Erikss. (R-FC02). Piceirhiza bicolorata (ARON3669.S) was obtained from ascomycete research

group in the Department of Biology at the University of Oslo, Oslo, Norway. Unknown bands were excised and placed in 100 mL of sterile ultrapure water and incubated at room temperature overnight. The solution was used as a template for amplification with reduced cycles (25 cycles) and rerun in DGGE (Korkama et al. 2007). Single-banded samples were amplified with an ITS1F–ITS2 primer pair, purified with the JET quick PCR product purification spin kit/250 (GENMED GmbH, Lohne, Germany) and sequenced with the quick start kit and a CEQ 8000 DNA analysis system (Beckman Coulter Inc., Fullerton, California). Sequences were comPublished by NRC Research Press

Note: Values are means ± SEs (n = 10 for the second year in nursery; n = 20 for the third year after outplanting). For each row, values with the same letters are not significantly different (one-way ANOVA, LSD test, p < 0.05). *Second year in nursery. { Third year after outplanting.

1.11±0.15a 0.97±0.08ab 0.9±0.08a 0.84±0.05ab 0.38±0.10c 0.64±0.07c 0.09±0.05d 0.89±0.06ab

0.95±0.13a 0.93±0.06a

0.75±0.1ab 0.8±0.05bc

0.21±0.06d 0.81±0.06ab

0.64±0.08bc 0.83±0.08ab

0.72±0.08ab 0.69±0.04abc 0.73±0.05ab 0.66±0.04bc 0.53±0.05bc 0.68±0.04abc 0.39±0.09bcd 0.57±0.06d 0.12±0.08d 0.79±0.04a

0.78±0.07a 0.76±0.02ab

0.72±0.07ab 0.63±0.03cd

0.23±0.07d 0.7±0.05abc

3.6±0.34ab 3.75±0.20abc 3.4±0.27b 3.65±0.33abc 3.2±0.39bc 3.7±0.23abc 3.4±0.37b 3.55±0.22abc 2.45±0.25c 3.2±0.20bc 1.7±0.15c 3.3±0.21bc

CW C

2.21±0.24c 3.45±0.23bc

CW C+

C

Richness Nursery* Field{ Evenness Nursery Field Shannon’s diversity index Nursery Field

Organic fertilization

C+

CWB Mineral fertilization

Table 4. Richness, evenness, and Shannon’s diversity index of ECM morphotypes associated with nursery substrates and fertilization programs.

4.44±0.38a 4.15±0.25a

71

CWB

Vaario et al.

pared with those deposited on the UNITE (Ko˜ljalg et al. 2005; http://hermes.zbi.ee), EMBL, DDBJ, and GenBank databases using the BLASTN algorithm. Data analysis The data were analyzed with an analysis of variance (ANOVA) and stepwise linear regression techniques using the SPSS statistical software package (version 15.0; SPSS Inc., Chicago, Illinois). Statistical significance (p < 0.05) of the differences between treatments was estimated using the least significant difference (LSD) test. Richness, evenness, and Shannon’s diversity index of ECM morphotypes associated with nursery substrates and fertilizer treatments were calculated using the PC-ORD software (version 5.0; MjM software, Gleneden Beach, Oregon).

Results Seedling growth During the first growth season in the nursery, seedlings grew most vigorously on conventional substrate (C) and the fertilized conventional substrate (C+), but no significant differences were observed between fertilization programs (Fig. 1a). After the second growth season in the nursery, shoots were significantly taller under the mineral fertilization program (M) than the organic fertilization program (O) in all substrates (Fig. 1b). Although, the tallest seedlings at the end of the second growth season were still those grown on conventional substrates (C and C+) with mineral fertilization, seedlings grown on substrates containing woody material and mineral fertilizers (CWM and CWBM) had approached or surpassed those grown on C+O and CO. C+ and C seedlings supplemented with mineral fertilization were the tallest after the first year of outplanting (Fig. 1c). However, the increased relative growth of seedlings on modified substrates containing organic fertilizers continued, and the growth of C+ seedlings apparently slowed during this time. After the second year of outplanting, seedling heights of those grown on other substrates approached the C+M and CM seedlings (Fig. 1d). Three growing seasons after outplanting, the CM shoots were still highest, but the difference was much smaller than at the time of outplanting (Fig. 1e). Among the organically fertilized seedlings, those grown on CW were the tallest, and those on C+ were the shortest at the end of our study (3 years after outplanting). Although not the tallest seedlings, the field growth rate (final shoot height/shoot height at outplanting) of seedlings cultivated in CWBO was the highest of those examined at the end of our study. Over all substrates after 3 years, the seedlings treated with organic fertilizers (O program) had higher field growth rates than those receiving mineral fertilizers (M program) (Table 2). Root and ECM characteristics The root/shoot ratios of seedlings in the M program were significantly lower than those in the O program in every substrate after the second growth season in the nursery (Table 2). In M-program seedlings, there was no significant difference in root/shoot ratio among substrates. The greatest root/shoot ratio was found in CWB seedlings in the O Published by NRC Research Press

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Can. J. For. Res. Vol. 39, 2009 Table 5. Regression analyses of shoot growth in the field and seedling characteristics in the nursery. Seedling characteristics* Aboveground performance Shoot height Shoot dry mass Belowground performance Root/shoot ratio ECM diversity Root dry mass Root tip frequency ECM fungal colonization intensity

Correlation with growth rate{

R2

p

n

Negative Negative

0.83 0.91

0.002 0.003

20 10–15

Positive Positive Positive Weakly positive Weakly positive

0.91 0.86 0.79 0.54 0.44

0.003 0.001 0.018 0.004 0.072

10–15 20 10–15 20 20

*Second year in nursery. { Growth rate was calcualted as seedling height in the third year after outplanting/height in the second year in the nursery.

program. Apparently, modified substrates in the O program had a stimulating effect on root tip frequency (Table 2). Low (46%–68%) ECM colonization intensities were observed in the C+ seedlings under both fertilization programs during the nursery period (Table 2). All others were at least 94%. A total of 12 different ECM morphotypes were observed in the sampled seedlings (Fig. 2a, Table 3). Thelephora terrestris was the dominant ECM fungus in all treatments except in CWB under the O program in the nursery (Fig. 2a). Seedlings grown in CW and CWB in both fertilization programs had a higher percentage of mycorrhizae other than T. terrestris than did the seedlings grown on C and C+. ECM diversity (the number of morphotypes per seedling) was significantly higher in modified substrates than in conventional substrates under both fertilization programs (Fig. 3a). Three years after outplanting, root tip frequencies of seedlings grown on CW and CWB substrates under both fertilization programs remained greater than of those grown on conventional substrates under either fertilization program (Table 2). By this stage, all the seedlings were nearly fully colonized by ECM (Table 2). The total number of different ECM morphotypes observed was still 12, and only one morphotype was different from those found in the nursery (Fig. 2b, Table 3). Three years after outplanting, T. terrestris was distributed more evenly among seedlings (46%–72%) than during nursery cultivation (25%–97%) (Figs. 2a and 2b). The highest ECM diversity remained in the CWB seedlings grown under the O program (Fig. 3b); however, in general, the number of morphotypes per seedling was becoming more even. Richness, evenness, and Shannon’s diversity index of ECM morphotypes also showed that colonization of the different types of seedlings was becoming more similar (Table 4). However, some differences were still apparent and more pronounced under the M program. Predicting shoot growth in the field from seedling characteristics in the nursery Shoot height after two growing seasons in the nursery did not correlate with shoot height in the field 3 years after outplanting. Moreover, regression analyses revealed that both shoot height and dry mass after two seasons in the nursery

were negatively correlated with field growth rate (Table 5). In contrast, root/shoot ratio, ECM diversity, and root dry mass were positively correlated with field growth rate. Additionally, root tip frequency and ECM colonization intensity in the nursery showed a positive trend with field growth rate (Table 5).

Discussion This study showed that the growth substrate and fertilization program have a strong influence on seedling growth of Norway spruce in the nursery and field. A conventional substrate of low-humified Sphagnum peat produced satisfactory shoot size in the nursery but this superior shoot growth did not transfer to the field. After outplanting, seedlings grown on conventional substrates (C and C+) suffered a reduction in relative growth rate compared with those grown on substrates modified by the addition of woody material. Modified substrates had a negative effect on shoot growth in the nursery, particularly seedlings cultivated in the CWBO treatment. The slow growth of seedlings on modified substrates containing woody material could be caused by heavy nutrient competition with wood-degrading microbes (Rayner and Boddy 1988). In spite of a lower nursery shoot height, adding woody material to the conventional substrate resulted in a well-developed root system and diverse assemblage of ECM that facilitated an increased growth rate of the seedling after outplanting. The regression analysis suggested that nursery shoot height correlated negatively with performance in outplanted Norway spruce seedlings, a finding that supports the earlier study (Thompson and Schultz 1995). In this study, nursery growth responses of the roots and associated ECM provided the best indication of seedling performance after outplanting. During the nursery period, we found that the root/shoot ratio was greater in seedlings grown on modified substrates than those on the conventional substrate and that root/shoot ratio in the nursery correlated positively with field growth rate. Rytter et al. (2003) investigated the field performance of containerized seedlings of silver birch (Betula pendula Roth) and Norway spruce and suggested that shoot size was of lesser importance to future growth than a large root/shoot ratio, which is known to be important in the growth of other coniferous species (Racey et al. 1983; Larsen et al. 1986). Furthermore, it has been rePublished by NRC Research Press

Vaario et al.

ported that the production of new roots can rapidly alleviate the reduced growth of seedlings caused by ‘‘transplant shock’’ (Davis and Jacobs 2005). We found that the addition of woody material into a conventional peat-based substrate made the root system more ramified and increased the root tip frequency during nursery cultivation, which remained detectable 3 years after outplanting. Furthermore, wood fibre and bark may improve the water-storage capacity of Sphagnum peat, thereby increasing its conductivity and minimizing its air-filled porosity (Heiskanen 1995). Thus, nursery techniques that promote root system growth can alleviate the risk of plantation failure due to drought during the outplanting period and encourage faster growth rates once established. In general, symbiotic associations with fungi are important to the structure, function, and health of plant communities (Smith and Read 1997). Substrate type is one of the principal factors influencing ECM colonization intensity of the seedlings (Rincoń et al. 2005). Recently, Aucˇina et al. (2007) reported that the addition of forest litter to the growth medium of Scots pine (Pinus sylvestris L.) seedlings in the nursery had a large effect on the relative proportions of ECM fungal symbionts. In this study, there was no significant difference in ECM colonization intensity between conventional and modified substrates during nursery cultivation. However, differences in ECM diversity were significant, and the regression analysis identified ECM diversity as one of the most important indicators of good performance in the field. A high diversity of ECM fungi is believed to benefit seedling growth and to affect plant responses such as changes in shoot–root biomass allocation and uptake of nutrients (Baxter and Dighton 2001), as well as reduce competition among conifers (Perry et al. 1989). Dighton et al. (1993) have shown that ECM fungi differ in their capacities to acquire essential nutrients from the soil and supply them to their plant hosts. In addition to the impact of ECM fungi, different growth rates among treatments may lead to physiological differences that influence the subsequent performance of seedlings. Unfortunately, we did not collect physiological measurements from the seedlings in our study. The presence of ECM fungi in the seedlings estimated by ECM morphotyping, DGGE analysis, and DNA sequencing indicated that the structure of ECM communities were rather similar in the nursery and field. This result is in accordance with a previous study (Dahlberg 1990) in which 80%–95% of the ECM fungi was of nursery origin following one growth season after outplanting. Similarly, Pennanen et al. (2005) found that most of the observed fungal sequences colonizing root tips after outplanting were the same as those found in the nursery. In our study, the high relative abundance of T. terrestris in the nursery (58%) and after 3 years of outplanting (57.5%) supports the same conclusions. However, the source population of root tip ECM fungi of outplanted seedlings is obscured by the fact that T. terrestris is the dominant ECM fungus in nurseries and clear-cut sites (Korkama et al. 2006). In the present study, it was shown that the shoot growth of Norway spruce seedlings was enhanced in the nursery through the addition of fertilizer, but these same seedlings suffered a reduction in relative growth rate after outplanting. Belowground, a high level of fertilization had a negative in-

73

fluence on ECM fungal diversity and root tip frequency, which resulted in a reduced number of different ECM root tips. This result was expected because many ECM fungi are sensitive to high nutrient conditions (Marx et al. 1977). ECM fungi represent a carbon drain on plants, and it is expected that in the presence of abundant nutrients ECM seedlings would be smaller than uninfected seedlings; however, their survival and growth in the field would be greater than those of seedlings with large shoots but few or no ECM fungi (Trofymow and van den Driessche 1991). In the present study, organic fertilization had a positive effect on the root/shoot ratio in the nursery especially when combined with woody substrates. Glatzel et al. (1990) also found organic fertilizer to be superior to mineral fertilizer because it stimulated better root growth as a result of its slow release of nitrogen, which resulted in enhancing the growth of spruce seedlings planted in forest conditions. Mineral fertilization programs are conventionally used in nurseries (Landis 1989; Donald 1991) including those in Finland (Juntunen and Rikala 2001). However, soluble nutrients can leach into underlying soil through irrigation or rain and escape into the environment where they may pose problems to local systems (Alexander 1993; Juntunen et al. 2002). Using slow-release organic fertilizers in nursery programs would be beneficial both to plant production and the natural environment. In conclusion, this study suggests that substrates containing woody material and organic fertilizers yield higher root/ shoot ratios and ECM diversities in the resulting seedlings, which in turn improve their growth rate for the first 3 years after outplanting. High root/shoot ratio and ECM diversity of the seedlings were the best predictors of seedling performance after outplanting. Thus, our study advocates nursery techniques based on these principles. Site preparation, such as inverting, has been shown to have a positive effect on the growth of spruce seedlings as early as 3 years after ¨ rlander et al. 1998; Pennanen et al. 2005). Thus, planting (O an optimal plantation program will involve modified substrates and organic fertilization in the nursery cultivation of seedlings possessing a high root/shoot ratio.

Acknowledgments This work was supported by the Finnish Forester Foundation. We thank Enni Flykt for collecting the samples from the nursery and Michael Hardman for checking the language.

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