adaptation strategies in wetland plants: links between ecology and physiology. proceedings of a...

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Root Symbioses of Alnus glutinosa (L.) Gaertn. and Their Possible Role in Alder Decline: A Preliminary Study Author(s): Silvie Struková, Miroslav Vosátka and Jan Pokorný Source: Folia Geobotanica & Phytotaxonomica, Vol. 31, No. 1, Adaptation Strategies in Wetland Plants: Links between Ecology and Physiology. Proceedings of a Workshop (1996), pp. 153-162 Published by: Springer Stable URL: http://www.jstor.org/stable/4181426 . Accessed: 12/06/2014 16:07 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . Springer is collaborating with JSTOR to digitize, preserve and extend access to Folia Geobotanica &Phytotaxonomica. http://www.jstor.org This content downloaded from 185.2.32.49 on Thu, 12 Jun 2014 16:07:41 PM All use subject to JSTOR Terms and Conditions

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Root Symbioses of Alnus glutinosa (L.) Gaertn. and Their Possible Role in Alder Decline: APreliminary StudyAuthor(s): Silvie Struková, Miroslav Vosátka and Jan PokornýSource: Folia Geobotanica & Phytotaxonomica, Vol. 31, No. 1, Adaptation Strategies in WetlandPlants: Links between Ecology and Physiology. Proceedings of a Workshop (1996), pp. 153-162Published by: SpringerStable URL: http://www.jstor.org/stable/4181426 .

Accessed: 12/06/2014 16:07

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

Springer is collaborating with JSTOR to digitize, preserve and extend access to Folia Geobotanica&Phytotaxonomica.

http://www.jstor.org

This content downloaded from 185.2.32.49 on Thu, 12 Jun 2014 16:07:41 PMAll use subject to JSTOR Terms and Conditions

Folia Geobot. Phytotax. 31: 153-162, 1996

ROOT SYMBIOSES OF ALNUS GLUTINOSA (L.) GAERTN. AND THEIR POSSIBLE ROLE IN ALDER DECLINE: A PRELIMINARY STUDY

Silvie Strukov&~a, Miroslav Vosatka2) & Jan Pokorny3)

1) Department of Plant Physiology, Charles University, Vinicna 7, CZ-128 00 Praha 2, Czech Republic;

2) Institute of Botany, Academy of Sciences of the Czech Republic, CZ-252 43 Pruhonice, Czech Republic; tel. +42 2 67750028, fax +42 2 67750031, E-mail [email protected]

3) Institute of Botany, Academy of Sciences of the Czech Republic, Dukelskd 145, CZ-379 82 7rebo,i, Czech Republic; tel. +42 333 721127, fax +42 333 721136, E-mail HA [email protected] CZ

Keywords: Actinorhiza, Arbuscular mycorrhiza, Frankia, Eutrophication

Abstract: During the last few years alder has declined in South Bohemia. The possible role of mycorrhizal and actinorhizal symbioses is reviewed and some of the preliminary results from experiments testing the influence of these symbioses on alder growth and the influence of eutrophication on the development of these symbioses are reported. Seedlings of Alnus glutinosa were inoculated with arbuscular mycorrhizal (AM) fungi and the actinomycete Frankia in experiment 1, and with rhizosphere soil collected from field sites with different degrees of alder damage in experiment 2. In both experiments, a solution containing nitrate, ammonia and phosphorus in concentrations simulating eutrophic waters, was applied. Both symbioses markedly promoted the growth of the seedlings in experiment 1. The plants inoculated with the rhizosphere soil microflora in experiment 2 were larger than the control plants. Response of the seedlings to the inoculation with the soil from the rhizosphere of damaged alder trees from six field sites differs, even though no correlation was found relating growth to the health status of the trees. Nutrient treatment did not have any effect on the growth of seedlings in either experiment. The dry weight of Frankia was greater in mycorrhizal plants compared to nonmycorrhizal plants and mycorrhizal colonization is reduced in Frankia inoculated plants supplemented with phosphorus in experiment 1. Nitrogen enhanced mycorrhizal colonization in nodulated plants which were not supplemented with phosphorus no effect of nitrogen on actinorhiza was observed.

INTRODUCTION

Alder (Alnus glutinosa) is the most common tree of river banks, and one of the most flood tolerant woody species in Central Europe. Beginning in the 1980's the alder has declined in South Bohemia and foresters have observed several phenomena in connection with this decline (JANCAkIK 1993): (a) presence of pathogenic fungi from the genus Ophiostoma, known as "tracheomycotic fungi", the occurrence of which is often related to the decline of other tree species such as oak, elm, spruce; (b) the decline of reddish roots bearing nodules with nitrogen fixing symbionts, and (c) the occurrence of leaf feeding insects.

No intensive study of alder decline has yet been undertaken and the reasons of alder decline are not understood since not all the above mentioned phenomena are always present on dying alders. Alder decline is generally attributed to weakening of the trees due to a combination

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154 S. Strukovc et al.

of environmental factors (JANCAPiK 1993), such as pathogens, 02 deficiency and toxic compounds in the soil formed under anaerobic conditions resulting from flooding. When the effect of anthropogenic impact is added, the tree can lose its resistance. The predominant anthropogenic stress factor in the agricultural region of South Bohemia is eutrophication of water and soil, particularly elevated concentrations of phosphates, nitrates and ammonia.

Root symbioses are reported to be sensitive to high nutrient concentration, so they might suffer from eutrophication (Huss-DANELL 1990, AzcoN-AcQUILAR 1994). Alder forms three types of symbioses with microorganisms: arbuscular mycorrhiza (AM), ectomycorrhiza (ECM) and actinorhiza (AR) (HARLEY & HARLEY 1987). We have focused on arbuscular mycorrhiza and actinorhiza.

Arbuscular mycorrhiza is formed by soil fungi and the roots of approximately 80% of plant species (TRAPPE 1987) and its main function is the enhancement of nutrient uptake, even though mycorrhiza has probably also other functions in the ecosystem. Mycorrhizae affect also plant photosynthesis, transpiration, hormonal balance, and root exudation (ALLEN 1991). Improved stress tolerance (SYLVIA & WILLIAMS 1992) and resistance to root pathogens (HOOKER et al. 1994) were reported for mycorrhizal plants. Mycorrhiza influences the competition between plants, plant community structure (FRANCIS & READ 1994, ALLEN 1991) and it can also affect the structure and function of other rhizosphere associative microorganisms (PuppI et al. 1994).

Actinorhiza is the symbiosis between the actinomycete Frankia and root of mostly woody shrubs or trees, belonging to approximately 200 species of 8 families (BAKER & SCHWINTZER 1990). The main function of actinorhiza is nitrogen fixation, accomplished by nitrogenase localized in the nodules which are similar to the nodules of Rhizobia. The rate of nitrogen fixation is estimated to range from 60 to 320 kg per ha per year for Alnus spp. Actinorhizal plants also seem to have a different nitrogen distribution pattern throughout the season; the nitrogen is not resorbed from Alnus leaves into the stem before defoliation, which results in the release of N to the soil through mineralization of the litter, estimated at 48 - 185 kg per ha per year (DAWSON 1990). There are also other functions associated with actinorhiza e.g. improved tolerance to some root pathogens (DAwsON 1990) and changes in hormonal (IAA) content (WHEELER et al. 1984).

Interaction of AM fungi and Frankia resulted in better plant growth of the host plant compared to the effect of a single organism (e.g. GARDNER et al. 1984, CHATARPAUL et al. 1989, JHA et al. 1993). JHA et al. (1993) found enhancement of nodule dry weight and nitrogenase activity by mycorrhiza probably due to better uptake of phosphorus by mycorrhizal plants (CERVANTES & RODRIQUEZ-BARRUECO 1992).

Both AR and AM symbioses are sensitive to many environmental factors, including high nutrient content and flooding. High phosphorus levels usually inhibit mycorrhizal development (e.g. ALLEN 1991), but the role of nitrogen is not clear. Both stimulation and inhibition of mycorrhizal colonization of the roots were observed at high N concentration (Azc6N-AcQUILAR 1994). The N:P ratio in particularly is probably an important factor in mycorrhiza inhibition and this ratio often decreases with water eutrophication. AR symbiosis was found to be sensitive to nitrogen in both ammonia and nitrate forms (HUSS-DANELL 1990). AM development might be inhibited under anaerobic conditions (ILAG et al. 1987). However, the presence of endomycorrhiza was documented in flooded soils, e.g. on Populus and Salix (LODGE 1989, WERNER 1992) reports that actinorhiza is aerobic and does not occur on

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Root symbioses of Alnus glutinosa 155

Table 1. The health status of five alder trees at the field sites. Health status estimation scale: I = healthy tree: all branches with leaves of normal size, rich green colour, 2 = slightly damaged tree: drying of single thin branches, the canopy is slightly thinned, defoliation up to 20%. 3 = medium damaged tree: thinned canopy with drying branches, defoliation over 45%, 4 = strongly damaged tree: even main branches are defoliated, defoliaton over 50%, 5 = dying or dead tree: almost whole canopy is dry, defoliation over 70% or dead tree. Each number represents the health status of one tree.

Site Tree No.

1 2 3 4 5

A Vlcetin 5 5 5 5 5 B Rosicka 4 4.5 3 3.5 4 C Bednarecek 3.5 3 3.5 4.5 4 D Vajgar 2.5 5 3 3 3 E Cejnuiv mlyn 4.5 2.5 3 2.5 2.5 F Hlubokodol 2.5 2.5 4 3 1.5

extremely wet habitats. In general, both organisms are aerobic to a certain extent and probably can be affected by extremely flooded conditions.

Both of these stress factors, flooding and excessive nutrients due to water and soil eutrophication, are present in South Bohemia, thus it is suggested that root symbioses might be affected. Because the role of these symbioses in the ecosystem is important, their impairment might have some impact on the health status of the host tree.

Two questions were addressed in our study: (1) what is the effect of the symbioses on alder seedling growth, and (2) are these symbioses affected by the nutrient concentration

in eutrophic soils. We tested (a) the effect of AM and AR inoculation on alder seedlings growth and (b) the effect of high nutrient content on the development of the symbioses. In experiment 2 we tested the effect on alder growth of rhizosphere soil microflora from the field site under different degrees of damage.

SITE DESCRIPTION

Six field sites with different degrees of alder damage were chosen for rhizosphere soil sampling and health status of five trees surrounding the sampling site was estimated (Tab. 1). The plots are situated in South Bohemia, northeast of Jindfichu'v Hradec, in the catchment of the Nezarka river in the Ceskomoravska' vrchovina uplands (average altitude about 500-600 m). All sites are non managed banks of river flood plains several meters wide in the neighbourhood of fields or meadows. The water in the river is eutrophic: BOD (biological oxygen demand) is 5-10, CODCr (chemical oxygen demand) is 10-20, concentration of N-NH4 is about 0.5-1 mg/l, concentration of N-NO3 -is 3-10 mg/l and total P about 0.1 - 0.5 mg/l.

The alders are approximately 20-50 years old. The prevailing plant species in the field layer are: Phalaris arundinacea L., Urtica dioica L., Galium aparine L., Anthriscus sylvestris (L.) HOFFM., Chaerophyllum hirsutum L., Aegopodium podagraria L., Humulus lupulus L., Padus avium MILL., Filipendula ulmaria (L.) MAXIM., Symphytum officinale L., Glechoma hederacea L., Heracleum sphondylium L., Poa palustris L., sometimes with Impatiens glandulifera ROYLE, Veronica chamaedrys L., Lamium album L., Alopecurus pratensis L., Dactylis glomerata L. and different species of Salix (S. triandra L., S. cinerea L., S. fragilis L.). Populus alba L. and Acer pseudoplatanus L. were found on site F and Rubus idaeus L. and Solanum dulcamara L. on sites D and F.

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156 S. Strukova et al.

Table 2. ANOVA's for Alnus glutinosa seedlings - experiment 1, harvest 1. ns = not significant, * P < 0.05, **P < 0.01, *** P < 0.001.

Treatment Stem Total Mycorrhizal Nodule d.f. height (F) biomass (F) infection (F) weight (F)

Frankia (A) 1 101.8 *** 31.3 *** 2.6 ns -

Mycorrhiza (B) 1 207.0 *** 57.9*** - 26.8 AxB 1 54.6*** 10.5** -

Phosphorus (C) 1 0.1 ns 0.5 ns 27.9 *** 0.1 ns A xC 1 2.1 ns 0.1 ns 54.2** Nitrogen (D) 2 4.3 * 0.2 ns 0.2 ns 0.2 ns AxD 2 0.4ns 0.1 ns 4.5 * -

AxCxD 2 1.8ns 1.0ns 8.01** Error d.f. 108 105 45 41

MATERIAL AND METHODS

Alnus glutinosa (L.) GAERTN. seeds were pregerminated for one month in sand and perlite substratum (1: l,v:v). One month old seedlings were transplanted to 750 ml containers with steam sterilized peat based substratum (peat, clay, sand, 4:1:1, v:v) and grown in a greenhouse with regulated, computer controlled ventilation and shading.

In experiment 1 seedlings were inoculated with Frankia and endomycorrhizal inocula. Frankia nodules were collected from healthy trees on the field sites and stored in the freezer at -5 ?C for six weeks. Immediately before inoculation, the nodules were thawed, separate lobes were washed with water, their surface sterilized with sodium hypochlorite (5% for 5 min.) and homogenized in a blender. The suspension (50 g nodules per liter) was filtered through filter paper (Whatman No 1) and 5 ml of the filtrate added to each pot. Inoculum of AM fungi (Glomus etunicatum - isolate S329 from the University of Florida, Gainesville, U.S.A., Glomusfasciculatum - from Rothamsted Experimental Station, U.K. and Gigaspora margarita - from Laboratoire de Phytoparasitologie, INRA, Dijon, France), containing infected roots, spores and mycelium, was grown on maize in a sand and perlite mixture (1: 1, v:v) for four months. The inoculum was added in amounts of 15 ml per plant.

Experiment 1 was divided into four treatments: 1 - control, 2 - plants inoculated with AM fungi, 3 - plants inoculated with Frankia, 4 - plants inoculated with both AM fungi and Frankia. All plants were supplemented with mycorrhiza inoculum filtrate (100 g of inoculum/l of distilled water; 5 ml per plant) with saprophytic microflora from mycorrhizal inoculum. Each treatment was divided into six nutrient subtreatments: A - control, B - ammonia, C - nitrate, D - phosphorus, E - ammonia and phosphorus, F - nitrate and phosphorus. Nutrients were added in amounts of 50 ml per pot twice a month in the following forms and concentrations: KNO3 650 mg/I, (NH4)2CO3 50 mg/l and KH2PO4 17 mg/l. The nutrient concentrations were chosen according to the long term measurement of the surface water (POKORNY et al. 1994). Nutrient treatments started in the 12th week of cultivation. The seedlings (5 for each subtreatment) were harvested after 18 and 23 weeks and the following growth parameters were evaluated: stem height, root collar diameter, root and shoot dry weight and total leaf area (TLA). Biomass was determined after drying at 90 ?C and TLA was

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Root symbioses of Alnus glutinosa 157

Table 3. ANOVA's for Alnus glutinosa seedlings - experiment 2, harvest 1. ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001.

Treatment Stem height (F) Total biomass (F) d.f.

Site (A) 5 5.7 *** 3.7 ** Nutrients (B) 1 0.0 ns 4.6 ns AxB 5 1.9ns 2.8ns Error d.f. 48 41

measured with a light areameter (LI-3 100, LI-COR, inc., Lincoln, Nebraska, USA). Actinorhizal nodules were excised and their dry biomass weighed. Roots were stained with 0.05% trypan blue in lactophenol (PHILLIPS & HAYMAN 1970) and mycorrhizal colonization was

estimated by the segment method (GIOVANNETrI & MOSSE 1980). In experiment 2, we tested the effect of rhizosphere soil microflora from alder trees with

different degrees of damage from six field sites. The soil was taken from five trees on each plot, mixed (for each plot separately) and 15 ml of it was added to each container. The experiment was divided into two nutrient treatments: 1 - control and 2 - ammonia, nitrate and phosphorus application. The nutrients were applied in the same amounts and forms as in experiment 1. Plants were harvested after 18 and 23 weeks (5 plants per subtreatment) and basic growth parameters were evaluated, as described in experiment 1.

The data were analyzed by ANOVA (with Frankia, mycorrhiza, phosphorus and nitrogen as factors in the experiment 1 and field site and treatment as factors in the experiment 2) and Duncan's test. The program SOLO (BMDP software) was used. Significance level was set at P < 0.05 (Tabs. 2 and 3). The data were summarized over nitrogen and/or phosphorus treatments in case these were not significant.

RESULTS

The data from experiment 1 showed that both symbionts significantly increased the growth of plants compared to the uninoculated plants (Tab. 2). Plants inoculated with both symbionts separately or together were higher (Fig. 1) and had greater dry biomass (Fig. 2). Measured parameters not presented showed a similar distribution. Dual inoculation showed synergistic effect on stem height at the second harvest. The most pronounced differences were found at the first harvest where the seedlings in all inoculated treatments were 300 - 380% higher and had 340 - 450% greater dry biomass compared to the control plants. These differences decreased by the second harvest where the stem height of inoculated plants was 170 - 240% and their dry biomass 120 - 170% of that of the control plants (Figs. 1 and 2). Application of nutrients did not show any effect on the growth of the seedlings.

The dry weight of Frankia nodules was significantly higher in the plants inoculated with both Frankia and mycorrhizal fungi compared to plants inoculated with Frankia only (Fig. 4). Mycorrhizal colonization was lower in the roots of plants supplemented with phosphorus, but only if both Frankia and mycorrhiza were present. Nitrogen supplemented plants had greater mycorrhizal colonization in case of Frankia presence and phosphorus absence (Fig. 3).

In experiment 2, the growth of seedlings was enhanced by inoculation with rhizosphere soil from the field sites, compared to the control plants. The reaction of the seedlings inoculated with the rhizosphere soil collected on different sites was different (Figs. 5 and 6). Alder seedlings inoculated with soil from Site E (tejnuv mlyn) showed the most reduced growth

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158 S. Strukova et al.

stem height [cml 100

C

80a

a Y

60 b,

40

20 C

CONTHOL FRANKIA MYCORRHIZA FRAN. +MYC.

| harvest 1 E2harvest 2

Fig. 1. The effect of Frankia and mycorrhiza inoculation (experiment 1) on stem height. Columns marked with the same letters are not significantly different. Harvest I and harvest 2 were tested separately (a-d for harvest 1, x-z for harvest 2). There were 30 replicates per one inoculation treatment when summarized over all nutrient subtreatments.

total biomass [g] 12

a x

10

8 Y ab

b 6

2 C . .

0 CONTROL FRANKIA MYCORRHIZA FRAN. + MYC.

*harvest 1 0harvest 2

Fig. 2. The effect of Frankia and mycorrhiza inoculation (experiment 1) on the total dry biomass. Columns marked with the same letters are not significantly different. Harvest I and harvest 2 were tested separately (a-d for harvest 1, x-z for harvest 2). There were 30 replicates per one inoculation treatment when summarized over all nutrient subtreatments.

compared to the others. Decay of the seedlings root systems was also observed in this treatment. No significant differences in the growth of seedlings with different nutrient treatment were found (Tab. 3).

DISCUSSION

Both AM fungi and Frankia significantly affect alder growth; this is in agreement with the results of other studies (GARDNER et al. 1984, JHA et al. 1993, CHATARPAUL 1989). Unlike these studies, which showed a synergistic effect of both organisms, alder growth was enhanced only to a limited extent. Plants inoculated with both organisms did not grow significantly more than those inoculated with either mycorrhiza or Frankia separately, with stem height measured at the second harvest as the exception. The fact that control plants grew faster than the inoculated plants between the first and second harvest was possibly caused by growth limitation of larger inoculated plants in the containers and/or by Frankia contamination, which occurred on some control plants during the second half of the experiment.

Mycorrhizal infection was lower in nodulated plants supplemented with phosphorus even though neither dual inoculation nor phosphorus treatment itself had any effect (Fig. 3). This result is not in agreement with the results of JHA et al. (1993), CHATARPAUL et al. (1989), GARDNER et al. (1984) and ROSE & YOUNBERG (1980) who observed increased mycorrhizal colonization in the nodulating plants. Our data indicate that there may be competition between symbionts on the

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Root symbioses of Alnus glutinosa 159

mycorrhizal Infection 1%

100I

a

bc

cd

20

MYCOR. MYCOR. +P FRANKIA FRAN.+P

I VIControi RNH4+ ON03-

Fig. 3. The effect of Frankia inoculation and N and P treatments on the mycorrhizal colonization of alder roots. Columns represents means from analysis of variance; 5 replicates per one column. Columns marked with the same letters are not significantly different.

nodule weight (g) 50 50

40 b 40

30 . .. 30

20 . . . . . . . . . .20 a

10 10

0 0 FRANKIA FRAN. + MYC.

Fig. 4. Effect of mycorrhizal inoculation on the dry biomass of Frankia nodules. Columns represents means over all nutrient subtreatments. 24 or 29 replicates per one inoculation treatment. Columns marked with the same letters are not significantly different.

roots, probably for assimilates. This competition occurs only when P level is increased which points to the possibility that plant regulation is also involved in the control of mycorrhizal development. Increase of the dry weight of nodules in the mycorrhizal plants was found in experiment 1 (Fig. 4), which confirms the results of the authors cited above, but nitrogenase activity measured as acetylene reduction (data not presented here) was lower in the mycorrhizal plants, which also suggests competition between symbionts. Similar results (higher fresh weight of nodules together with lower acetylene reduction) were obtained by Russo (1989) on plants grown with a medium level of P applied. However, in our experiment no difference in the nodule weight or acetylene reduction between P treated and P untreated plants was observed. The application of N had no effect on the development of actinorhiza in the forms and concentrations used in our experiment, even though nodulation suppression by supplied potassium nitrate was reported by KOHLS & BAKER (1989) and decrease of nitrogenase activity and vesicle damage after addition of ammonia was observed by HUSS-DANELL et al. (1982). The stimulation of mycorrhiza was observed in the plants which were supplied with N both from nitrogen fixation and experimental treatment and at the same time not supplied with

phosphorus (Fig. 3). The possible interpretation of this fact can be that the plant keeps the balance between P and N uptake through the regulation of mycorrhizal infection.

The alders in experiment 2 showed a different growth response. Because the amount of

soil added was very small compared to the container volume, we can attribute the effect to

soil organisms and neglect the effect of nutrients in the added inoculum. However, the

differences between treatments are not usually very distinct (except treatment E) which may

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160 S. Strukova et al.

stem height [cm] 100

a x ab

b ba

60

40 .... ...y .... y..-

20.C

0 A B C D E F CONTR.

field sites

I *harvest 1I 3 harvest 2

Fig. 5. The effect of inoculation with rhizosphere soil on the height of stem. Soil collected on the sites with different degrees of alder damage (experiment 2). Columns marked with the same letters are not significantly different. Harvest 1 and harvest 2 were tested separately (a-d for harvest 1, x-z for harvest 2). There were 10 replicates per one treatment when summarized over nutrient subtreatment. Contr. means control (uninoculated) plants (average from 30 values).

total biomass [g] 14

X X 12

10 -a .. X a

y 6

4 .. . . . . . .

2 b

0 A B C D E F CONT.

field sites

U harvest 1 0 harvest 2

Fig. 6. The effect of inoculation with rhizosphere soil on the total biomass. Soil collected on the sites with different degrees of alder damage (experiment 2). Columns marked with the same letters are not significantly different. Harvest 1 and harvest 2 were tested separately (a-d for harvest 1, x-z for harvest 2). There were 7 - 1O replicates per one treatment when summarized over nutrient subtreatment. Cont. means control (uninoculated) plants (average from 30 values).

be the result of mixing the soil from the trees on each site. Each site contains at least one relatively healthy (degree 4) tree. To avoid this, soil samples from the separate trees should be tested. Root decay in alders inoculated by soil from plot E occurred, indicating the presence of some root pathogen. These alders also show significantly less vigorous growth at the end of the experiment. We do not know whether the response to rhizosphere soil inoculation is connected with the presence of symbiotic organisms or if it is caused by some other compound of the rhizosphere microflora; quantification of the symbionts and their correlation to plant growth is thus necessary. As in experiment 1, nutrient application did not show any effect on the growth of seedlings.

The data presented in this article indicate that both mycorrhiza and actinorhiza markedly enhanced alder seedling growth. Seedlings positively reacted to the rhizosphere soil microflora collected under alder trees, even though no correlation was found between growth response and degree of damage of the trees on the site. Competition between symbionts occurred, influenced by phosphorus treatment. It can be concluded that even quite low nutrient concentration which does not have any effect on plant growth can influence the interaction of the symbionts on the roots, which might, with respect to the multiple function of the symbioses, have some impact on the health status of the host tree. The efficiency of both symbionts has also been estimated in experiment 1, using specific physiological probes e.g. nitrogenase activity of AR

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Root symbioses of Alnus glutinosa 161

measured as the potential of nodules on the roots to reduce acethylene, and for mycorrhiza the metabolic dehydrogenase activity of extraradical fungal structures (data not presented here). To highlight the role of root symbioses in alder growth, with respect to the effects of eutrophication, a long-term experiment is needed using a wider range of nutrients concentration. For field sampling of the rhizosphere soil from mature trees it seems to be necessary to sample each tree separately and when possible several times during the growing season.

Acknowledgement: This work was supported from Grant Agency of the Czech Republic (No. 206/94/1821 - Wetlands and their ecological role and efficiency evaluation of artificial wetlands in waste water treatment). Authors would like to thank to Mrs J. Rouckova for technical assistance, Dr M. Gryndler for consultation during the work.

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