Cultivatio...n organic nitrogen improves

Forest Ecology and Management 276 (2012) 118–124

Contents lists available at SciVerse ScienceDirect

Forest Ecology and Management journal homepage: www.elsevier.com/locate/foreco

Cultivation of Norway spruce and Scots pine on organic nitrogen improves seedling morphology and field performance Linda Gruffman a, Takahide Ishida b, Annika Nordin b, Torgny Näsholm a,⇑ a b

Swedish University of Agricultural Sciences, Department of Forest Ecology and Management, SE-901 83 Umeå, Sweden Swedish University of Agricultural Sciences, Department of Forest Genetics and Plant Physiology, SE-901 83 Umeå, Sweden

a r t i c l e

i n f o

Article history: Received 3 February 2012 Received in revised form 30 March 2012 Accepted 31 March 2012 Available online 26 April 2012 Keywords: Organic nitrogen Root: shoot ratio Biomass allocation Picea abies (L.) Karst. Pinus sylvestris (L.)

a b s t r a c t Nitrogen availability exerts a significant control on biomass allocation of plants including Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris (L.)) in boreal forest ecosystems. Recent studies suggest, however, this control differs for inorganic and organic nitrogen sources. The importance of the chemical form of nitrogen (inorganic or organic) for the morphology and growth of conifer seedlings was studied during production of seedlings in a forest nursery and subsequently in a field trial in northern Sweden. Seedlings were supplied with two different nutrient solutions; an inorganic conventional fertilizer and an organic, amino acid-based fertilizer. Seedlings cultivated on the organic nitrogen source displayed larger root systems resulting in a higher root: shoot ratio than did seedlings cultivated on the inorganic nitrogen source. The proportion of fine roots to lateral roots and the root tip proportion colonized by mycorrhiza were positively affected by the organic nitrogen source. Norway spruce seedlings cultivated on organic nitrogen displayed significantly increased shoot growth compared to seedlings cultivated on inorganic nitrogen. Our results suggest that the chemical form of nitrogen influences the allocation of biomass in conifer seedlings. The shift in allocation of resources to root biomass further leads to a competitive advantage in field conditions, resulting in a significant increase in shoot growth one year following transplant. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction The long rotation times of northern coniferous forests and high costs for forest regeneration underscore the importance of successful seedling establishment. In Scandinavia, conifer seedlings are mainly transplanted from the nursery to the field during a short period in spring and early summer before soil conditions become too dry (Helenius et al., 2002, 2005) and irradiance is supra-optimal for seedling establishment. One of the most common stresses to recently transplanted seedlings is therefore water deficiency (Burdett, 1990; Haase and Rose, 1993; Helenius et al., 2002, 2005). The water stress may be caused by factors such as poor root-soil contact and low root permeability (Burdett, 1990). Water stress and insufficient supply of photosynthate from the shoot to the root of the newly transplanted seedling might lead to limited root establishment which in turn restricts photosynthesis. Thus, there is a mutual dependency between root growth and current photosynthesis (Burdett, 1990). Successful seedling establishment is hence dependent on the seedling ability to acquire enough water to support a transpiring shoot, and the size and distribution of the

⇑ Corresponding author. Tel.: +46 90 786 82 05; fax: +46 90 786 81 63. E-mail address: [email protected] (T. Näsholm). 0378-1127/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.foreco.2012.03.030

root system are important seedling traits in overcoming postplanting stress (Grossnickle, 2005). It is well documented that plants preferentially allocate growth to shoots at high nitrogen availability and when nitrogen availability is limited allocation to below-ground structures is favored (Brouwer, 1962; Ingestad and Kähr, 1985; Ericsson, 1995; Rytter et al., 2003; Kaakinen et al., 2004; Hermans et al., 2006). Traditionally, seedling size and root-collar diameter have been important criteria in the evaluation of seedling quality. However, aboveground seedling traits might not be optimal in predicting seedling performance in the field (Davis and Jacobs, 2005). Moreover, seedling height is acquired at the expense of a well developed root system as cultivated seedlings receive high concentrations of fertilizer in the form of mineral nitrogen in forest nurseries (Juntunen and Rikala, 2001). The above described nitrogen responses of biomass allocation in plants entail a potential problem concerning forest regeneration. Newly transplanted seedlings with restricted root systems, and with a high proportion of transpiring shoot area, would probably be susceptible to desiccation, which might lead to lower survival rates of planted seedlings. The scope of producing seedlings with a large proportion of roots and a high root: shoot ratio could be achieved by a decrease in the nitrogen fertilizer in forest nurseries. However, such restriction of nitrogen leads to stressed plants, deprived of the most important plant nutrient

L. Gruffman et al. / Forest Ecology and Management 276 (2012) 118–124

and in the field they might be more susceptible to other stresses, biotic as well as abiotic. Field performance of planted seedlings has been shown to be positively correlated with their root: shoot ratios (cf. Davis and Jacobs, 2005). In a recent study, Vaario et al. (2009) showed that organic fertilization in the form of composted chicken manure had a significant positive effect on the root: shoot ratio of Norway spruce (Picea abies (L.) Karst.) seedlings in the nursery, which was suggested to be due to the presumed slow release of nitrogen from the organic fertilizer. In this study, the frequency of root tips and ectomycorrhizal (ECM) colonization in the nursery showed a positive trend with growth rate. Moreover, the root: shoot ratio, ECM diversity and root dry mass were positively correlated with growth rate of seedlings following plantation in the field. Mycorrhization of seedlings expands the surface area of the root system and therefore increases the plant ability to acquire water and nitrogenous compounds (Finlay et al., 1989; Ek et al., 1994; Turnbull et al., 1995). In boreal forests, nitrogen is usually the limiting factor for growth. This limitation was presumed to result mainly from a slow mineralization rate of soil organic nitrogen (Vitousek and Howarth, 1991). However, it has been shown repeatedly that plants possess the ability to circumvent the mineralization step by the direct uptake of organic nitrogen (Hutchinson and Miller, 1911; Virtanen and Linkola, 1946; Chapin et al., 1993). Amino acids, one of the most prevalent forms of organic nitrogen, are efficiently taken up by an array of different plants growing in boreal forests including conifers, and the uptake has been shown to be effective in mycorrhizal as well as non-mycorrhizal forest plants (Persson and Näsholm, 2001). It has also been shown that Norway spruce and Scots pine (Pinus sylvestris (L.)) seedlings have the capacity for intact uptake of the amino acids and perform as well as, or better when grown on such nitrogen forms, compared to seedlings grown on inorganic nitrogen sources (Öhlund and Näsholm, 2001). Plant root transporters responsible for amino acid uptake have also been identified (Hirner et al., 2006; Lee et al., 2007; Svennerstam et al., 2007, 2008; Näsholm et al., 2009) further verifying the ability of plants to acquire amino acids regardless of mycorrhizal colonization. In a recent study, Cambui et al. (2011) showed that the root mass fraction of the small herb Arabidopsis thaliana was positively affected by the presence of an amino acid in the growth media. This stimulation of root growth was documented for plants grown at identical nitrogen concentrations, and resulting in similar nitrogen concentrations of plants, suggesting amino acids had a direct positive effect on root growth. The objective of the current study was to investigate whether the chemical form of nitrogen (inorganic or organic) affects the morphology and growth of seedlings cultivated in a forest nursery, and if so, to what extent this potential effect influences seedling performance after planting in the field. In light of the study by Cambui et al. (2011) we hypothesized that seedlings supplied with organic nitrogen would allocate more resources to root biomass compared to seedlings cultivated on an inorganic nitrogen source. Further, we hypothesized that seedlings with a more well-developed root system would show an improved capacity for establishment following outplanting in the field.

2. Methods 2.1. Plant material and growth conditions Scots pine and Norway spruce seedlings were grown in Gideå conifer nursery in Gideå, N. Sweden as part of their commercial cultivation during one growing season, with pine seeds originating from seed orchard Våge 125 (location of seed orchard: 63°180 N,

119

10 m.a.s.l.; mean clonal origin: 65°480 N, 440 m.a.s.l.) and spruce seeds originating from seed orchard Domsjöänget 130 (location of seed orchard: 63°120 N, 30 m.a.s.l.; mean clonal origin: 64°240 N, 375 m.a.s.l.). Two different nutrient solutions differing only with respect to the chemical form of nitrogen were used; a conventional inorganic fertilizer containing nitrate (NO3) and ammonium (NH4+) which is commonly used in forest nurseries (WallcoÒ, Cederroth International AB), and an organic amino acid-based fertilizer with arginine as the sole nitrogen source. The composition of the inorganic fertilizer was (g/L): N, 100 of which NO3 –N, 60 and NH4+ –N, 40; K, 65; P, 13; S, 9; Mg, 4; Fe, 0.7; Mn, 0.4; B, 0.2; Zn, 0.06; Cu, 0.03; Mo, 0.007. The composition of the organic fertilizer was identical except for the nitrogen source (g/L): N, 100 originating exclusively from Arginine-N. Hence, the N:K:P ratios of the fertilizers were 100:65:13 for both the inorganic- and organic fertilizer. One kg dolomite (CaMg(CO3)2) m3 peat was also added to the growth substrate. The plant material was sown in containers (StarpotÓ 50–60, Panth produkter AB, Sweden) 19–20th of April 2006 and cultivated in greenhouse conditions at the forest nursery during the first period (Temp. 20 °C, with supplemental light between 10 p.m. and 4.30. a.m.). The 12–13th of June the containers were moved for further growth outdoors. Liquid fertilizer was applied through the irrigation system by mobile booms. The fertilizer addition rates were designed to produce seedlings of the respective species with a total biomass of 2 g per seedling and with a nitrogen concentration of 2% DW. Routines for addition of conventional fertilizer had been developed over several years to meet these goals. Accordingly, the need for fertilizer addition was determined by measurements of the electrical conductivity (EC) of press-water from the growth substrate of the containers, where threshold values varied from 0.5 S in the early season to 0.7 S later in the season. This resulted in total addition rates of conventional fertilizer of 56.5 and 70.4 mg nitrogen seedling1, for spruce and pine seedlings respectively during the growing season and fertilizer was distributed on 37 and 42 occasions (i.e. 2–3 times a week) for spruce and pine seedlings, respectively. Arginine is a strong cation in the relevant pH-interval and hence binds to the growth substrate, minimizing nitrogen losses caused by leaching (Öhlund and Näsholm, 2002). This allows for a scheduled fertilizing regime which is not possible to practice with an inorganic fertilizer with nitrate as the major nitrogen source. Earlier estimates of recovery of nitrogen in seedlings concluded that 80% of added arginine-nitrogen was recovered (Öhlund and Näsholm, 2002). Hence, with the same targets of 2 g biomass per seedling and 2% nitrogen DW the estimated addition rate of the arginine fertilizer was 50 mg nitrogen seedling1. The arginine-treated spruce and pine seedlings received equal amounts of fertilizer corresponding to 50.6 mg nitrogen seedling1, distributed on 14 occasions. The last fertilization event took place the 15th of September and seedlings were thereafter entering dormancy and stored in a cold room at 0.5–1 °C throughout the winter. 2.2. Field trial sites characteristics and experimental design The following spring (2007), field trials were established on three sites located in the area of the forest nursery, hereafter referred to as site 1, 2 and 3. The dominating tree covers before clear-cut were stands of Norway spruce and Scots pine and the field layers were dominated by Vaccinium spp. The forest soils were classified as sandy glacial till Haplic Podzol (FAO, 1998) and the soil surface was mounded before planting. Average annual precipitation measured at SMHI meteorological station Hemling is 587 mm and the annual mean air temperature 1.8 °C (SMHI, 1991). (Locations of the sites are specified in Table 1.) The sites were planted in a complete randomized block design on 16th, 18th and 28th of May 2007, respectively. On each site, 400 seed-

120

L. Gruffman et al. / Forest Ecology and Management 276 (2012) 118–124

Table 1 Locations of the three field trial sites. Location Site 1 Site 2 Site 3

Latitude 0

63°28 63°290 63°270

Longitude 0

18°34 18°350 19°60

Altitude (m.a.s.l.) 221–253 217 103

lings from each species with the two different fertilizing regimes were planted in 10 blocks (i.e. in total 800 seedlings in 20 blocks on each site), the exception from this being site 1, where 8 blocks and hence 320 pine seedlings were planted. Seedlings were planted with a stocking rate of 2300 seedlings ha1. Any seedlings affected by pine weevils, (Hylobius abietis) and any planting points containing two seedlings (caused by sowing machine error in the nursery) were excluded from further analysis. 2.3. Morphology, nutrient- and chemical analyses Seedlings of each species were characterized before planting in the field and twenty seedlings of each species and treatment were harvested at the forest nursery for this purpose. The seedlings were kept in a cold-room for two-four days and then analyzed visually and molecularly for ecto mycorrhizal fungi (EMF) as described below. The seedlings were thereafter freeze-dried for 72 h. Seedling total shoot lengths and dry weights were measured on seedlings from each species and treatment which had previously been separated into shoots and roots at the uppermost positioned lateral root. Needles were milled to a fine powder in a bead mill and subsequently analyzed with an elemental analyzer FLASH EA 1112, Thermo Fisher Scientific, to determine the nitrogen content. In order to measure the fine root fraction, three lateral roots from each root system were removed, the fine roots and lateral roots were separated, dried and weighed. The fine root fraction was calculated as the percentage of the total lateral root biomass. After one growing-season in the field, survival and the current year shoot lengths were measured. Analysis of mycorrhizal colonization was done on root systems from each species and treatment that were sampled and carefully washed with tap water. Each root tip was assigned to an EMF morphotype by examination with a dissecting microscope. However, since fungal sheaths were not well developed on many of the inspected root tips, morphological characteristic was poor. For the same reason, discrimination of EMF and non-EMF root tips was difficult. Presumed EMF root tips were placed individually in 2.0-ml tubes and dried for DNA extraction. In total, 6309 root tips from 71 root systems were examined under a dissecting microscope, and 56 EMF root tips were used for molecular identification. Extraction of DNA was performed according to Ishida et al. (2007). Briefly, each sample was pulverized and subjected to the modified cetyltrimethylammonium bromide (CTAB) method. The rDNA internal transcribed spacer (ITS) regions were amplified using Taq DNA polymerase (New England BioLabs, Herts, UK). The ITS1F primer (Gardes and Bruns, 1993) with a 50 D2 fluorescent label (Sigma–Aldrich, Sweden) and the ITS4 primer (White et al., 1990) with a 50 D3 fluorescent label (Sigma–Aldrich) were used. Two microliters of each PCR product were digested by either Hinf I or Alu I (Fermentas, St. Leon-Rot, Germany, or New England Biolabs, Herts, UK). The digested products were purified by ethanol precipitation and diluted with 25 ll of HiDi formamide (Beckman Coulter, Fullerton, CA, USA) containing 0.06 ll of CEQ 600 size standard (Beckman Coulter). Capillary gel electrophoresis was performed using a CEQ 8000 (Beckman Coulter). The terminal restriction fragment lengths were determined using FRAGMENTS implemented in a CEQ 8000 genetic analysis system (Beckman

Coulter). Four fragments were obtained for each sample. When samples had fragments within ±2 bp, they were considered to be the same terminal restriction fragment length polymorphism (TRFLP) type. When samples contained multiple fragments in comparable peak strength, a maximum of two T-RFLP types were assigned to single root tips. Each T-RFLP type was sequenced from the matching T-RFLP type in our mycorrhizal fungal cloning library. ITS region was re-amplified using ITS1F and/or ITS4, purified using presequencing kit (USB Co., Cleveland, OH, USA), then sequenced using CEQ 2000 or a commercially available company. The obtained sequences were compared with the sequences of known species in GenBank or UNITE by blastn. All sequences were deposited in the Genbank. 2.4. Statistical analyses Two-way analysis of variance, (ANOVAs) were used to test the effects of nitrogen source, tree species and the interaction between these two factors on seedling traits on 20 randomly selected seedlings before transition to field conditions. The following statistical model was used for seedling traits:

Y ij ¼ l þ ai þ bj þ ðabÞij þ eij where Y is the response (seedling traits), l is the overall mean, ai is the effect of the ith treatment, bj is the effect of the jth species and eij is the error. The factors were: treatment (i = 1, 2), species (j = 1, 2). Partly nested ANOVAs (separate for Norway spruce and Scots pine) were used to test the effects of nitrogen source (in the nursery), field site, blocks and the interaction between these factors on seedling current year shoot length following one growing season in the field. Similarly partly nested ANOVAs (separate for Norway spruce and Scots pine) were used to test the effect of nitrogen source (in the nursery), field site, blocks and the interaction between these factors on seedling survival. Potential nuisance variables were eliminated by constant-keeping. The following statistical model was used:

Y ijk ¼ l þ ai þ bj þ ðabÞij þ ckj þ eijk where Y is the response (current year shoot length or survival), l is the overall mean, ai is the effect of the ith treatment, bj is the effect of the jth site, ck(j) is the effect of the kth block within site, and eijk is the error. The factors were: treatment (i = 1, 2), site (j = 1, 2, 3), block (k = 1–10). Latin characters of the constants in the model indicate random factors (i.e. site and block). For all statistical analyses p-values 60.05 were considered as significant. Minitab 16 Statistical Software (Minitab Inc., State College, PA) was used for the analyses. 3. Results The different nitrogen sources had no significant effects on the shoot biomass nor on the seedling total biomass for any of the species, with average shoot biomasses ranging from 0.92 g DW1 seedling for the arginine-treated Norway spruce seedlings to 1.15 g DW1 seedling for the conventionally treated Scots pine seedlings and average total biomasses ranging from 1.37 g DW1 seedling for the arginine-treated spruce seedlings to 1.54 g DW1 seedling for the conventionally treated pine seedlings (Fig. 1A and B, and Table 2). However, root systems of seedlings fertilized with arginine had a significantly larger biomass than seedlings fertilized with inorganic nitrogen, as arginine fertilized spruce and pine seedling roots weighed 0.45 and 0.47 g DW1 seedling, respectively as compared to 0.37 and 0.39 g DW1 seedling for the conventional treatment (Fig. 1C and Table 2). The larger root systems of arginine-grown seedlings thus caused a significant shift of the root: shoot ratio in both species (Fig. 1D and Table 2).

L. Gruffman et al. / Forest Ecology and Management 276 (2012) 118–124

121

Fig. 1. Seedling traits at the end of nursery culture divided into shoot biomass (A), total biomass (B), root biomass (C), root: shoot ratio (D), total shoot length (E), needle nitrogen content (F), fine root proportion (G) and root tip proportion colonized by mycorrhiza (H) of Norway spruce and Scots pine seedlings cultivated on a conventional inorganic fertilizer or a fertilizer based on the amino acid arginine. White columns indicate conventional nitrogen source, while black columns indicate arginine as the nitrogen source. Error bars indicate standard error, n = 10–20.

Seedling total shoot length before planting in the field differed between treatments so that conventionally-grown seedlings were significantly longer. Spruce seedlings grown on the conventional fertilizer had a shoot length of 16.3 cm and the shoot length was 12.8 cm for the arginine fertilizer. The corresponding shoot lengths

of the pines were 16.7 and 15.6 cm for the conventional and arginine fertilizer, respectively (Fig. 1E and Table 2). The needle nitrogen content of the seedlings did not differ significantly between the different fertilizers. The target nitrogen concentration of needles aimed for by the forest nursery in this study

122

L. Gruffman et al. / Forest Ecology and Management 276 (2012) 118–124

Table 2 Results (F- and P-values) from two-way ANOVAs testing the effects of nitrogen source, tree species and the interaction between nitrogen source and tree species on selected seedling traits at the end of nursery culture of Norway spruce and Scots pine seedlings cultivated on a conventional inorganic fertilizer or a fertilizer based on the amino acid arginine. Numbers in bold indicate significant differences (P < 0.05). Seedling trait

Shoot biomass Seedling total biomass Root biomass Root: shoot ratio Total shoot length Needle N content Fine root proportion Mycorrhizal root tip proportion

Effect of nitrogen source

Effect of species

Effect of interaction between nitrogen source and species

F-value

P-value

F-value

P-value

F-value

P-value

3.189 0.414 4.342 21.605 7.800 0.060 8.868 12.999

0.078 0.522 0.041 <0.001 0.007 0.814 0.004 0.001

0.994 0.835 0.277 0.495 3.800 27.370 0.210 2.373

0.334 0.364 0.600 0.484 0.055 <0.001 0.648 0.128

0.323 0.193 0.001 1.410 2.090 22.380 2.461 2.713

0.572 0.662 0.979 0.239 0.153 <0.001 0.121 0.104

Table 3 Frequency of mycorrhizal fungi obtained from root tips of Norway spruce and Scots pine seedlings cultivated in a nursery on a conventional inorganic fertilizer or a fertilizer based on the amino acid arginine. Species

Fertilizer

No. of observed seedlings

No. EMF molecular samples

Asco-mycota sp. 1

Asco-mycota sp. 2

Thelephora terrestris

Laccaria sp.

P. abies

Conventional Arginine

17 17

13 15

0 0

6 6

13 15

0 0

P. sylvestris

Conventional Arginine

19 18

9 19

1 0

3 8

4 14

1 0

was 2.0%; hence conventionally treated spruce seedlings were considered slightly low and conventionally treated pine seedlings were considered slightly high in their nitrogen status. The proportion of fine roots to lateral roots was significantly larger for arginine-grown seedlings than for seedlings grown on conventional nitrogen fertilizer. Arginine-grown spruce and pine seedlings had a fine root proportion of 71% and 68%, respectively as compared to 59% and 64% for the conventional treatment. The

arginine fertilization also showed a significant effect on mycorrhizal colonization as the root tip proportion colonized by mycorrhiza was 80% and 82% on spruce and pine, respectively. Corresponding values for the conventional treatment were 62% and 32% (Fig. 1G and H, and Table 2). T-RFLPs were obtained from 55 root tip samples. Among them, five different mycorrhizal genotypes were identified of which two were pooled due to the high sequence similarity (98%). The root tips were mainly colonized by Ascomycota sp. 2 (AB476471) and by Thelephora terrestris (AB476476) while Ascomycota sp. 1 (AB476468) and Laccaria sp. (AB612229) were found on roots of single seedlings (Table 3). The nitrogen source used for seedling growth in the nursery had a significant effect on the current year shoot length of Norway spruce seedlings after the first season in the field (Fig. 2 and Table 4). The shoot increment of seedlings cultivated on arginine was 15.3% higher compared to seedlings supplied with inorganic nitrogen fertilizer. The current year shoot lengths of spruce seedlings were 9.9 and 11.5 cm on average for the conventional- and arginine-treatments, respectively. Corresponding shoot lengths of the Scots pine seedlings were 7.9 and 8.5 cm. Hence, there was no significant effect of the nitrogen source on shoot length of Scots pine seedlings (Fig. 2 and Table 4).

Table 4 Results from partly nested ANOVAs testing the effects of nitrogen source in the nursery, site and their interaction on current year shoot length and seedling survival one season following outplanting in the field on Norway spruce and Scots pine seedlings cultivated on a conventional inorganic fertilizer or a fertilizer based on the amino acid arginine. Numbers in bold indicate significant differences (P < 0.05). Species

Fig. 2. Current year shoot length of Norway spruce and Scots pine seedlings after one growing-season in three different field trial sites. White columns indicate seedlings cultivated on a conventional inorganic nitrogen source, black columns indicate seedlings cultivated on the amino acid arginine as the only nitrogen source. Error bars indicate standard error, N = 8–10.

Shoot length

Survival

Effect of

df

F-value

P-value

F-value

P-value

P. abies

N source Site N source  site Block (site)

1 2 2 27

1286.49 52.17 0.01 1.51

<0.001 0.013 0.991 0.144

1.10 6.53 2.83 1.96

0.404 0.068 0.077 0.043

P. sylvestris

N source Site N source  site Block (site)

1 2 2 25

1.24 2.64 1.45 0.87

0.381 0.315 0.253 0.631

0.14 7.98 0.25 1.85

0.742 0.021 0.782 0.065

L. Gruffman et al. / Forest Ecology and Management 276 (2012) 118–124 Table 5 Survival of Norway spruce and Scots pine seedlings cultivated on a conventional inorganic fertilizer or a fertilizer based on the amino acid arginine one season following outplanting in three different field sites in northern Sweden. Location

P. abies seedling survival (%)

P. sylvestris seedling survival (%)

Conventional

Arginine

Conventional

Arginine

Site 1 Site 2 Site 3

89.4 54.8 69.2

85.0 67.6 76.3

48.3 63.3 64.4

45.1 60.8 67.4

Average (±SE)

71.1 10.0

76.3 5.0

58.7 5.2

57.8 6.6

The nitrogen source in the nursery had no significant effect on seedling survival after one season in the field (Tables 4 and 5). At site 1, 89.4% of the spruce seedlings had survived but only 48.3% of the pines (Table 5). The great losses in both species investigated were caused by pine weevils on all sites, although the losses were not as severe as for site 1. 4. Discussion The objective of the current study was to investigate the potential effects of different nitrogen sources on morphology and growth of seedlings cultivated in forest nursery conditions and to what extent these potential effects would influence seedling performance in the field. The results show that both shoot biomass and total biomass of seedlings were similar for the arginine and conventional fertilizer but the shoot length was significantly higher in conventionally fertilized seedlings and the root biomass of seedlings cultivated on arginine as nitrogen source was significantly greater (Fig. 1 and Table 2). These differences logically resulted in higher root: shoot ratios for arginine-cultivated seedlings (Fig. 1D and Table 2). General allocation theory states that plant biomass allocation is responsive to nutrient-, and in particular nitrogen supply. For Scots pine and Norway spruce seedlings, there is a positive relationship between the internal nitrogen concentration of plants and the fraction of biomass allocated to shoots (Ingestad and Ågren, 1991). In our study, spruce seedlings cultivated on the conventional, inorganic fertilizer displayed lower nitrogen concentrations than those cultivated on arginine as nitrogen source while this pattern was reversed for pine seedlings. However, irrespective of these differences, biomass allocation to roots was, for both species, higher for seedlings treated with the organic fertilizer. Our results do not directly contradict the general model for nitrogen effects on biomass allocation but suggest the allocation response may also depend on the actual nitrogen source. Studies on Arabidopsis thaliana suggest that, at identical rates of nitrogen supply and at similar nitrogen contents of plants, organic nitrogen promotes allocation of growth to roots (Cambui et al., 2011). The exact mechanisms by which organic (in comparison with inorganic) nitrogen sources may stimulate biomass allocation to roots and development of ectomycorrhizal symbioses cannot be deduced from the current study. However, two major differences between the organic and inorganic fertilizer may be considered in this context. Firstly, the actual concentrations of the organic fertilizer in the soil solution may be much lower than those of the inorganic fertilizer as a result of strong binding of arginine to the peat substrate (Öhlund and Näsholm, 2002). Secondly, the organic fertilizer will provide carbon to roots and mycorrhiza making them less dependent on allocation of photosynthate (cf. Näsholm et al., 2009). Seedlings supplied with the organic fertilizer displayed a significantly enhanced proportion of fine roots in their root systems (Fig. 1G and Table 2). The enhanced proportion of fine roots should also lead to increased possibilities for colonization by ectomycor-

123

rhizal fungi and estimates of the fraction of colonized root tips suggest this was also the case (Fig. 1G and H, and Table 2). Successful seedling establishment is closely dependent on the capacity of seedlings for quick acclimation to field conditions. One of the most critical factors is the ability of the seedling to acquire water for the demands of a transpiring shoot (Burdett, 1990). The size and distribution of the root system (Grossnickle, 2005) as well as the balance between root and shoot biomass (Davis and Jacobs, 2005) are thus important factors in overcoming post-planting stress. It has been shown that successful root establishment directly influences the shoot growth of newly planted seedlings (Burdett, 1990). In the present study we hypothesized that seedlings with a higher root: shoot ratio would be better equipped to establish in field conditions. Results from the inventory of seedlings after one growing-season in the field suggest the organic nitrogen source used for seedling growth in the nursery had a significant positive effect on the current year shoot length. This emphasizes the importance of good seedling quality, including a well developed root system, and shows that arginine-treated seedlings despite their smaller initial shoot length were able to acclimate quickly in the field and invest more resources to current year shoot length. In a study of loblolly pine (Pinus taeda L.), Larsen et al. (1986) showed that the root: shoot ratio and the number of new roots explained 80% of the variation in survival one year following plantation in the field but no correlation was found between foliar nutrient content and survival (Larsen et al., 1986). In the present study, no obvious effects of nitrogen source on seedling survival rates were found after one season in the field (Tables 4 and 5). However, the main cause of death of seedlings in this experiment was not related to drought but rather to a severe attack by the pine weevil. Damage caused by pine weevil on both species may, therefore, have over-shadowed any potential positive effects of increased root fraction of seedlings on seedling survival in this study (Tables 4 and 5). Conifer seedlings in forest nurseries are heavily fertilized with inorganic nitrogen in the form of ammonium and nitrate (Juntunen and Rikala, 2001). Therefore, there is reason to believe that both the high nitrogen concentration and nitrogen form (nitrate) actually work concomitantly to stimulate shoot growth at the expense of root growth. It would therefore be of great interest for the forestry sector to produce seedlings displaying desired plant traits such as high root: shoot ratio and high fine root fraction and with retained optimal nutrient concentrations. Here we show that the allocation of biomass is not only controlled by the addition rates, and internal concentrations of nitrogen, but also by the chemical form of nitrogen supplied. Clear morphological differences were found between plants grown on the nitrogen source arginine compared to plants grown on a mixture of ammonium and nitrate, differences that have been shown to be of importance for seedling growth and survival when planted in the field. Acknowledgements The authors wish to thank Margit Vesterlund, Yvonne Hedman and other personnel at Gideå conifer nursery for their help with cultivating seedlings. We also thank Jonas Öhlund for help with the field experiment and Sören Holm for statistical advice. This study was financed by grants from the Swedish research council, FORMAS and the Swedish foundation for strategic research, SSF. References Brouwer, R., 1962. Distribution of dry matter in the plant. Neth. J. Agric. Sci. 10, 361–376. Burdett, A.N., 1990. Physiological processes in plantation establishment and the development of specifications for forest planting stock. Can. J. For. Res. 20, 415– 427.

124

L. Gruffman et al. / Forest Ecology and Management 276 (2012) 118–124

Cambui, C.A., Svennerstam, H., Gruffman, L., Nordin, A., Ganeteg, U., Näsholm, T., 2011. Patterns of plant biomass partitioning depend on nitrogen source. PLoS ONE 6, e19211. Chapin, F.S., Moilanen, L., Kielland, K., 1993. Preferential use of organic nitrogen for growth by a nonmycorrhizal arctic sedge. Nature 361, 150–153. Davis, A.S., Jacobs, D.F., 2005. Quantifying root system quality of nursery seedlings and relationship to outplanting performance. New Forest. 30, 295–311. Ek, H., Andersson, S., Arnebrant, K., Söderström, B., 1994. Growth and assimilation of NH4+ and NO3 by Paxillus involutus in association with Betula pendula and Picea abies as affected by substrate pH. New Phytol. 128, 629–637. Ericsson, T., 1995. Growth and shoot: root ratio of seedlings in relation to nutrient availability. Plant Soil 168–169, 205–214. FAO, 1998. World Reference Base for Soil Resources. World Soil Resources Reports, Rome. Finlay, R.D., Ek, H., Odham, G., Söderström, B., 1989. Uptake, translocation and assimilation of nitrogen from 15N-labeled ammonium and nitrate sources by intact ectomycorrhizal systems of Fagus sylvatica infected with Paxillus involutus. New Phytol. 113, 47–55. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Mol. Ecol. Notes 2, 113–118. Grossnickle, S.C., 2005. Importance of root growth in overcoming planting stress. New Forest. 30, 273–294. Haase, D.L., Rose, R., 1993. Soil-moisture stress induces transplant shock in stored and unstored 2 + 0 Douglas-fir seedlings of varying root volumes. For. Sci. 39, 275–294. Helenius, P., Luoranen, J., Rikala, R., 2005. Physiological and morphological responses of dormant and growing Norway spruce container seedlings to drought after planting. Ann. For. Sci. 62, 201–207. Helenius, P., Luoranen, J., Rikala, R., Leinonen, K., 2002. Effect of drought on growth and mortality of actively growing Norway spruce container seedlings planted in summer. Scand. J. For. Res. 17, 218–224. Hermans, C., Hammond, J.P., White, P.J., Verbruggen, N., 2006. How do plants respond to nutrient shortage by biomass allocation? Trends Plant Sci. 11, 610– 617. Hirner, A., Ladwig, F., Stransky, H., Okumoto, S., Keinath, M., Harms, A., Frommer, W.B., Koch, W., 2006. Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll. Plant Cell 18, 1931–1946. Hutchinson, H., Miller, N., 1911. The direct assimilation of inorganic and organic forms of nitrogen by higher plants. J. Agric. Sci. 4, 282–302. Ingestad, T., Kähr, M., 1985. Nutrition and growth of coniferous seedlings at varied relative nitrogen addition rate. Physiol. Plant. 65, 109–116. Ingestad, T., Ågren, G.I., 1991. The influence of plant nutrition on biomass allocation. Ecol. Appl. 1, 168–174. Ishida, T.A., Nara, K., Hogetsu, T., 2007. Host effects on ectomycorrhizal fungal communities: insight from eight host species in mixed conifer-broadleaf forests. New Phytol. 174, 430–440.

Juntunen, M.-L., Rikala, R., 2001. Fertilization practice in Finnish forest nurseries from the standpoint of environmental impact. New Forest. 21, 141–158. Kaakinen, S., Jolkkonen, A., Iivonen, S., Vapaavuori, E., 2004. Growth, allocation and tissue chemistry of Picea abies seedlings affected by nutrient supply during the second growing season. Tree Physiol. 24, 707–719. Larsen, H.S., South, D.B., Boyer, J.M., 1986. Root growth potential, seedling morphology and bud dormancy correlate with survival of loblolly pine seedlings planted in December in Alabama. Tree Physiol. 1, 253–263. Lee, Y.-H., Foster, J., Chen, J., Voll, L.M., Weber, A.P.M., Tegeder, M., 2007. AAP1 transports uncharged amino acids into roots of Arabidopsis. Plant J. 50, 305– 319. Näsholm, T., Kielland, K., Ganeteg, U., 2009. Uptake of organic nitrogen by plants. New Phytol. 182, 31–48. Öhlund, J., Näsholm, T., 2001. Growth of conifer seedlings on organic and inorganic nitrogen sources. Tree Physiol. 21, 1319–1326. Öhlund, J., Näsholm, T., 2002. Low nitrogen losses with a new source of nitrogen for cultivation of conifer seedlings. Environ. Sci. 36, 4854–4859. Persson, J., Näsholm, T., 2001. Amino acid uptake: a widespread ability among boreal forest plants. Ecol. Lett. 4, 434–438. Rytter, L., Ericsson, T., Rytter, R.M., 2003. Effects of demand-driven fertilization on nutrient use, root: plant ratio and field performance of Betula pendula and Picea abies. Scand. J. For. Res. 18, 401–415. SMHI (Swedish Meteorological and Hydrological Institute), 1991. Temperaturen och nederbörden i Sverige 1961–1990 Referensnormaler. Rapport 81 (in Swedish). Svennerstam, H., Ganeteg, U., Bellini, C., Näsholm, T., 2007. Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids. Plant Physiol. 143, 1853–1860. Svennerstam, H., Ganeteg, U., Näsholm, T., 2008. Root uptake of cationic amino acids by Arabidopsis depends on functional expression of amino acid permease. New Phytol. 180, 620–630. Turnbull, M.H., Goodall, R., Stewart, G.R., 1995. The impact of mycorrhizal colonization upon nitrogen source utilization and metabolism in seedlings of Eucalyptus grandis Hill ex Maiden and Eucalyptus maculata Hook. Plant Cell Environ. 18, 1386–1394. Vaario, L.M., Tervonen, A., Haukioja, K., Haukioja, M., Pennanen, T., Timonen, S., 2009. The effect of nursery substrate and fertilization on the growth and ectomycorrhizal status of containerized and outplanted seedlings of Picea abies. Can. J. For. Res. 39, 64–75. White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M. A., et al. (Eds.). Pcr Protocols: A Guide to Methods and Applications. Xviii+482p. Academic Press Inc., San Diego, California, USA, London, England, Uk. Illus, pp. 315–322. Virtanen, A.I., Linkola, H., 1946. Organic nitrogen compounds as nitrogen nutrition for higher plants. Nature 158, 515. Vitousek, P.M., Howarth, R.W., 1991. Nitrogen limitation on land and in the sea – how can it occur? Biogeochemistry 13, 87–115.