Trees (2001) 15:319–326 DOI 10.1007/s004680100106

O R I G I N A L A RT I C L E

Marjoriitta Möttönen · Tarja Lehto · Pedro J. Aphalo

Growth dynamics and mycorrhizas of Norway spruce (Picea abies) seedlings in relation to boron supply

Received: 25 February 2001 / Accepted: 18 April 2001 / Published online: 12 June 2001 © Springer-Verlag 2001

Abstract The effects of three boron levels on the growth dynamics and ectomycorrhizas of seedlings of Norway spruce [Picea abies (L.) Karst.] were studied in a growth room experiment. The seedlings were grown in forest humus mixed with quartz sand for 16 weeks. The B treatment was applied in the nutrient solution. Stem height, dry weight, number of root tips, mycorrhizas as well as B and N concentrations in the seedlings were monitored in sequential harvests. By the last harvest, in week 16, needle B concentrations were 6.6 mg kg–1 at the lowest B level and 17.5 mg kg–1 and 26.5 mg kg–1 at the two higher levels. Boron slightly increased the stem height and the total dry weight, but did not affect N content of the seedlings. Low internal B reduced the number of root tips and mycorrhizas as well as mycorrhizal percentage and root dry weight, which indicates the importance of B for root growth in Norway spruce seedlings. The seedlings grown with adequate internal B had more root tips than those receiving the two lower B treatments as early as week 9, when needle B concentrations at the lowest B supply were 16.0–17.3 mg kg–1, which has previously been considered a sufficient B level. Therefore, the critical needle B concentrations should perhaps be re-examined. Keywords Boron · Ectomycorrhiza · Growth · Norway spruce · Roots

Introduction Boron is an essential micronutrient for plants, and boron deficiency is known to inhibit or prevent the growth of vegetative and reproductive plant parts, depending on the timing and extent of the deficiency. The soils with a minimum of maritime influence are susceptible to B defiM. Möttönen (✉) · T. Lehto · P.J. Aphalo University of Joensuu, Faculty of Forestry, P.O. Box 111, 80101 Joensuu, Finland e-mail: [email protected] Tel.: +358-13-2514423, Fax: +358-13-2513590

ciency (Wikner 1983; Stone 1990; Shorrocks 1997). There are areas with low B availability in many countries and especially in exotic plantations of eucalypts and pines, and in plantations and natural stands of native species on soils altered by macronutrient fertilisation, liming, N deposition, fire or erosion (Stone 1990; Shorrocks 1997). The deficiency symptoms are usually characteristic and include most commonly discoloration and dieback of the leader, swelling of leading shoots and multileadered bushy crowns (Kolari 1979; Raitio 1983; Stone 1990). These growth disturbances in apical meristems are probably mainly due to the function of B in cell walls (Matoh 1997). The critical boron concentrations in foliage depend on the species and growing conditions. Needle B concentrations below 4 mg kg–1 in mature Norway spruce [Picea abies (L.) Karst.] have been reported to indicate deficiency and concentrations above 8 mg kg–1 to correspond to an adequate B supply (Brække 1977, 1979; Aronsson 1983; Jukka 1988). Suboptimal needle B concentrations (below 8 mg kg–1) are common in Finnish forests (Derome et al. 1986; Mälkönen et al. 1990), and in such cases even a small decrease in availability can induce deficiency symptoms as reported by Lehto and Mälkönen (1994). Boron deficiency is known to lessen shoot height growth in many coniferous species (Hopmans and Flinn 1984; Veijalainen et al. 1984; Stone 1990; Hopmans and Clerehan 1991). Measurement of the height of B-deficient trees is somewhat questionable, however, as those trees usually have multileadered crowns due to the effects of the condition on apical dominance. The most rapid growth effect of B has been observed to be on roots, e.g. in tomato, sunflower and squash plants (Dugger 1983; Dell and Huang 1997). B fertilisation has also been reported to increase vesiculararbuscular mycorrhizal colonisation in rough lemon (Citrus jambhiri Lush.) seedlings (Dixon et al. 1989) as well as in red clover (Trifolium pratense L.) and alfalfa (Medicago sativa L.) (Lambert et al. 1980). However, there is only one study on the effects of B on roots of co-

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nifer seedlings. Mitchell et al. (1987) reported enhanced root dry weight growth and ectomycorrhizal colonisation after addition of B in shortleaf pine (Pinus echinata Mill.) seedlings. The needle B concentrations in this study were, however, very high, about 140 mg kg–1 even in the control seedlings (Mitchell et al. 1987). Moreover, shortleaf pine is adapted to different climate and soils than Norway spruce and might not react in the same way to different B levels as Norway spruce. Conifers tend to have lower B requirements than dicotyledons (Marschner 1986; Hu et al. 1996). In field experiments, a large increase in the number of ectomycorrhizas in B-deficient mature Norway spruce trees after boron fertilisation suggested that root growth and ectomycorrhiza formation in conifers was inhibited by a lack of boron (Lehto 1994). It is difficult in the field, however, to assess whether B affects more the formation of root tips or their colonisation by mycorrhizal fungi, as almost all the root tips of Norway spruce trees in boreal forest soils are mycorrhizal. It is important to quantify when and how trees respond to different levels of boron, because of their possible effects on growth, mycorrhizas and nutrient uptake. Furthermore, it is important to know which responses occur first and which later, because the earliest response observed is likely to be a direct one while later responses could follow from the earlier ones. To our knowledge, however, there are no published reports on the response of the growth, roots and mycorrhizas of conifers to low and optimal rates of boron supply. The aim of this work was therefore to compare the effects of three addition rates of boron from a low level up to optimal on the growth dynamics and ectomycorrhizas of Norway spruce seedlings over a growing period of 16 weeks. Sequential harvests enabled comparisons of the timing of the different effects.

Materials and methods Cultivation of Norway spruce seedlings Norway spruce seeds from central Finland were soaked in cold water overnight, treated with 30% H2O2 for 15 min and then rinsed five times with distilled water. They were sown in 165 cm3 plastic pots (Ray-Leach Cone-tainer SC-10, Stuewe & Sons, Corvallis, Ore., USA), four seeds per pot, in sieved mor humus from a low-B spruce forest (30% vol) mixed with quartz sand (70% vol, particle size 0.5–1.5 mm). The initial total B concentration in the soil mixture was low, under 0.5 mg kg–1 [B concentration determined with an inductively coupled plasma atomic emission spectrometer (ARL 3580, Interface Design, Houston, Tex., USA) from dry ash material digested in HNO3 (suprapur, Merck K, Darmstadt, Germany)]. The seedlings were raised in a walk-in growth room (Conviron GR 77, Conviron, Controlled Environments, Winnipeg, Manitoba, Canada) for a total of 16 weeks (113 days) after germination. The seedlings were thinned to one per pot 8 days after germination. Day length (photoperiod) in the growth room was 20 h with a day/night temperature regime of 25/15°C, cooling/warming at 5°C h–1 and a relative humidity of 60/80% (water vapour pressure deficit 12.7/3.4 mmol mol–1). Day/night irradiance was 350/0 µmol m–2s–1 PAR from incandescent lamps (60 W, Oy

Airam AB, Finland) and fluorescent tubes (VHO 215 W, Sylvania Cool White, Sylvania, USA). The red: far-red photon ratio was 1.16 (LI-1800 spectroradiometer, LI-COR, Lincoln, Neb., USA). Light intensity was reduced/increased in three 1-h steps. The pots were watered daily with deionised water (contains no B) before germination and for 15 days after germination. Afterwards the seedlings were fertilised with a modified Ingestad nutrient solution (Riddoch et al. 1991) to make sure that the seedlings would not suffer nutrient imbalances other than B deficiency. Three boron treatments were used in all the fertilisations. The nutrient solutions contained 0%, 30% or 100% of the total amount of boron in the complete Ingestad solution while the proportions of the other nutrients did not vary. In the nutrient solutions the proportions of N, P and K were 100:16:55 by weight, the ratio of nitrate-N to ammonium-N was 7:5 and pH was 4.0. The nutrient solution used in the first fertilisation (week 2) contained 5.80 mg N dm–3, and 33 µg H3BO3 dm–3 in the case of B100, 30% of this B in B30 treatment and no B at B0. After this the concentrations of the solutions were increased each time following a relative nutrient addition rate (RAR) of 4% day–1 (Ingestad 1979). The seedlings were fertilised twice a week (10 ml per seedling) with an excess of nutrient solution to ensure that all the growth medium was flushed with fresh solution. After 39 days (week 6), the fertilisation frequency was increased to three times per week and RAR to 6% day–1. At the last fertilisation (day 113) the solution contained 357 mg N dm–3, and 2.04 mg H3BO3 dm–3 in the case of B100. In addition, the seedlings were watered with deionised water if required. Experimental design and statistics The experiment had a complete randomised block design with three treatments (boron levels of 0, 30 and 100%) and six blocks (true replicates). Each block had 48 seedlings in each B treatment in individual pots. The means of data for all seedlings measured at a given time and receiving the same treatment within each block were used in the statistical analysis, as the individual seedlings were not true replicates but subsamples. Statistical analyses were performed with SPSS (SPSS for Windows, Release 10.0.5, SPSS). Angular transformations were used for proportions and ln(x) for the height, dry weight and nutrient content data to improve normality and reduce heterogeneity in the variances. The back-transformed values for dry weight and B and N contents are shown in the figures and tables. A multivariate analysis of variance (MANOVA) for repeated measurements was used when assessing growth and mycorrhiza data. The significance of the differences between treatments were tested with the Pillai’s Trace test. Furthermore, when analysing weekly data the data were subjected to an analysis of variance (ANOVA), and Tukey’s test was used when there were significant differences between the treatment means. In statistical analyses the number of degrees of freedom for error (dferror) was 10, except in analysing B and N concentrations and contents before week 16 (samples pooled). The time dependencies of the relative growth rate (RGR) and the relative stem elongation rate (SER) were calculated from the first derivative of a third degree polynomial fitted to the block means of ln-transformed values in each B treatment for whole plant dry weight and stem height, respectively (dry weight: 0.943≤R2≤0.951, height: 0.981≤R2≤0.985). Growth of the seedlings, roots and mycorrhizas Two randomly selected seedlings per treatment per block were harvested every week starting 7 weeks after germination until the end of the experiment (16 weeks after germination). Altogether there were 10 harvests, providing 36 seedlings at each. After harvesting, the dry weights of the needles, stems and roots were determined (40°C with forced ventilation for a minimum of 3 days). Dry weight ratios for different parts of the plants were calculated

321 by dividing their dry weight by the total dry weight. In addition, there were 180 height measurement seedlings (10 per treatment per block) which were not harvested, and on which height was measured to the nearest millimetre with a ruler repeatedly every week between weeks 3 and 16. Root tips and mycorrhizas were counted and classified according to their morphology and colour under a stereomicroscope every second week between weeks 7 and 15. Ponceau S [0.1% Ponceau S (Acid red 112, Sigma Chemical) in 10% acetic acid] was used for mycorrhizal determination (Daughtridge et al. 1986; Holopainen and Vaittinen 1988). A mycorrhizal root tip was defined as a short root tip with a fully developed mantle. The mycorrhizal root tips were separated into two groups: root tips with Cenococcum geophilum and root tips with other mycorrhizas with a mantle. All other root tips, i.e. long root tips, root tips without a mantle and developing mycorrhizas (de la Rosa et al. 1998) were counted together and termed “root tips without a mantle”. Nutrient analyses Boron concentrations were determined with an inductively coupled plasma atomic emission spectrometer (ARL 3580, Interface Design, Houston, Tex., USA) from dry ash material digested in HNO3 (suprapur, Merck K, Darmstadt, Germany). Nitrogen concentrations were determined by the micro-Kjeldahl method (Kubin 1978; ISO 7150/1 1984) using a spectrophotometer (Lambda 11, Perkin-Elmer, Norwalk, Conn., USA). The nutrient contents in the different organs were calculated by multiplying their dry weights by the respective nutrient concentrations. Boron concentrations were determined from seedlings (2 seedlings per treatment per block) that were harvested every second week between weeks 4 and 16 and nitrogen concentrations (similarly 2 seedlings per treatment per block) from seedlings harvested every second week between weeks 6 and 15. As the sample sizes were small at the beginning of the experiment, all the replicates within each harvest were pooled for both B (until week 10) and N determinations (until week 10/11), and the stems of the two harvests for weeks 4 and 6 were pooled for B determinations. Between weeks 12 and 14 the samples from the first three blocks were pooled and likewise the last three ones. In weeks 15 and 16 only subsamples were pooled.

Results Boron concentrations in the needles were slightly lower at B0 than at B30 and B100 as early in the experiment as in week 4 and were already slightly different between all three B levels in week 10 (Table 1). It was not possible to test these differences statistically due to lack of replicates before week 12. The sampling was replicated, however, only the chemical analyses were not replicated. By week 12, needle B concentrations at B100 were 108% and 61% higher than those at B0 and B30 and were statistically different. The corresponding ratios by week 16 were 302% and 51%. Differences in needle B content (concentration × dry weight) also increased in accordance with the B treatments, so that the boron content of the needles at B100 was 159% and 44% higher than at B0 and B30, respectively, in week 12 and 355% and 63% higher by week 16. The boron concentrations in the roots did not differ clearly between the treatments before week 12 (Table 1). By week 14 they were 121% and 156% higher at B100 than at B0 and B30 and by week 16 44% and 19% higher, respectively. Root B content differed between the

Fig. 1 Boron allocation, expressed as B content ratios in Norway spruce seedlings grown at different levels of B supply [0% (B0), 30% (B30) and 100% (B100)] (n=6, number of seedlings=36) between weeks 8 and 16 (data for weeks 4 and 6 not shown). a=B0, b=B30 and c=B100; roots: cross-hatching, stems: solid white, and needles: diagonal hatching

treatments more than did the concentrations, as the content at B100 was 76% and 35% higher than at B0 and B30 in week 16. The B concentrations in the stems were rather variable and relatively high throughout the experiment (Table 1). The stem boron content at B100 was 106% higher than at B0 in week 14 however, and 50% higher in week 16. Boron was allocated mainly to the stems at B0 at the expense of the needles, as seen in weeks 12 and 16 (Fig. 1). The total B content of the whole plant was not different between the treatments early in the experiment. During weeks 12–14 B0 contained about 70% of the B accumulated by plants in B100, and by the end of the experiment, still more than 30% (Table 1). The nitrogen concentration and content in the roots, stems and needles were not affected significantly by the B treatments (Table 2). In general the N concentration in roots and needles and the N content of the roots, stems and needles increased with time. B/N ratios were not presented as there were no differences between the B treatments in the N concentrations and therefore the differences in B/N ratios would have been similar to those ones presented for B concentrations. The stem heights of the seedlings increased slowly until week 10 and more rapidly thereafter (data not shown). Boron increased the stem height (P=0.018 for B treatment, MANOVA, for weeks 7–16). When analysing weekly data, the stem height of the seedlings was higher at B100 than at B0 in weeks 11 (P=0.006) and 12 (P=0.005). By week 16 the height of the seedlings was 74 mm at B0, 68 mm at B30 and 77 mm at B100. The relative SER were 0.75% day–1 (B0), 0.72% day–1 (B30) and 0.86% day–1 (B100) on day 37 (week 5) and 3.3% day–1 (B0), 3.3% day–1 (B30) and 3.2% day–1 (B100) on

322 Table 1 Boron concentrations (means) and content per seedling (back-transformed means) in Norway spruce seedlings grown at different levels of B supply [0% (B0), 30% (B30) and 100% (B100)] between weeks 4 and 16 after germination. In weeks 4–10 n=1, as all the replicate samples were pooled, as also were the stems from both harvests in weeks 4 and 6. In weeks 12–14 n=2, as the samples from the first three blocks and those from the last three ones were pooled. In week 16 n=6 (95% confidence intervals: both upper and lower bounds are presented in parentheses). Means followed by the same letter are not significantly different (Tukey’s test). (NA, not available)

Treat- Concentrations ment Roots Stems (mg kg–1) (mg kg–1)

Needles (mg kg–1)

Roots (µg)

Stems (µg)

Needles (µg)

Total (µg)

Week 4 B0 14.9 B30 13.6 B100 10.1

49.1 40.2 NA

16.9 20.4 19.4

0.20 0.13 0.13

0.09 0.06 NA

0.20 0.22 0.27

0.49 0.41 NA

Week 6 B0 19.5 B30 9.5 B100 8.4

49.1 40.2 NA

17.7 18.0 20.9

0.47 0.19 0.20

0.12 0.24 NA

0.31 0.29 0.35

0.90 0.72 NA

Week 8 B0 8.5 B30 7.7 B100 8.4

59.5 50.1 33.5

16.0 19.9 21.0

0.19 0.17 0.22

0.18 0.15 0.12

0.33 0.37 0.46

0.70 0.69 0.80

Week 10 B0 8.5 B30 11.3 B100 9.9

28.3 34.1 42.9

17.3 22.7 27.5

0.22 0.33 0.32

0.09 0.11 0.14

0.52 0.66 0.78

0.83 1.10 1.24

Week 12 B0 12.0 B30 10.1 B100 11.0

87.3 72.1 67.6

14.4a 18.6a 30.0b

0.47 0.48 0.49

0.74a 0.51ab 0.36b

0.70a 1.26b 1.81b

1.91 2.25 2.66

Significance of B treatment 0.415 0.235

0.005

0.970

0.023

0.000

0.101

Week 14 B0 10.4 B30 9.0 B100 23.0

10.5a 16.2a 28.3b

0.58a 0.75ab 1.74b

0.34a 0.58ab 0.70b

1.00a 2.08b 3.57c

1.92a 3.41a 6.01b

0.059

0.016

0.000

0.003

6.6a (3.5, 9.6) 17.5b (14.4, 20.5) 26.5c (23.4, 29.5)

0.90a (0.77, 1.07) 1.17a (0.99, 1.38) 1.58b (1.34, 1.86)

1.66a (1.35, 2.05) 1.84ab (1.49, 2.27) 2.49b (2.02, 3.08)

1.55a (1.28, 1.86) 4.33b (3.59, 5.21) 7.06c (5.86, 8.51)

4.11a (3.56, 4.95) 7.34b (6.28, 8.75) 11.13c (9.59, 13.36)

0.000

0.001

0.031

0.000

0.000

29.1 35.5 42.5

Significance of B treatment 0.481 0.160 Week 16 B0 7.5a (5.8, 9.1) B30 9.1ab (7.5, 10.8) B100 10.8b (9.1, 12.5)

32.0 (25.8, 38.2) 33.2 (27.0, 39.4) 38.4 (32.1, 44.6)

Significance of B treatment 0.032 0.276

day 84 (week 12). According to MANOVA, the B treatments did not affect the relative rate of increase in height of the stems, however. In addition, no visible signs of any growth disturbances were observed in the seedlings even at the lowest B level. The total dry weight of the seedlings increased slowly until week 11, after which the dry weight increased more rapidly (Fig. 2 A). The RGR for the different B treatments were 1.7% day–1, 2.1% day–1 and 2.1% day–1 for B0, B30 and B100 respectively on day 37 (week 5). At that time RAR (Ingestad 1979) was 4% day–1. On day 84 (week 12) the RGR were 5.8% day–1, 5.4% day–1, and 5.6% day–1 at

Content per seedling

0.009

B0, B30 and B100, respectively. At that time RAR was 6% day–1. The total dry weight between weeks 7 and 16 was slightly affected by boron (P=0.078 for B treatment, MANOVA), but the relative rate of increase in total dry weight (RGR) was not affected by B. The needle and stem dry weights were not affected by boron. However, the root dry weight between weeks 7 and 16 was increased by boron (P=0.001 for B treatment, MANOVA), but the relative rate of increase in root dry weight was not affected by B. When the weekly data were analysed the root dry weight at B100 was 46% and 40% higher than at B0 in weeks 13 and 14 (P=0.002, P=0.001).

323 Table 2 Nitrogen concentrations (means) and content per seedling (back-transformed means) in Norway spruce seedlings grown at different levels of boron supply [0% (B0), 30% (B30) and 100% (B100)] between weeks 6–15 after germination. Roots are from the seedlings harvested in weeks 6, 8, 10, 12, and 14, and needles and stems from those harvested in weeks 7, 9, 11, 13, and 15. In weeks 6–10 n=1, as all the replicate samples were pooled. In weeks 12–14 n=2, as the samples from the first three blocks and those from the last three ones were pooled. In week 15 n=6 (95% confidence intervals: both upper and lower bounds are presented in parentheses)

Treatment

Concentrations

Content per seedling

Roots (g kg–1)

Stems (g kg–1)

Needles (g kg–1)

Roots (mg)

Stems (mg)

Needles (mg)

Week 6/7 B0 B30 B100

13.7 15.7 13.5

14.5 12.2 12.1

18.0 15.6 17.6

0.33 0.33 0.34

0.043 0.029 0.038

0.32 0.30 0.26

Week 8/9 B0 B30 B100

14.7 15.5 15.8

14.5 12.2 12.1

18.2 21.2 19.7

0.32 0.33 0.41

0.051 0.032 0.039

0.45 0.51 0.45

Week 10/11 B0 B30 B100

16.8 17.3 17.3

21.0 19.0 21.0

20.7 23.6 24.4

0.44 0.50 0.56

0.064 0.076 0.105

0.77 0.80 1.00

Week 12/13 B0 B30 B100

21.1 20.5 20.9

20.8 22.5 22.1

22.9 23.7 22.8

0.83 0.99 0.92

0.192 0.175 0.191

1.73 1.74 1.66

0.721

0.486

0.780

0.904

23.2 (22.3, 24.1) 22.8 (21.9, 23.7) 23.2 (22.3, 24.1)

1.25

0.580 (0.477, 0.705) 0.551 (0.453, 0.670) 0.584 (0.480, 0.710)

4.27 (3.78, 4.83) 3.94 (3.49, 4.45) 4.14 (3.66, 4.67)

0.879

0.583

Significance of B treatment 0.169 0.595 Week 14/15 B0

22.3

B30

22.9

B100

21.4

19.8 (18.4, 21.2) 19.7 (18.2, 21.1) 20.5 (19.0, 21.9)

Significance of B treatment 0.789 0.677

Needle weight ratio between weeks 7 and 16 was decreased by B (P=0.033 for B treatment, MANOVA), but the rate of change in needle weight ratio was not affected by B (Fig. 2B). Stem weight ratio was not dependent on B treatments, but again the root weight ratio was increased by B between weeks 7 and 16 (P=0.024 for B treatment, MANOVA). The change in time of root weight ratio was not affected by B, however. When analysing weekly data the root weight ratio of the seedlings grown at B100 was usually highest, although significantly different only in one harvest: it was 31% higher in week 13 than that of seedlings grown at B0 (P=0.006). Correspondingly, the needle weight ratio was lower at B100 than at B0 in week 13 (P=0.002). The number of all root tips was increased by B between weeks 7 and 15 (P=0.003 for B treatment, MANOVA), and the rate of increase was slightly faster with high B treatments (P=0.102 for B treatment × time, MANOVA) (Fig. 3 A). There were no differences between B treatments in the number of all root tips in week 7, but this number was 35% higher at B100 than at B30 in week 9 (P=0.042). It was also 67% higher at B100 than at B0 in week 11 (P=0.017), and 53% higher in

0.709

1.91 1.96

0.123

week 13 (P=0.009). In week 15 the number of all root tips at B100 was 37% higher than at B0 (P=0.062). The number of root tips expressed per unit of root dry weight was not affected by B, however (data not shown). The number of Cenococcum geophilum mycorrhizas did not significantly differ between the B treatments between weeks 7 and 15 (Fig. 3A). The number of other mycorrhizas with a mantle was increased by B, however (P=0.001 for B treatment, MANOVA), but the rate of increase in time was the same in all B treatments. The number of other mycorrhizas with a mantle was higher at B100 than at B0 in week 11 (P=0.013) and in week 15 (P=0.029). The mycorrhizal percentage increased from 22% (week 7) to 43% (week 15) at B0, from 17% to 40% at B30 and from 24% to 49% at B100, respectively (Fig. 3B). Boron did not affect the percentage of Cenococcum geophilum, but the percentage of other mycorrhizas with a mantle was consistently highest at B100 (P<0.001 for B treatment, MANOVA), but the rate of increase was not affected by B. The difference in the percentage of other mycorrhizas with a mantle was significant in two occasions: 78% and 86% higher at B100 than

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Fig. 2 Dry weight growth (back-transformed values and 95% confidence intervals, n=6, number of seedlings=36) of roots, stems and needles (A) and dry weight ratios (B) of Norway spruce seedlings grown at different levels of B supply [0% (B0), 30% (B30) and 100% (B100)] between weeks 7 and 16. a=B0, b=B30 and c=B100; roots: cross-hatching, stems: solid white and needles: diagonal hatching

Fig. 3 Number of root tips (A) and number of mycorrhizas expressed as percentages of all root tips (B) in Norway spruce seedlings grown at different levels of B supply [0% (B0), 30% (B30) and 100% (B100)] (means and 95% confidence intervals, n=6, number of seedlings=36) between weeks 7 and 15. a=B0, b=B30 and c=B100; root tips with Cenococcum geophilum mycorrhizas: crosshatching, root tips with other mycorrhizas with a mantle: horizontal hatching and root tips without a mantle: diagonal hatching

at B0 in weeks 11 (P=0.035) and 15 (P=0.029). Furthermore, the number of mycorrhizas expressed per unit of root dry weight was increased by B (P=0.044 for B treatment, MANOVA), but the rate of increase was not (data not shown).

relatively high until week 12, but after that they were at suboptimal or deficiency levels (Brække 1977, 1979; Aronsson 1983; Jukka 1988). Most of the B in the seedlings had been taken up by roots, as the total B content of a seedling from week 4 after germination onwards was greater than the storage of B in the seed (B concentration 19 mg kg–1, content 0.11 µg seed–1). During the experiment, nutrients were added in relation to the expected RGR of the seedlings (Ingestad 1979). The RGR was smaller than the RAR for most of the time, however, and therefore the B and N concentrations in the B100 seedlings somewhat increased with time. Boron increased the total dry weight of the seedlings only slightly. Mitchell et al. (1987) reported the total dry weight of shortleaf pine seedlings to be approximately 27% higher with B fertilisation, but we found the differences in dry weight between the B treatments to be very small. However, the root dry weight was highest at B100, but it was not as much affected by B as the number of root tips.

Discussion Seedlings grown at three boron levels had slightly different boron concentrations in their needles as early as week 10 after germination, when the fertilisation treatments had been going on for 8 weeks, after which the differences became more pronounced with time. At the two highest B levels the needle boron concentrations were optimal for Norway spruce as stated by Brække (1977, 1979), Aronsson (1983) and Jukka (1988). Their critical needle concentrations were determined for mature trees, however, and may differ from those for seedlings. Needle B concentrations at the lowest B level were

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The different B levels had the most pronounced effect on the number of root tips, the seedlings grown with adequate B having more root tips than those receiving the two lower B treatments as early as week 9. The B concentrations in the roots did not differ between the B treatments before week 14, however. B concentrations in the needles, on the other hand, already differed between the B treatments in week 10. It seems, therefore, that the roots responded to different rates of B addition in the nutrient solution and to the needle B concentrations at a stage when the concentrations in the roots did not yet differ between the B levels and the needle B concentrations were optimal at all three B levels. In a recent study by Lehto et al. (2000) it was shown that in Norway spruce B can be translocated in the phloem from shoots to roots and, therefore, some B in roots may have been translocated from shoots in the present study. Redistribution of B is likely to diminish the importance of the B concentration of roots and the external B supply on root growth at any particular moment, as B can be moved from the shoot for root growth. Our results suggest that the critical needle B concentrations for seedlings should perhaps be re-evaluated, as B increased the number of root tips even within the B levels that have been earlier considered as optimal (see e.g. Brække 1979; Jukka 1988). In shortleaf pine, B addition increased the growth of seedlings, nutrient accumulation and mycorrhizal colonisation of roots, although needle B concentrations in control seedlings were 140 mg kg–1 and in fertilised seedlings needle B increased up to 850 mg kg–1 (Mitchell et al. 1990). These concentrations are considerably higher than the B concentrations usually reported as being normal for shortleaf pine (Mitchell et al. 1990), however, no symptoms of toxicity were observed in their study. In many areas, needle B concentrations are very low compared to figures given by Mitchell et al. (1990). In Finland, the needle B concentrations are reported to vary between 6 and 16 mg kg–1 in pine (Pinus sylvestris L.) and Norway spruce stands according to the results of extensive field experiments (Derome et al. 1986; Mälkönen et al. 1990). In Sweden, Wikner (1983) reported needle B concentrations in Norway spruce forests to be 5–20 mg kg–1. The percentage of mycorrhizas with a mantle was increased by B, although the enhancement of mycorrhizal colonisation in our study was not as large as found by Mitchell et al. (1987) on Pisolithus tinctorius mycorrhizas of shortleaf pine. The role of boron in the mycorrhizal colonisation of roots is still poorly understood, and may result from an interaction with phenolics (Pilbeam and Kirkby 1983; Sword and Garrett 1994). In the present study, the number of root tips and mycorrhizas were both increased by B, but the root dry weight was not increased as much. If the response is similar in mature trees, the increased mycorrhizal colonisation coupled with increased short root formation could lead to improved nutrient uptake. The way in which boron improves root growth is connected to its role in both cell

enlargement and division in the root meristematic region (Dell and Huang 1997). In this experiment boron did not affect nitrogen dynamics of the seedlings, whereas Hopmans and Clerehan (1991) found increased foliar N concentrations in radiata pine (Pinus radiata D. Don) 6 years after B fertilisation and suggested that this had been caused by increased root growth. In contrast, Lehto and Mälkönen (1994) observed that boron fertilisation reduced needle N concentrations in mature Norway spruce and suggested that this may have been due to a dilution effect. In the present study, the nutrient solution used contained adequate amounts of N and the increased number of root tips and increased mycorrhizal percentage did not increase N uptake of the seedlings. Although the seedlings grown at adequate B had an increased number of root tips, and increased root dry weight, adequate B nutrition increased the stem height only slightly. Many other studies have shown an increase in height growth as a result of the application of borax in mature trees (e.g. Hopmans and Flinn 1984; Veijalainen et al. 1984; Hopmans and Clerehan 1991). However, Mitchell et al. (1987) did not observe increased height growth in shortleaf pine seedlings According to our study, boron was observed to accumulate more in stems at the expense of other parts of the seedlings at the lowest B level, which is consistent with the report by Wolf (1940) that cauliflower plants growing in soils receiving no borax had the largest percentage of their boron in the stems. This is consistent with the importance of B for stem formation and elongation through its effect on both cell division and cell elongation (Dugger 1983) and/or through the amount of B needed for the lignification of xylem (Lewis 1980). In conclusion, we show that the most pronounced effect of low B was a lower number of root tips and mycorrhizas with only a slight impact on the above-ground growth of the seedlings. Moreover, as the addition of boron affected the roots when needle concentrations were well above the critical B levels quoted in the literature, it can be concluded that B nutrition can affect the fitness of trees through root formation even when needle concentrations are “adequate”. The effect of B on the number of root tips may become more pronounced and may have a more significant effect on the growth and survival of the seedlings in the longer term and/or under conditions of environmental stress, e.g. drought or excess nitrogen. Acknowledgements We would like to thank Ms. Maini Mononen and Ms. Rauni Oksman for their skilful technical assistance and for the nutrient analyses. We are also very grateful to Mr. Malcolm Hicks, M.A., who carefully revised the language of an earlier version of this paper. This research was made possible by grants from Academy of Finland (Project 40896) and the Maj and Tor Nessling Foundation, Helsinki, Finland.

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