Journal of Chemical Ecology, Vol. 18, No. 10, 1992
I N H I B I T I O N OF SCOTS PINE S E E D L I N G E S T A B L I S H M E N T BY Empetrum hermaphroditum
M A R I E - C H A R L O T T E N I L S S O N * and O L L E Z A C K R I S S O N Swedish University of Agricultural Sciences Faculty of Forestry, Department of Forest Vegetation Ecology S-901 83 Ume~, Sweden (Received March 2, 1992; accepted June 11, 1992) Abstract--Poor establishment and reduced seedling growth of Scots pine (Pinus silvestris L.) in northern Sweden is related to an allelopathic inhibition by the dwarf shrub Empetrum hermaphroditum Hagerup. Indoor bioassays with green and brown leaves of Empetrum have strong negative effects on rooting ability, radicle elongation, and growth of Scots pine seedlings. Bioassays with soil samples show that phytotoxic substances leached from Empetrum foliage accumulate in the soil. Field experiments reveal that chemical inhibition by Empetrum, causing high mortality and slow growth of pine seedlings, can be reduced by adding activated carbon to the soil. Key Words--Allelopathy, Pinus silvestris, Empetrum hermaphroditum, succession, regeneration failures, soil toxicity, activated carbon, forest fires. INTRODUCTION Empetrum hermaphroditum Hagerup is a 5- to 15-cm-high evergreen dwarf shrub forming extensive clones in postfire successions on acid m o r humus soils with high C / N ratios in northern Scandinavia (Haapasaari, 1988; Odell and Drakenberg, 1991). Regeneration o f Scots pine (Pinus silvestris L.) from seed trees and from planting operations on clear-cut areas dominated by Empetrum have often been unsuccessful (Arnborg, 1943; Malmstr6m, 1949; Sarvas, 1950; Ebeling, 1979; Hagner, 1984). Traditional interpretations of these regeneration problems and slow growth have focused mostly on harsh climate, unfavorable soils, and dense plant cover. Recently, the possibility of chemical inhibition by Empetrum has been suggested (Zackrisson and Nilsson, 1992). Empetrum leaves *To whom correspondence should be addressed. 1857 0098-0331/92/1000-1857506.50/0 9 1992 Plenum Publishing Corporation
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NILSSON AND ZACKRISSON
were found to possess glands that are responsible for the production of watersoluble toxins easily leached out by rain. Chemical identification revealed that 3-methoxy-5,3'-dihydroxy, dihydrostilbene, or batatasin III (B-III) is one substance in water extracts of Empetrum that strongly inhibits seed germination under controlled indoor conditions (Odrn et al., 1992). In this paper we have assessed the allelopathic potential of Empetrum on early seedling growth of Scots pine in both field experiments and indoor bioassays.
METHODS AND MATERIALS
Green leaves of living Empetrum were collected during late autumn at Rov~tgem (63~ air-dried at 20~ and stored at - 1 8 ~ before use in the bioassays. A stock 5 % solution of aqueous extract was prepared by soaking 50 g of dry green leaves in 1 liter of distilled water while slowly stirring for 48 hr. One leaf is about 3 • 7 mm and has a dry weight of 0.4 mg. The extract was filtered (Munktell No. 3 filter paper) to yield a clear, yellowish brown solution with a characteristic odor. The solution had an osmotic potential of 43 mmol/kg and a pH of 4.2. Scots pine seeds (Skaholma, 66~ half-sib seeds with a viability of 92%) were used in all experiments, unless otherwise noted.
Analysis of Data Variation among treatments was tested with analysis of variance or Student's t test. Tukey's multiple a posteriori range test and the least significant difference (LSD) (Sokal and Rohlf, 1981) were used to determine significant (P _< 0.05) differences between treatments.
Experiment 1. Bioassays of Aqueous Leaf Extracts Method 1. Empetrum extract (2 ml of 5 % solution) was placed into 50mm-diameter Petri dishes lined with a sheet of filter paper (Munktell No. 3). In seven replicate dishes for each treatment, seven seeds with a pregerminated radicle of 1 mm were exposed to the extract or to distilled water controls for 7, 19, 25, 48, 72, or 240 hr, and then rinsed in distilled water and transferred to Petfi dishes lined with filter paper and containing 2 ml of distilled water. Petri dishes were maintained in a growth chamber under 20 hr illumination at 20~ The length of the radicle and, later, total seedling length (root plus hypocotyl) were measured daily with a digital ruler. Method 2. Scots pine seeds were germinated on moist, oven-sterilized (130~ 12 hr) industrial quartz sand (Silversand 90) in the greenhouse. After 12 days, one seedling was transferred to each of 80 0. l-liter plastic pots containing sterile sand watered to field capacity, and placed in the greenhouse under
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18 hr illumination and alternating temperatures of 20~ (day) and 15~ (night). All pots were watered every second day with a complete nutrient solution (5 mg NH4-N/liter) recommended by Ingestad (1979). Twenty days after transfer, 4 ml of three concentrations (5%, 2.5%, and 1.25%) of aqueous extracts of Empetrum or distilled water was given twice a week. Seedling height, measured to the longest primary or secondary needle, and mortality were recorded at the start of the treatments and every fifth day thereafter. At harvest after 46 days of treatment, sand was rinsed off the roots, seedlings were dried at 70~ for 72 hr, dry weights of shoots and roots were determined separately, and root growth characteristics were recorded. All extracts were kept at 2~ during the experiment. Each treatment was replicated 20 times.
Experiment 2. Bioassays of Empetrum Leaves Method 1. Scots pine seeds were germinated on industrial quartz sand watered to field capacity with distilled water in 10 90-mm-diameter 0.7-liter plastic pots. When seedlings reached 10 mm in height, they were thinned to 20 seedlings per pot. Then, 3.0 g of air-dried green leaves of Empetrum was added to five pots and 3.0 g of dry commercial greenhouse peat (unlimed, unfertilized and finely ground) was added to five pots as a control. Pots were misted several times a day with tapwater to maintain a high moisture level. On days 4, 7, 11, 14, and 17, seedling height and mortality were recorded. After 17 days of treatment, sand was rinsed off the roots, the seedlings were dried at 70~ for 72 hr, and the main root length and dry weights of shoots and roots were determined. Method 2. Scots pine seeds were pregerminated in distilled water. When a radicle of 1 mm emerged, 10 seeds were carefully transferred, without changing their orientation, onto each of five replicate 0.7-liter pots containing moistened quartz sand and either 0.48, 0.12, 0.06 g or no dry green leaves and 3 g peat on the surface. Plastic bags allowing airflow were placed over the pots to maintain the moisture level. When the radicle penetrated the substrate and the hypocotyl grew above the substrate, the seedling was considered rooted. Method 3. Green leaves and brown leaves, still connected to the three-year shoots, were removed and air-dried at 20~ Ten Scots pine seeds, pregerminated in distilled water with a radicle of 1 mm length, were transferred to 60-mm-diameter 0.15-liter plastic pots half-filled with sterile quartz sand and 2 g of one of the following substrates on top: (1) green leaves, (2) brown leaves, (3) a control of soaked green leaves, (4) a control of soaked brown leaves and (5) peat. "Soaked" leaves were 25 g leaves in 1 liter of distilled water, repeatedly changed during eight days, to detoxify the leaves, yet maintain physical similarity in surface structure of the seed beds. As this experiment was intended to simulate leaves and litter exposed to natural wet conditions (heavy rainfall or
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NILSSON AND ZACKRISSON
snow meltwater), the leaves and peat were soaked in 10 ml of distilled water for 48 hr before transfer to the pots. Ten replicates of each treatment were sealed with parafilm to maintain uniform moisture and placed in a climate chamber at 20~ and with 20 hr illumination. The numbers of rooted seedlings and seedling mortality were recorded daily for five days, after which the experiment was terminated and total length of seedlings (root plus hypocotyl) was measured.
Experiment 3. Bioassay of Freshly Collected Soil To investigate the accumulation of phytotoxins in soils, six replicate soil samples were taken beneath a homogeneous clone of Empetrum at the Akkangdke field site (lat 65~ in May, August, and October 1990, May and July 1991, and in May 1992. There were no trees, or ground layer of mosses or lichens at the sampling site. The sampling in May 1991 and May 1992 coincides with recent snow melt on frozen soil, but in May 1990 the frozen soil had thawed during unseasonably warm weather. Each sample included a complete humus soil profile plus the uppermost mineral soil (A00-A2) (Rieger, 1983). A similar fine-textured leached moraine soil (A2 horizon), where plant cover and humus had been removed three years earlier, was sampled nearby as a control. Soil samples were transferred to plastic bags and immediately transported at 2~ to the laboratory, where each sample was separated into A00 (litter), A1 (humus), and A2 (leached mineral soil) portions. The uppermost 0.5 cm of each layer was transferred to 50-mm-diameter Petri dishes. Industrial quartz sand was used as an additional control substrate in the bioassay. Fifty aspen (Populus tremula L.) seeds were sown on the soil in each dish because aspen germinates more quickly than pine, and a quick germination response is needed when diminishing levels of toxins are expected in samples brought indoors. The dishes were sealed with parafilm and placed in a growth chamber. Seed germination was recorded for seven days. Six replicates per treatment were used.
Experiment 4. Field Experiment Three field sites on acid mor humus soils with cover dominated by Empe700 m asl), Kiutisvaara (67~ 300 m asl), and Salmisj/irvi (68~ 430 m asl) in northem Sweden. At each site 40 level plots (0.75 • 0.75 m) were selected where Empetrum cover was about 80% and no mosses, lichens, or trees were present. On 20 randomly chosen plots, 280 g of fine-powdered (pro analysi) activated carbon (Labasco) was added (Zackrisson and Nilsson, 1992). In the central part of each plot, 100 seeds of Scots pine were sown in a 60 x 60-cm plastic exclosure with 10 mm mesh, at Akkan~lke and Kiutisvaara in May 1988 and at Salmisj/irvi in May 1989. Additional amounts of activated carbon were applied in the springs of t988 (140 g/plot), 1989 (70 g/plot), and 1990 (45 g/plot). Germination, and
trum were selected at Akkan/~lke (65~
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the survival and the stage of development of emerged seedlings were counted two to four times a year during 1988-1991. Seedling height to the apical bud was measured in September 1991.
Experiment 5. Bioassays of Activated Carbon Method 1. To determine the effect of activated carbon on seed germination of Scots pine, 20 g of industrial quartz sand or 2.5 g of well-decomposed humus were added to 10 50-mm-diameter Petri dishes. One gram of fine-powdered activated carbon was added to five dishes, each filled with sand and humus. Each dish was watered to field capacity with distilled water and 50 Scots pine seeds were sown. When radicles reached seed length, they were counted as germinated and removed from the dishes daily until there was no further germination. Method 2. Ten Scots pine seeds germinating on wet industrial quartz sand in the greenhouse were transferred when seedlings reached 10 mm length into each of 10 plastic pots filled with wet sand. Five grams of activated carbon (equivalent to field applications) was added to the sand surface of five pots. Shoot heights of the seedlings were measured every third day until termination at day 28, when shoot height and main root length were measured.
RESULTS
Experiment ! Growth of germinating seedlings exposed to the 5 % aqueous extract of
Empetrum was significantly inhibited compared to controls exposed to distilled water (Figure 1). Degree of inhibition increased with time of exposure. Although exposed seedlings grew, inhibition after exposure was persistent and growth as a percent of control continued to decline. Seedlings continuously exposed or exposed 48 hr or 72 hr almost stopped growing--an indication of severe physiological damage. In addition, Empetrum aqueous extract significantly decreased growth of pine seedlings when application of extracts was initiated after depletion of the cotyledonary reserves (Table 1). As Empetrum extract concentration increased, mean dry weight of seedlings and elongation of roots decreased. In the 5 % treatment, seedlings started to lose turgor after six days of treatment, and mortality reached 10% at day 22 of treatment, 40% at day 31, and 100% by day 46. Other treatments exhibited little mortality. Seedlings exposed to Empetrum extract had dark, discolored primary roots and strongly reduced side roots, chiefly occurring near the pot bottoms. The abnormalities appeared to be related to extract concentration.
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NILSSON AND ZACKRISSON 10o1
80"
7h
~
60.
19h
9
40-
0 20. ~
~
h Continuous
o
2~
4'8
z~
,~o
,~2
2~o
Hours
FIG. 1. Radicle growth as a percent o f controls o f Scots pine germinating seedlings exposed to 5% aqueous extract o f Empetrum leaves for different times. All growth reductions are statistically different from controls except for the dots marked with " o n e . " Arrows indicate w h e n exposure to Empetrum ended.
TABLE 1. MEAN AND q- SE FOR SCOTS PINE SEEDLINGS AFTER 46 DAYS OF EXPOSURE TO DIFFERENT CONCENTRATIONS OF AQUEOUS LEAF EXTRACT OF Empetrum OR DISTILLED WATER CONTROLS a
Empetrum treatment
Shoot height (Am) Shoot weight (rag) Root weight (rag) Shoot-root ratio Total weight (rag) Main root length (Am) Mortality (% of control)
1.25 %
2.50 %
5%
Control
44.4 __. 1.5a 13.8 + 0.8a 15.6 • 1.ga 1.2a 29.4 • 2.4a 110.8 • 7.6a 5
41.1 • 1.8a 13.2 • 0.7a 10.0 • 0.9b 1.5a 23.2 • 1.4b 102.9 • 23.3a 0
41.8 • 1.2a 7.3 • 0.4b 2.6 • 0.2c 3.2b 9.8 • 0.4c 56.6 • 2.6b 100
46.4 • 1.2a 19.5 • 1.0c 20.1 • 2.2d 1. la 39.6 • 3.1d 121.2 + 8.0a 0
"Values are means of 20 seedlings. Values in a row followed by the different letters are significantly different at P < 0.05 in Tukey's multiple range test.
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TABLE 2. MEAN AND d- SE OF DRY WEIGHT OF SHOOTS AND ROOTS, SHOOT LENGTHS, ROOT LENGTHS, AND MORTALITY OF SCOTS PINE SEEDLINGS GROWN IN CONTACT WITH 3.0 GRAMS OF GREEN LEAVES OF EmpetrumOR PEAT (CONTROL)a
Treatment
Shoot weight (mg) Root weight (mg) Shoot-root ratio Shoot length (mm) Main root length (mm) Mortality (%)
Empetrum
Control
129.6 + 2.2 b 30.4 + 7.40 4.7 + 0.5 31.8 _ 0.7 b 44.8 + 2.9 b 57.6 b
229.8 + 4.5 72.0 + 1.2 3.2 + 0.1 35.0 + 0.5 88.5 ___ 1.8 12.8
~Seedlings were harvested after 17 days of treatment. Values are means of 100 seedlings per treatment. Shoot and root dry weight values are means of five replicates. bSignificantly different from control at P _< 0.05.
Experiment 2 In the first bioassay of Empetrum leaves, seedlings grown with air dried green Empetrum leaves had significantly higher mortality (58 %) than the control (13 %) (Table 2). Surviving seedlings in the Empetrum treatment had dry weights of shoots and roots and lengths of main roots about half those in the control (Table 2). Differences in shoot length were less exaggerated. A higher shootroot ratio was also observed for Empetrum treated seedlings. Seedlings in the Empetrum treatment had dark-colored, dwarfed root systems whereas control seedlings were light-colored and healthy. In the second bioassay of Empetrum leaves, rooting of pregerminated Scots pine seeds was inversely related to leaf weights in pots even after one day of treatment (Figure 2). In the 0.48-g treatment most radicles were stunted and died before rooting. Convergence of the treated seedlings over time is due to mortality. Variation in number of rooted seedlings is explained by mortality and delayed rooting. In the third bioassay of Empetrum leaves, rooting of germinating Scots pine seedlings was shown to be severely affected by green leaves of Empetrum (Figure 3). Brown leaves also strongly inhibited the rooting. Soaked green and brown leaves revealed lower inhibition than the respective unsoaked leaves, although all treatments caused statistically significant growth reductions compared to the control. Mortality o f germlings was 100% in the green leaf treatment, but they grew in the other treatments.
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NILSSON AND ZACKRISSON
10-
9"
Control
8-
"8
c
5.
3-
i"06g
2.
1
3.12g
0 i
9 0.48g
1
2
~
~
7
;o
Days
FIG. 2. Rooting ability of pregerminated Scots pine seeds grown in pots with different quantities of green leaves of Empetrum or peat (control). Vertical bars give the least significant differences (LSD) among treatments.
d
b b b
8o u "~
~
j~/~~/~/ 40 ::i::i::i:~// :i::i 2o
b .
/
9 Green unsoaked r--1Greensoaked [] B.... u. . . . ked [] Brown soaked
iiiii!iill
No.Rooted' Seedling Seedlings length
% Survival
FrG. 3. Rooting ability and seedling length (root plus hypocotyl) of pregerminated Scots pine seeds grown in contact with soaked and unsoaked green and brown leaves of Empetrum during five days of treatment. Bars topped with the same letter do not differ significantly in Tukey's multiple-range test.
Experiment 3 Seed germination of aspen on different layers (A00, A1, A2) of Empetrum mor humus showed that inhibition was strongest during May 1991 and May 1992, and generally increased with depth in the soil profile from the litter layer
SCOTS PINE INHIBITION
BY
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(A00) to the leached mineral soil (A2) (Figure 4). The mineral soil (A2) sampled from the soil uncovered with humus and the industrial quartz sand (data not shown) had no inhibitory effect on seed germination.
Experiment 4 A significantly smaller number of Scots pine seeds germinated and fewer seedlings became established in untreated Empetrum vegetation compared with the activated carbon-treated vegetation at all three field sites (Table 3). Gera
100-
a
a
80 A
6 0 84
E.
a
b ~ ~ 7 ~
----a-- -layer ] ? A2-layer --*--
AO0- layer A1
/
Uncovered A2- layer
40
0
20-
May
Aug
Oct
May
Jul
May
90
90
90
91
91
92
FIG. 4. Seed germination of aspen in soil collected at different depths and at different times in natural Empetrum vegetation. Means on the same date followed by the same letters do not differ in Tukey's multiple-range test. TABLE 3. SEED GERMINATION, SURVIVAL, AND GROWTH OF 2000 SCOTS PINE PER PLOT IN UNTREATED AND ACTIVATED (A) CARBON-TREATED Empetrum VEGETATION AT THREE FIELD SITES IN INTEP,IOR NORTHERN SWEDEN, 1988-1991 a
Surviving in September 1991 (%)
Seedling height (mm) in September 1991 (Mean _+ SE)
Site
Treatment
Germination observed (%)
Akkanfilke
Untreated A carbon
36.6 a 52.2
11.4 15.0
39.7 + 0.4 a 45.7 + 0.7
Kiutisvaam
Untreated A carbon
21.3 ~ 35.9
3.1 a 12.4
36.3 + 1.0 ~ 39.6 -+_ 0.7
Salmisjhrvi
Untreated A carbon
11.0" 45.3
1.0 ~ 6.8
43.9 + 1.4 49.1 + 1.0
~Significantly different from plots treated with activated carbon at P _< 0.05 level.
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NILSSONAND ZACKRISSON
mination was also delayed and seedlings grew more slowly in the untreated plots compared to the carbon-treated plots during the first season (data not shown). Seedling survival and seedling height were significantly higher in activated carbon-treated plots at the end of the experiment. Seedling density was compared to seedling height in order to check for interspecific competition in the plots, but no correlations were found.
Experiment 5 Activated carbon alone did not have any positive effect on seed germination. No differences in seed germination were evident between seeds grown in humus (96.8%), sand (96.8%), humus treated with carbon (99.2%), or sand treated with carbon (99.6%). There were also no differences in the rate of germination among the treatments (data not shown). For seedlings in pots, shoot height did not show any significant differences when grown in sand (39.1 _+ 0.7 mm) or in sand treated with activated carbon (36.7 _+ 0.7 mm). Main root lengths were 38.5 _+ 1.4 mm and 39.2 + 1.4 ram, respectively. DISCUSSION Indoor bioassays showed strongly increased mortality and reduced growth of Scots pine seedlings when grown in pots together with green or brown leaves of Empetrum, or when watered with different dosages of aqueous leaf extracts. Water-soluble phytotoxins may be leached from leaves of Empetrum and taken up by the developing radicle and primary roots, leading to stagnant growth and high mortality. The different results of growth and rooting ability between germinating seedlings grown in contact with soaked and unsoaked leaves could not be attributed to physical dissimilarities of the seed bed. Soaked leaves are probably less inhibitory than unsoaked leaves because allelochemicals were leached from the former during soaking. Soaking, however, did not totally reduce the inhibitory effect, indicating that all toxins are not easily washed away despite repeated soaking in large quantities of water. Compared to green leaves, brown leaves were also less inhibitory to rooting ability. When a seed lands on the upper soil surface in an Empetrum-dominated microhabitat, it comes in direct contact with newly fallen toxic litter and is exposed to phytotoxins released from the green foliage of Empetrum by rain and snowmelt water. During the imbibition process, the seeds absorb water, and if the water contains toxins from Empetrum, the germination will be delayed or totally inhibited, depending on the toxin concentrations (Zackrisson and Nilsson, 1992). Before rooting occurs, the early radicle may be exposed in the same way. In this paper we have shown that very short exposure times of germinating seedlings to aqueous leaf extracts of Empetrum have a persistent inhibitory effect
SCOTS PINE INHIBITION BY
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1867
on radicle growth. Osmotic pressure or pH in extracts are not responsible for the observed inhibition (unpublished data). Our field data provide empirical evidence of a possible allelopathic inhibition by Empetrumon Scots pine. The treatments used in the field studies were designed to subject pine seeds and seedlings to the interference of Empetrum, without removing competitive influence, and to reveal possible allelopathic effects. Pine responded to this treatment by displaying significantly better establishment and growth when the phytotoxins were removed by activated carbon. The activated carbon will adsorb inhibiting substances produced by leaves and litter leached into the soil. The reduction in germination and lower seedling shoot growth is certainly due to chemical inhibition as the experimental design ruled out the possibilities that conditions of illumination, root competition, humidity, and nutritional conditions of the soil could differ between the two treatments. There is no reason to assume that seed or seedling predation would differ between the treatments. The activated carbon in itself does not contribute to any positive effects, as shown in our indoor bioassays. Similarly, Eliasson (1959) showed that activated carbon, when present in a nutrient solution, did not stimulate the growth of wheat roots. Low amounts of activated carbon effectively detoxify aqueous extracts of Empetrum (Zackrisson and Nilsson, 1992). Smaller amounts than presently used in the field experiment are preferable in future studies, as an excess of carbon may retard growth of seedlings by blackening the photosynthetic tissues. Activated carbon can only be expected to detoxify substances originating from leaves and litter leaching into the soil. Toxins accumulated in the soil by Empetrum before the experiment started or through the decomposition process cannot be reduced by activated carbon on the surface. It was also found that germination and seedling growth were delayed in the untreated plots during the first growing season. This delay in growth probably also contributes to the lower total shoot height found after three (Salmi@irvi) and four growing periods (Akkan~tlke and Kiutisvaara). Indoor experiments with pine seeds also show that germination is delayed when exposed to low amounts of extracts (Zackrisson and Nilsson, 1992). Slow seedling growth in the first season (data not shown) may also cause a delay in the winter hardening process, giving rise to the increased mortality demonstrated in the untreated plots. Allelopathy could also cause seedlings to suffer from reduced mycorrhizal infection (Olsen et al., 1971; Brown and Mikola, 1974; Rose et al., 1983, Perry and Choquette, 1987; Cot6 and Thibault, 1988), which may inhibit the seedling's ability to compete for nutrients. No seedlings were harvested in our field experiments and conclusions could not be drawn about the below-ground effects of inhibition on pine root development. In the greenhouse experiment, however, we noted a greater negative impact of Empetrum on root growth than on shoot growth. Restricted root growth may result in mortality of seedlings due to desiccation. Desiccation interpreted in a wide sense is one of
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NILSSONAND ZACga~SSON
the major causes of mortality registered during periods with spring and summer droughts. The snowmelt water contains biologically inhibiting levels of toxins produced by Empetrum through the natural soaking of green vegetative parts and litter (Zackrisson and Nilsson, 1992). The accumulation of toxins in soil may be greatest during early spring when the snow has just melted, and ground ice still persists deep into the soil, preventing the snowmelt water from draining into the ground. The microbial detoxification in this situation is probably slow because of the low soil temperatures and anaerobic conditions. After the snow has melted away, the soil may remain frozen for weeks, as in May 1991 and 1992, when the toxicity level at all soil depths studied was high. In other years, such as May 1990, with hardly any frozen soil and rapid snowmelt (statistics from the Swedish Meteorological Institute), the water drained quickly into the soil and the threshold situation with persistently high toxin levels in the soil was probably very short. As pointed out by Jalal and Read (1983), Yoder-Williams and Parker (1987), Cast et al. (1990), and Molina et al. (1991), allelopathy could be operative in a specific environment only for a short period of the growing season or under specific environmental conditions. The most profound effects of pine seedling mortality are registered during the spring. Allelopathic interference is one likely explanation of conifer seed regeneration failures found in Empetrum-dominated vegetation in northern Sweden. There are reasons to suspect that the substances leached from Empetrum could also affect more long-term growth of pine saplings. Martinsson (unpublished data) has shown that young coniferous forest stands established on Empetrumdominated clear-cuts were less productive than nearby prescribed burned areas of the same age on sites previously influenced by Empetrum. Empetrum is fire sensitive and normally does not resprout vegetatively after burning. Empetrum is a slow seed recolonizer, and thus seldom reaches a dominance in the postfire forest floor vegetation before crown closure. After cutting without burning, Empetrum expands vegetatively and can form a dense field layer vegetation. In virgin pine stands, fire return intervals normally were short (Zackrisson, 1977, 1981)7 and Empetrum seldom reached the distribution and densities found today due to fire protection. Fire elimination will therefore indirectly contribute to the regeneration failures attributed to expansion of Empetrum vegetation. Whether allelopathy is the major factor responsible for the regeneration problems described cannot be fully concluded from this study. However, regeneration failures and reduced growth under allelopathic influence of Empetrum may exacerbate an already stressful boreal environment. Although other environmental factors may contribute to regeneration failure, our study indicates allelopathy by Empetrum may be an important factor.
Acknowledgments--Thisproject was funded by The Swedish Council of Forestry and Agricultural Research. We thank Anders J~iderlund, Gisela Norberg, and Anita Wennstr6m for skillful field and laboratory assistance. We also owe Stephen B. Horsley many thanks for valuable methodological discussions over the years.
SCOTS PINE INHIBITION BY E m p e t r u m
1869 REFERENCES
ARNBORG, T.
1943. Granberget. En v~ixtbiologisk unders6kning av ett sydlappl~indskt granskogsomrfide med s~irskild h/insyn till skogstyper och frryngring. Almqvist & Wiksell, Uppsala. BROWN, R.T., and MIKOLA, P. 1974. The influence of fructicose soil lichens upon the mycorrhizae and seedling growth of forest trees. Acta For. Fenn. 141:1-22. CAST, K.G., MCPHERSON,J.K., POLLARD, A.J., KRENZER, E.G., JR., and WALLER,G.R. 1990. Allelochemicals in soil from no-tillage versus conventional-tillage wheat (Triticum aestivum) fields. J. Chem. Ecol. 16:2277-2289. COT(~, J.-F., and THIBAULT, J.-R. 1988. Allelopathic potential of raspberry foliar leachates on growth of ectomycorrhizal fungi associated with black spruce. Am. J. Bot. 75(5):966-970. EBELING, F. 1979. Anvisningar for skogssk6tsel i kyliga klimatl/igen. Sver. Skogsvardsfoerb. Tidskr. 77:4. ELIASSON, L. 1959. Inhibition of the growth of wheat roots in nutrient solutions by substances exuded from the roots. K. Lantbrukshoegs. Ann. 25:285-293. HAAPASAARI,H. 1988. The oligotrophic heath vegetation of northern Fennoscandia and its zonation. Acta Bot. Fenn. 135:1-219. HAGNER, M. 1984. Hur p~verkas plantor av sin n~irmaste milj6 p~ hygget? in P.O. B~ickstr0m (ed.). Skogsfrryngring i fj/illn/ira skogar. Rapport. ISBN 91-576-2162-4. Skogsvetenskapliga fakulteten. Sveriges Lantbruksuniversitet, Umeh. INOESTAD, T. 1979. Mineral nutrient requirements of Pinus silvestris and Picea abies seedlings. Physiol. Plant. 45:373-380. JALAL,M.A.F., and READ,D.J. 1983. The organic acid composition of Calluna heathland soil with special reference to phyto- and fungitoxicity. If. Monthly quantitative determination of the organic acid content of Calluna and spruce dominated soils. Plant Soil 70:273-286. MALMSTROM,C. 1949. Studier av skogstyper och trfidslagsf0rdelning inom V/isterbottens l/in. Medd. Statens skogsforskningsinst. 37(11): 1-231. MOLINA, A., REIGOSA, M.J., and CARBALLEIRA,A. 1991. Release of allelochemical agents from litter, throughfall, and topsoil in plantations of Eucalyptus globulus labitl in Spain. J. Chem. Ecol. 17:147-160. ODELL, G., and DRAKENBERG,B. 1991. Atlas 6ver skogsmarksv~ixters f0rekomst i Sverige. Rapport 64. Sveriges Lantbruksuniversitet, Uppsala. ODI~N, P.C., BRANDTBERG, P.O., ANDERSSON,R., GREF, R., ZACKRISSON,O., and NILSSON, M.C. 1992. Isolation and characterization of a germination inhibitor from leaves of Empetrum hermaphroditum (Hagemp.). Scand. J. For. Res. 7. OLSEN, R., ODHAM, G., and LINDEBERG,G. 1971. Aromatic substances in leaves ofPopulus tremula as inhibitors of mycorrhizal fungi. Physiol. Plant 25:122-129. PERRY,D.A., and CHOQUETTE,C. 1987. Allelopathic effects on mycorrhizae. Influence of structure and dynamics of forest ecosystems, pp. 185-194, in G.R. Waller (ed.). Allelochemicals. Role in Agriculture and Forestry. ACS Symposium Series 330. American Chemical Society, Washington, D.C. RIEGER~S. 1983. The Genesis and Classification of Cold Soils. Academic Press. New York. ROSE, S.L., PERRY, D.A., PILZ, D., and SCHOENEBEROER,M.M. 1983. Allelopathic effects of litter on the growth and colonization of mycorrhizal fungi. J. Chem. Ecol. 9: 1153-1162. SARVAS,R. 1950. Mets~tieteellisen tutkimuslaitoksen. Julkasisuja. Commun. Inst. For. Fenn. 38:8595. SOKAL, R.R., and ROHLF, F.J. 1981. Biometry. The Principles and Practice of Statistics in Biological Research. W.H. Freeman and Company, New York.
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NILSSON AND ZACKRISSON
YODER-WILLIAMS, M.P., and PARKER, V.T. 1987. Allelopathic interference in the seedbed of Pinus jeffreyi in the Sierra Nevada, California. Can. J. For. Res. 17:991-994. ZACKRISSON, O. 1977. Influence of forest fires on the north Swedish boreal forest. Oikos 29:2232. ZACKRISSON, 0 . 1981. Forest fire history: Ecological significance and dating problems in the north Swedish boreal forest. Proceedings of the Fire History Workshop, 1980, Tucson. USDA General Technical Report RM-81. ZACKRISSON, O., and NILSSON, M.-C. 1992. Allelopathic effects by Empetrum hermaphroditum on seed germination of two boreal tree species. Can. J. For. Res. Sept. issue.
I N H I B I T I O N OF SCOTS PINE S E E D L I N G E S T A B L I S H M E N T BY Empetrum hermaphroditum
M A R I E - C H A R L O T T E N I L S S O N * and O L L E Z A C K R I S S O N Swedish University of Agricultural Sciences Faculty of Forestry, Department of Forest Vegetation Ecology S-901 83 Ume~, Sweden (Received March 2, 1992; accepted June 11, 1992) Abstract--Poor establishment and reduced seedling growth of Scots pine (Pinus silvestris L.) in northern Sweden is related to an allelopathic inhibition by the dwarf shrub Empetrum hermaphroditum Hagerup. Indoor bioassays with green and brown leaves of Empetrum have strong negative effects on rooting ability, radicle elongation, and growth of Scots pine seedlings. Bioassays with soil samples show that phytotoxic substances leached from Empetrum foliage accumulate in the soil. Field experiments reveal that chemical inhibition by Empetrum, causing high mortality and slow growth of pine seedlings, can be reduced by adding activated carbon to the soil. Key Words--Allelopathy, Pinus silvestris, Empetrum hermaphroditum, succession, regeneration failures, soil toxicity, activated carbon, forest fires. INTRODUCTION Empetrum hermaphroditum Hagerup is a 5- to 15-cm-high evergreen dwarf shrub forming extensive clones in postfire successions on acid m o r humus soils with high C / N ratios in northern Scandinavia (Haapasaari, 1988; Odell and Drakenberg, 1991). Regeneration o f Scots pine (Pinus silvestris L.) from seed trees and from planting operations on clear-cut areas dominated by Empetrum have often been unsuccessful (Arnborg, 1943; Malmstr6m, 1949; Sarvas, 1950; Ebeling, 1979; Hagner, 1984). Traditional interpretations of these regeneration problems and slow growth have focused mostly on harsh climate, unfavorable soils, and dense plant cover. Recently, the possibility of chemical inhibition by Empetrum has been suggested (Zackrisson and Nilsson, 1992). Empetrum leaves *To whom correspondence should be addressed. 1857 0098-0331/92/1000-1857506.50/0 9 1992 Plenum Publishing Corporation
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NILSSON AND ZACKRISSON
were found to possess glands that are responsible for the production of watersoluble toxins easily leached out by rain. Chemical identification revealed that 3-methoxy-5,3'-dihydroxy, dihydrostilbene, or batatasin III (B-III) is one substance in water extracts of Empetrum that strongly inhibits seed germination under controlled indoor conditions (Odrn et al., 1992). In this paper we have assessed the allelopathic potential of Empetrum on early seedling growth of Scots pine in both field experiments and indoor bioassays.
METHODS AND MATERIALS
Green leaves of living Empetrum were collected during late autumn at Rov~tgem (63~ air-dried at 20~ and stored at - 1 8 ~ before use in the bioassays. A stock 5 % solution of aqueous extract was prepared by soaking 50 g of dry green leaves in 1 liter of distilled water while slowly stirring for 48 hr. One leaf is about 3 • 7 mm and has a dry weight of 0.4 mg. The extract was filtered (Munktell No. 3 filter paper) to yield a clear, yellowish brown solution with a characteristic odor. The solution had an osmotic potential of 43 mmol/kg and a pH of 4.2. Scots pine seeds (Skaholma, 66~ half-sib seeds with a viability of 92%) were used in all experiments, unless otherwise noted.
Analysis of Data Variation among treatments was tested with analysis of variance or Student's t test. Tukey's multiple a posteriori range test and the least significant difference (LSD) (Sokal and Rohlf, 1981) were used to determine significant (P _< 0.05) differences between treatments.
Experiment 1. Bioassays of Aqueous Leaf Extracts Method 1. Empetrum extract (2 ml of 5 % solution) was placed into 50mm-diameter Petri dishes lined with a sheet of filter paper (Munktell No. 3). In seven replicate dishes for each treatment, seven seeds with a pregerminated radicle of 1 mm were exposed to the extract or to distilled water controls for 7, 19, 25, 48, 72, or 240 hr, and then rinsed in distilled water and transferred to Petfi dishes lined with filter paper and containing 2 ml of distilled water. Petri dishes were maintained in a growth chamber under 20 hr illumination at 20~ The length of the radicle and, later, total seedling length (root plus hypocotyl) were measured daily with a digital ruler. Method 2. Scots pine seeds were germinated on moist, oven-sterilized (130~ 12 hr) industrial quartz sand (Silversand 90) in the greenhouse. After 12 days, one seedling was transferred to each of 80 0. l-liter plastic pots containing sterile sand watered to field capacity, and placed in the greenhouse under
SCOTS PINE INHIBITION BY
Empetrum
1859
18 hr illumination and alternating temperatures of 20~ (day) and 15~ (night). All pots were watered every second day with a complete nutrient solution (5 mg NH4-N/liter) recommended by Ingestad (1979). Twenty days after transfer, 4 ml of three concentrations (5%, 2.5%, and 1.25%) of aqueous extracts of Empetrum or distilled water was given twice a week. Seedling height, measured to the longest primary or secondary needle, and mortality were recorded at the start of the treatments and every fifth day thereafter. At harvest after 46 days of treatment, sand was rinsed off the roots, seedlings were dried at 70~ for 72 hr, dry weights of shoots and roots were determined separately, and root growth characteristics were recorded. All extracts were kept at 2~ during the experiment. Each treatment was replicated 20 times.
Experiment 2. Bioassays of Empetrum Leaves Method 1. Scots pine seeds were germinated on industrial quartz sand watered to field capacity with distilled water in 10 90-mm-diameter 0.7-liter plastic pots. When seedlings reached 10 mm in height, they were thinned to 20 seedlings per pot. Then, 3.0 g of air-dried green leaves of Empetrum was added to five pots and 3.0 g of dry commercial greenhouse peat (unlimed, unfertilized and finely ground) was added to five pots as a control. Pots were misted several times a day with tapwater to maintain a high moisture level. On days 4, 7, 11, 14, and 17, seedling height and mortality were recorded. After 17 days of treatment, sand was rinsed off the roots, the seedlings were dried at 70~ for 72 hr, and the main root length and dry weights of shoots and roots were determined. Method 2. Scots pine seeds were pregerminated in distilled water. When a radicle of 1 mm emerged, 10 seeds were carefully transferred, without changing their orientation, onto each of five replicate 0.7-liter pots containing moistened quartz sand and either 0.48, 0.12, 0.06 g or no dry green leaves and 3 g peat on the surface. Plastic bags allowing airflow were placed over the pots to maintain the moisture level. When the radicle penetrated the substrate and the hypocotyl grew above the substrate, the seedling was considered rooted. Method 3. Green leaves and brown leaves, still connected to the three-year shoots, were removed and air-dried at 20~ Ten Scots pine seeds, pregerminated in distilled water with a radicle of 1 mm length, were transferred to 60-mm-diameter 0.15-liter plastic pots half-filled with sterile quartz sand and 2 g of one of the following substrates on top: (1) green leaves, (2) brown leaves, (3) a control of soaked green leaves, (4) a control of soaked brown leaves and (5) peat. "Soaked" leaves were 25 g leaves in 1 liter of distilled water, repeatedly changed during eight days, to detoxify the leaves, yet maintain physical similarity in surface structure of the seed beds. As this experiment was intended to simulate leaves and litter exposed to natural wet conditions (heavy rainfall or
1860
NILSSON AND ZACKRISSON
snow meltwater), the leaves and peat were soaked in 10 ml of distilled water for 48 hr before transfer to the pots. Ten replicates of each treatment were sealed with parafilm to maintain uniform moisture and placed in a climate chamber at 20~ and with 20 hr illumination. The numbers of rooted seedlings and seedling mortality were recorded daily for five days, after which the experiment was terminated and total length of seedlings (root plus hypocotyl) was measured.
Experiment 3. Bioassay of Freshly Collected Soil To investigate the accumulation of phytotoxins in soils, six replicate soil samples were taken beneath a homogeneous clone of Empetrum at the Akkangdke field site (lat 65~ in May, August, and October 1990, May and July 1991, and in May 1992. There were no trees, or ground layer of mosses or lichens at the sampling site. The sampling in May 1991 and May 1992 coincides with recent snow melt on frozen soil, but in May 1990 the frozen soil had thawed during unseasonably warm weather. Each sample included a complete humus soil profile plus the uppermost mineral soil (A00-A2) (Rieger, 1983). A similar fine-textured leached moraine soil (A2 horizon), where plant cover and humus had been removed three years earlier, was sampled nearby as a control. Soil samples were transferred to plastic bags and immediately transported at 2~ to the laboratory, where each sample was separated into A00 (litter), A1 (humus), and A2 (leached mineral soil) portions. The uppermost 0.5 cm of each layer was transferred to 50-mm-diameter Petri dishes. Industrial quartz sand was used as an additional control substrate in the bioassay. Fifty aspen (Populus tremula L.) seeds were sown on the soil in each dish because aspen germinates more quickly than pine, and a quick germination response is needed when diminishing levels of toxins are expected in samples brought indoors. The dishes were sealed with parafilm and placed in a growth chamber. Seed germination was recorded for seven days. Six replicates per treatment were used.
Experiment 4. Field Experiment Three field sites on acid mor humus soils with cover dominated by Empe700 m asl), Kiutisvaara (67~ 300 m asl), and Salmisj/irvi (68~ 430 m asl) in northem Sweden. At each site 40 level plots (0.75 • 0.75 m) were selected where Empetrum cover was about 80% and no mosses, lichens, or trees were present. On 20 randomly chosen plots, 280 g of fine-powdered (pro analysi) activated carbon (Labasco) was added (Zackrisson and Nilsson, 1992). In the central part of each plot, 100 seeds of Scots pine were sown in a 60 x 60-cm plastic exclosure with 10 mm mesh, at Akkan~lke and Kiutisvaara in May 1988 and at Salmisj/irvi in May 1989. Additional amounts of activated carbon were applied in the springs of t988 (140 g/plot), 1989 (70 g/plot), and 1990 (45 g/plot). Germination, and
trum were selected at Akkan/~lke (65~
SCOTS PINE INHIBITION BY
Empetrum
1861
the survival and the stage of development of emerged seedlings were counted two to four times a year during 1988-1991. Seedling height to the apical bud was measured in September 1991.
Experiment 5. Bioassays of Activated Carbon Method 1. To determine the effect of activated carbon on seed germination of Scots pine, 20 g of industrial quartz sand or 2.5 g of well-decomposed humus were added to 10 50-mm-diameter Petri dishes. One gram of fine-powdered activated carbon was added to five dishes, each filled with sand and humus. Each dish was watered to field capacity with distilled water and 50 Scots pine seeds were sown. When radicles reached seed length, they were counted as germinated and removed from the dishes daily until there was no further germination. Method 2. Ten Scots pine seeds germinating on wet industrial quartz sand in the greenhouse were transferred when seedlings reached 10 mm length into each of 10 plastic pots filled with wet sand. Five grams of activated carbon (equivalent to field applications) was added to the sand surface of five pots. Shoot heights of the seedlings were measured every third day until termination at day 28, when shoot height and main root length were measured.
RESULTS
Experiment ! Growth of germinating seedlings exposed to the 5 % aqueous extract of
Empetrum was significantly inhibited compared to controls exposed to distilled water (Figure 1). Degree of inhibition increased with time of exposure. Although exposed seedlings grew, inhibition after exposure was persistent and growth as a percent of control continued to decline. Seedlings continuously exposed or exposed 48 hr or 72 hr almost stopped growing--an indication of severe physiological damage. In addition, Empetrum aqueous extract significantly decreased growth of pine seedlings when application of extracts was initiated after depletion of the cotyledonary reserves (Table 1). As Empetrum extract concentration increased, mean dry weight of seedlings and elongation of roots decreased. In the 5 % treatment, seedlings started to lose turgor after six days of treatment, and mortality reached 10% at day 22 of treatment, 40% at day 31, and 100% by day 46. Other treatments exhibited little mortality. Seedlings exposed to Empetrum extract had dark, discolored primary roots and strongly reduced side roots, chiefly occurring near the pot bottoms. The abnormalities appeared to be related to extract concentration.
1862
NILSSON AND ZACKRISSON 10o1
80"
7h
~
60.
19h
9
40-
0 20. ~
~
h Continuous
o
2~
4'8
z~
,~o
,~2
2~o
Hours
FIG. 1. Radicle growth as a percent o f controls o f Scots pine germinating seedlings exposed to 5% aqueous extract o f Empetrum leaves for different times. All growth reductions are statistically different from controls except for the dots marked with " o n e . " Arrows indicate w h e n exposure to Empetrum ended.
TABLE 1. MEAN AND q- SE FOR SCOTS PINE SEEDLINGS AFTER 46 DAYS OF EXPOSURE TO DIFFERENT CONCENTRATIONS OF AQUEOUS LEAF EXTRACT OF Empetrum OR DISTILLED WATER CONTROLS a
Empetrum treatment
Shoot height (Am) Shoot weight (rag) Root weight (rag) Shoot-root ratio Total weight (rag) Main root length (Am) Mortality (% of control)
1.25 %
2.50 %
5%
Control
44.4 __. 1.5a 13.8 + 0.8a 15.6 • 1.ga 1.2a 29.4 • 2.4a 110.8 • 7.6a 5
41.1 • 1.8a 13.2 • 0.7a 10.0 • 0.9b 1.5a 23.2 • 1.4b 102.9 • 23.3a 0
41.8 • 1.2a 7.3 • 0.4b 2.6 • 0.2c 3.2b 9.8 • 0.4c 56.6 • 2.6b 100
46.4 • 1.2a 19.5 • 1.0c 20.1 • 2.2d 1. la 39.6 • 3.1d 121.2 + 8.0a 0
"Values are means of 20 seedlings. Values in a row followed by the different letters are significantly different at P < 0.05 in Tukey's multiple range test.
SCOTS PINE INHIBITIONBY Empetrum
1863
TABLE 2. MEAN AND d- SE OF DRY WEIGHT OF SHOOTS AND ROOTS, SHOOT LENGTHS, ROOT LENGTHS, AND MORTALITY OF SCOTS PINE SEEDLINGS GROWN IN CONTACT WITH 3.0 GRAMS OF GREEN LEAVES OF EmpetrumOR PEAT (CONTROL)a
Treatment
Shoot weight (mg) Root weight (mg) Shoot-root ratio Shoot length (mm) Main root length (mm) Mortality (%)
Empetrum
Control
129.6 + 2.2 b 30.4 + 7.40 4.7 + 0.5 31.8 _ 0.7 b 44.8 + 2.9 b 57.6 b
229.8 + 4.5 72.0 + 1.2 3.2 + 0.1 35.0 + 0.5 88.5 ___ 1.8 12.8
~Seedlings were harvested after 17 days of treatment. Values are means of 100 seedlings per treatment. Shoot and root dry weight values are means of five replicates. bSignificantly different from control at P _< 0.05.
Experiment 2 In the first bioassay of Empetrum leaves, seedlings grown with air dried green Empetrum leaves had significantly higher mortality (58 %) than the control (13 %) (Table 2). Surviving seedlings in the Empetrum treatment had dry weights of shoots and roots and lengths of main roots about half those in the control (Table 2). Differences in shoot length were less exaggerated. A higher shootroot ratio was also observed for Empetrum treated seedlings. Seedlings in the Empetrum treatment had dark-colored, dwarfed root systems whereas control seedlings were light-colored and healthy. In the second bioassay of Empetrum leaves, rooting of pregerminated Scots pine seeds was inversely related to leaf weights in pots even after one day of treatment (Figure 2). In the 0.48-g treatment most radicles were stunted and died before rooting. Convergence of the treated seedlings over time is due to mortality. Variation in number of rooted seedlings is explained by mortality and delayed rooting. In the third bioassay of Empetrum leaves, rooting of germinating Scots pine seedlings was shown to be severely affected by green leaves of Empetrum (Figure 3). Brown leaves also strongly inhibited the rooting. Soaked green and brown leaves revealed lower inhibition than the respective unsoaked leaves, although all treatments caused statistically significant growth reductions compared to the control. Mortality o f germlings was 100% in the green leaf treatment, but they grew in the other treatments.
1864
NILSSON AND ZACKRISSON
10-
9"
Control
8-
"8
c
5.
3-
i"06g
2.
1
3.12g
0 i
9 0.48g
1
2
~
~
7
;o
Days
FIG. 2. Rooting ability of pregerminated Scots pine seeds grown in pots with different quantities of green leaves of Empetrum or peat (control). Vertical bars give the least significant differences (LSD) among treatments.
d
b b b
8o u "~
~
j~/~~/~/ 40 ::i::i::i:~// :i::i 2o
b .
/
9 Green unsoaked r--1Greensoaked [] B.... u. . . . ked [] Brown soaked
iiiii!iill
No.Rooted' Seedling Seedlings length
% Survival
FrG. 3. Rooting ability and seedling length (root plus hypocotyl) of pregerminated Scots pine seeds grown in contact with soaked and unsoaked green and brown leaves of Empetrum during five days of treatment. Bars topped with the same letter do not differ significantly in Tukey's multiple-range test.
Experiment 3 Seed germination of aspen on different layers (A00, A1, A2) of Empetrum mor humus showed that inhibition was strongest during May 1991 and May 1992, and generally increased with depth in the soil profile from the litter layer
SCOTS PINE INHIBITION
BY
Empetrum
1865
(A00) to the leached mineral soil (A2) (Figure 4). The mineral soil (A2) sampled from the soil uncovered with humus and the industrial quartz sand (data not shown) had no inhibitory effect on seed germination.
Experiment 4 A significantly smaller number of Scots pine seeds germinated and fewer seedlings became established in untreated Empetrum vegetation compared with the activated carbon-treated vegetation at all three field sites (Table 3). Gera
100-
a
a
80 A
6 0 84
E.
a
b ~ ~ 7 ~
----a-- -layer ] ? A2-layer --*--
AO0- layer A1
/
Uncovered A2- layer
40
0
20-
May
Aug
Oct
May
Jul
May
90
90
90
91
91
92
FIG. 4. Seed germination of aspen in soil collected at different depths and at different times in natural Empetrum vegetation. Means on the same date followed by the same letters do not differ in Tukey's multiple-range test. TABLE 3. SEED GERMINATION, SURVIVAL, AND GROWTH OF 2000 SCOTS PINE PER PLOT IN UNTREATED AND ACTIVATED (A) CARBON-TREATED Empetrum VEGETATION AT THREE FIELD SITES IN INTEP,IOR NORTHERN SWEDEN, 1988-1991 a
Surviving in September 1991 (%)
Seedling height (mm) in September 1991 (Mean _+ SE)
Site
Treatment
Germination observed (%)
Akkanfilke
Untreated A carbon
36.6 a 52.2
11.4 15.0
39.7 + 0.4 a 45.7 + 0.7
Kiutisvaam
Untreated A carbon
21.3 ~ 35.9
3.1 a 12.4
36.3 + 1.0 ~ 39.6 -+_ 0.7
Salmisjhrvi
Untreated A carbon
11.0" 45.3
1.0 ~ 6.8
43.9 + 1.4 49.1 + 1.0
~Significantly different from plots treated with activated carbon at P _< 0.05 level.
1866
NILSSONAND ZACKRISSON
mination was also delayed and seedlings grew more slowly in the untreated plots compared to the carbon-treated plots during the first season (data not shown). Seedling survival and seedling height were significantly higher in activated carbon-treated plots at the end of the experiment. Seedling density was compared to seedling height in order to check for interspecific competition in the plots, but no correlations were found.
Experiment 5 Activated carbon alone did not have any positive effect on seed germination. No differences in seed germination were evident between seeds grown in humus (96.8%), sand (96.8%), humus treated with carbon (99.2%), or sand treated with carbon (99.6%). There were also no differences in the rate of germination among the treatments (data not shown). For seedlings in pots, shoot height did not show any significant differences when grown in sand (39.1 _+ 0.7 mm) or in sand treated with activated carbon (36.7 _+ 0.7 mm). Main root lengths were 38.5 _+ 1.4 mm and 39.2 + 1.4 ram, respectively. DISCUSSION Indoor bioassays showed strongly increased mortality and reduced growth of Scots pine seedlings when grown in pots together with green or brown leaves of Empetrum, or when watered with different dosages of aqueous leaf extracts. Water-soluble phytotoxins may be leached from leaves of Empetrum and taken up by the developing radicle and primary roots, leading to stagnant growth and high mortality. The different results of growth and rooting ability between germinating seedlings grown in contact with soaked and unsoaked leaves could not be attributed to physical dissimilarities of the seed bed. Soaked leaves are probably less inhibitory than unsoaked leaves because allelochemicals were leached from the former during soaking. Soaking, however, did not totally reduce the inhibitory effect, indicating that all toxins are not easily washed away despite repeated soaking in large quantities of water. Compared to green leaves, brown leaves were also less inhibitory to rooting ability. When a seed lands on the upper soil surface in an Empetrum-dominated microhabitat, it comes in direct contact with newly fallen toxic litter and is exposed to phytotoxins released from the green foliage of Empetrum by rain and snowmelt water. During the imbibition process, the seeds absorb water, and if the water contains toxins from Empetrum, the germination will be delayed or totally inhibited, depending on the toxin concentrations (Zackrisson and Nilsson, 1992). Before rooting occurs, the early radicle may be exposed in the same way. In this paper we have shown that very short exposure times of germinating seedlings to aqueous leaf extracts of Empetrum have a persistent inhibitory effect
SCOTS PINE INHIBITION BY
Empetrum
1867
on radicle growth. Osmotic pressure or pH in extracts are not responsible for the observed inhibition (unpublished data). Our field data provide empirical evidence of a possible allelopathic inhibition by Empetrumon Scots pine. The treatments used in the field studies were designed to subject pine seeds and seedlings to the interference of Empetrum, without removing competitive influence, and to reveal possible allelopathic effects. Pine responded to this treatment by displaying significantly better establishment and growth when the phytotoxins were removed by activated carbon. The activated carbon will adsorb inhibiting substances produced by leaves and litter leached into the soil. The reduction in germination and lower seedling shoot growth is certainly due to chemical inhibition as the experimental design ruled out the possibilities that conditions of illumination, root competition, humidity, and nutritional conditions of the soil could differ between the two treatments. There is no reason to assume that seed or seedling predation would differ between the treatments. The activated carbon in itself does not contribute to any positive effects, as shown in our indoor bioassays. Similarly, Eliasson (1959) showed that activated carbon, when present in a nutrient solution, did not stimulate the growth of wheat roots. Low amounts of activated carbon effectively detoxify aqueous extracts of Empetrum (Zackrisson and Nilsson, 1992). Smaller amounts than presently used in the field experiment are preferable in future studies, as an excess of carbon may retard growth of seedlings by blackening the photosynthetic tissues. Activated carbon can only be expected to detoxify substances originating from leaves and litter leaching into the soil. Toxins accumulated in the soil by Empetrum before the experiment started or through the decomposition process cannot be reduced by activated carbon on the surface. It was also found that germination and seedling growth were delayed in the untreated plots during the first growing season. This delay in growth probably also contributes to the lower total shoot height found after three (Salmi@irvi) and four growing periods (Akkan~tlke and Kiutisvaara). Indoor experiments with pine seeds also show that germination is delayed when exposed to low amounts of extracts (Zackrisson and Nilsson, 1992). Slow seedling growth in the first season (data not shown) may also cause a delay in the winter hardening process, giving rise to the increased mortality demonstrated in the untreated plots. Allelopathy could also cause seedlings to suffer from reduced mycorrhizal infection (Olsen et al., 1971; Brown and Mikola, 1974; Rose et al., 1983, Perry and Choquette, 1987; Cot6 and Thibault, 1988), which may inhibit the seedling's ability to compete for nutrients. No seedlings were harvested in our field experiments and conclusions could not be drawn about the below-ground effects of inhibition on pine root development. In the greenhouse experiment, however, we noted a greater negative impact of Empetrum on root growth than on shoot growth. Restricted root growth may result in mortality of seedlings due to desiccation. Desiccation interpreted in a wide sense is one of
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NILSSONAND ZACga~SSON
the major causes of mortality registered during periods with spring and summer droughts. The snowmelt water contains biologically inhibiting levels of toxins produced by Empetrum through the natural soaking of green vegetative parts and litter (Zackrisson and Nilsson, 1992). The accumulation of toxins in soil may be greatest during early spring when the snow has just melted, and ground ice still persists deep into the soil, preventing the snowmelt water from draining into the ground. The microbial detoxification in this situation is probably slow because of the low soil temperatures and anaerobic conditions. After the snow has melted away, the soil may remain frozen for weeks, as in May 1991 and 1992, when the toxicity level at all soil depths studied was high. In other years, such as May 1990, with hardly any frozen soil and rapid snowmelt (statistics from the Swedish Meteorological Institute), the water drained quickly into the soil and the threshold situation with persistently high toxin levels in the soil was probably very short. As pointed out by Jalal and Read (1983), Yoder-Williams and Parker (1987), Cast et al. (1990), and Molina et al. (1991), allelopathy could be operative in a specific environment only for a short period of the growing season or under specific environmental conditions. The most profound effects of pine seedling mortality are registered during the spring. Allelopathic interference is one likely explanation of conifer seed regeneration failures found in Empetrum-dominated vegetation in northern Sweden. There are reasons to suspect that the substances leached from Empetrum could also affect more long-term growth of pine saplings. Martinsson (unpublished data) has shown that young coniferous forest stands established on Empetrumdominated clear-cuts were less productive than nearby prescribed burned areas of the same age on sites previously influenced by Empetrum. Empetrum is fire sensitive and normally does not resprout vegetatively after burning. Empetrum is a slow seed recolonizer, and thus seldom reaches a dominance in the postfire forest floor vegetation before crown closure. After cutting without burning, Empetrum expands vegetatively and can form a dense field layer vegetation. In virgin pine stands, fire return intervals normally were short (Zackrisson, 1977, 1981)7 and Empetrum seldom reached the distribution and densities found today due to fire protection. Fire elimination will therefore indirectly contribute to the regeneration failures attributed to expansion of Empetrum vegetation. Whether allelopathy is the major factor responsible for the regeneration problems described cannot be fully concluded from this study. However, regeneration failures and reduced growth under allelopathic influence of Empetrum may exacerbate an already stressful boreal environment. Although other environmental factors may contribute to regeneration failure, our study indicates allelopathy by Empetrum may be an important factor.
Acknowledgments--Thisproject was funded by The Swedish Council of Forestry and Agricultural Research. We thank Anders J~iderlund, Gisela Norberg, and Anita Wennstr6m for skillful field and laboratory assistance. We also owe Stephen B. Horsley many thanks for valuable methodological discussions over the years.
SCOTS PINE INHIBITION BY E m p e t r u m
1869 REFERENCES
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