Occurrence of Alnus-Infective Frankia and Trifolium-Infective Rhizobium in Circumpolar Soils

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Occurrence of Alnus-Infective Frankia and Trifolium-Infective Rhizobium in CircumpolarSoilsAuthor(s): Kerstin Huss-Danell, Halldor Sverrisson, Ann-Sofi Hahlin and Kjell DanellSource: Arctic, Antarctic, and Alpine Research, Vol. 31, No. 4 (Nov., 1999), pp. 400-406Published by: INSTAAR, University of ColoradoStable URL: http://www.jstor.org/stable/1552588 .

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Arctic, Antarctic, and Alpine Research, Vol. 31, No. 4, 1999, pp. 400-406

Occurrence of Alnus-infective Frankia and Trifoliuminfective Rhizobium in Circumpolar Soils

Kerstin Huss-Danell,* Halldor Sverrisson, t Ann-Sofi Hahlin, * and

Kjell Danellt *Department of Agricultural Research for Northern Sweden, Crop Science Section, Swedish University of Agricultural Sciences, S-904 03 Umea, Sweden. Kerstin.Huss-Danell @njv.slu.se tAgricultural Research Institute, Keldnaholt, IS- 112 Reykjavik, Iceland. tDepartment of Animal Ecology, Swedish University of Agricultural Sciences, S-901 83 Umea, Sweden.

Abstract A survey was made of the occurrence of Frankia, infective on Alnus, in some 40 soils from the whole circumpolar area. Some of these soils were also tested for the occurrence of Rhizobium infective on Trifolium pratense. Infectivity tests were performed by growing test seedlings in soil or soil suspensions. Frankia was detected only in very few soils, in spite of extended experimental periods. When nodulation took place, nodulation was observed in few test plants. Several of nodulated test seedlings never turned green, suggesting that Frankia was ineffective in N2 fixation. An exception was soil from a site in the Faeroe Islands where nodulated Alnus had been introduced. This soil showed high nodulation ability and N2 fixation was likely. It is suggested that lack of infective Frankia in the circumpolar soils studied may be because Frankia had not been spread to these sites, but does not necessarily mean that soil conditions are negative for Frankia. Infective Rhizobium was rare in the soils studied. Lack of infective root nodule bacteria in potential sites for soil reclamation calls for the need to inoculate the plants and also provides the opportunity for introduction of selected bacterial strains without competition from an endogeneous soil microflora.

Introduction Actinorhizal plants form N2-fixing root nodules upon infec-

tion by Frankia sp. (Huss-Danell, 1997). Nearly all actinorhizal plants are woody plants and comprise species that often occur in early stages of natural vegetation successions. Because of these characteristics, actinorhizal plants, in particular alders (Al- nus spp.), are of potential use in soil reclamation (Wheeler and Miller, 1990). The natural distribution of Alnus is limited in ex- treme northern areas (Hult6n, 1990), but several species have, however, been introduced in Iceland and the Faeroe Islands. Frankia infective on Alnus are frequently found in soil within the natural distribution of Alnus and also at sites lacking host plants (Huss-Danell, 1997). Information on occurrence of Fran- kia outside the natural distribution of Alnus is scarce but has importance for the handling of plants when trying to establish the symbiosis at new sites. The present study was initially focussed on the occurrence of infective Frankia in soils from Ice- land and the Faeroe Islands; it was then extended to include sites from the whole circumpolar area. Occurence of Alnus-infective Frankia was the main question, but some of the soils also were tested for presence of infective Rhizobium leguminosarum bv. trifolii using Trifolium pratense as host plant. To indicate infective Frankia and Rhizobium, infectivity tests rather than molecular methods were used.

Material and Methods SOIL SAMPLES

The sampling program comprised one site in Spitsbergen, 18 in Siberia, five in Alaska, three in the Northwest Territories, six in Iceland, and two in the Faeroe Islands (Table 1). On each site vegetation and land use was recorded. In particular, presence of the actinorhizal genera Alnus, Myrica, Hippophae, and Dryas

400 / ARCTIC, ANTARCTIC, AND ALPINE RESEARCH

as well as Trifolium or any other legumes was recorded (Table 1). The genus Dryas is here designated as actinorhizal, even though only D. drummondii has been shown with certainty to be actinorhizal (Baker and Schwintzer, 1990). At each sampling point vegetation was removed and 1 to 2 L of soil were collected with a sterilized small spade and put into new plastic bags. Any stones or coarse roots were removed. To minimize contamination new disposable gloves were used. Samples were kept cold for up to 4 mo until analyzed.

PLANT MATERIAL FOR INFECTIVITY TESTS

Seeds of Alnus glutinosa (L.) Gaertn. were collected from one stand at Elsegem, Belgium, in 1990. Seeds of Alnus incana (L.) Moench. were half-siblings as the "A. incana II" described in Huss-Danell (1991). Seeds of Alnus sinuata Rydb. were from Skagway, Alaska, U.S.A. Seeds of Alnus rubra Bong were collected in 1988 from seed zone 042, elevation 300-450 m, and were purchased from Brown Seed Co., Vancouver, Washington, U.S.A. Seeds of Myrica gale L. were collected locally (Bygdea, Vasterbotten; KHD) in September 1994. Seeds of Trifolium pratense L. cv. Bjursele were obtained from Svalof-Weibull AB, Umea, Sweden.

ASEPTIC CONDITIONS

All seeds were surface sterilized by shaking for 15 min in 30% H202 and then rinsed four times for 5 to 10 min each in sterile distilled water. Throughout the experiment, new disposable gloves were used, and all solutions, substrates, tools, and glassware were sterilized.

INFECTIVITY TESTS

Plant species and growth systems during the tests as well as experimental times are summarized in Table 2.

? 1999 Regents of the University of Colorado 1523-0430/99 $7.00



Samples from Spitsbergen

Infective Frankia was first tested on seedlings grown in a hydroponics system in tubes with nutrient solutions and other growth conditions as described by Huss-Danell and Myrold (1994). The test plants were in a growth chamber with 17 h light at 25?C and darkness at 15?C. The photosynthetic photon flux was about 200 timol m-2 s-1 provided by Osram Power Star HQ-T 400 W lamps. Seeds were germinated in petri dishes containing perlite moistened with Evans solution as used in Huss- Danell and Myrold (1994) supplemented with ammonium nitrate to 10 mg N L-~. Duplicate dilution series were prepared from the soil available. Soil suspensions in water were diluted in six steps to contain 0.031-1.0 g FW (0.024-0.76 g DW) soil per test plant. There were four seedlings of each Alnus species for each dilution step. Negative and positive controls were as used in Huss-Danell and Myrold (1994).

A second test for infective Frankia was done by growing seedlings in soil cores kept in glass tubes (35 mm diameter, 195 mm deep). Gravel (2-5 mm) was added to make a 15 mm layer in the bottom of the tubes which were then capped with cellulose stoppers and autoclaved. Soil was added to about 50 mm depth on top of the gravel and seeds were added to the soil. Negative controls, to detect any contaminations from handling the tubes and plants, consisted of sterilized perlite instead of soil in the tubes. Positive controls, to verify that the growth conditions would permit nodulation, contained soil samples as above plus nodule homogenate of "the local source of Frankia" (Huss-Da- nell, 1991) added to the soil. Tubes with soil were given water only. Negative controls were watered with Evans solution (Huss- Danell and Myrold, 1994) supplemented with 10 mg N L-1 as ammonium nitrate. The lower part of glass tubes was covered with aluminium foil to keep soil and roots darkened. Tubes were kept in a greenhouse with supplemental lights for 17 h per day and at a temperature of 25?C in day and 15?C at night. Trays carrying the tubes were regularly moved on the greenhouse benches to reduce any differences in light availability.

Samples from Siberia

Infectivity tests were done in soil cores kept in glass tubes, as described for Spitsbergen (above). Positive controls were as above or consisted of Rhizobium leguminosarum bv. trifolii added to tubes with T. pratense. Frankia or Rhizobium was added 4 wk after planting the seeds into the tubes. The Rhizobium inoculum was a mix of strains 2080, 2098, and 2123 obtained from Baljvaxtlaboratoriet, Uppsala, Sweden. These strains were originally isolated from Abisko in northern Sweden (Ljunggren, pers. comm., 1994). Seeds were planted into the soil and thinned to one seedling per tube 2 wk later. Experimental time is counted from the thinning of seedlings.

Samples from Alaska, Northwest Territories, and the Iceland site at Hafnarffjr6our

Infectivity tests were performed in soil cores in glass tubes as described for Spitsbergen (above).

Samples from the Iceland Sites at Mogilsd

Soil was mixed with sterile perlite and put into ethanol- rinsed new pots (8 x 8 cm wide, 8 cm deep). Pots containing the same soil were kept together on carefully cleaned and ethanol-rinsed trays. Negative controls contained sterile perlite in the pot. Pots with single seedlings were watered with the Evans

solution supplemented with N (see above) and kept in the growth chamber (see above). Positive controls had nodule homogenate of "the local source of Frankia" (Huss-Danell, 1991) added 1 wk after planting the seedlings into pots. Following the first examination for nodules, after 8 wk, the nutrient solution was used without N until final examination 18 wk after planting. At examination of plants the content of a pot was carefully poured out and roots were inspected for nodules. Seedlings were then replanted into the same pot and substrate.

Samples from the Iceland Sites at Geysir

The samples 32 and 35 were studied in pots as described for Mogilsa (above). The samples 33 and 36 were first studied in a hydroponics system as described for Spitsbergen (above). Each seedling received 0.004-0.125 g FW (corresponding to 0.002-0.07 g DW) soil. All species grew in the Evans solution. A second test of samples 33 and 36 was performed as above but with twice the amount of inoculum. Alnus incana was grown in Evans nutrient solution and A. rubra was in the Hoagland-type solution as described in Huss-Danell and Myrold (1994). A third test of samples 33 and 36 and tests of samples 34 and 37 were performed in soil cores kept in glass tubes as described for Spits- bergen (above).

Samples from the Faeroe Islands

Presence of infective Frankia was first studied in a hydroponics growth system as described for Spitsbergen (above). Sus- pensions containing 0.03-1.0 g FW (corresponding to 0.012- 0.40 g DW) soil were added to each plant. A second test was performed in soil cores kept in glass tubes as described for Spits- bergen (above).

Results Table 2 summarizes the nodulation observed on test plants

as well as on control plants. The negative controls never formed nodules, and their leaves were yellowish or pale green.

Spitsbergen

None of the A. incana or the A. rubra seedlings grown in hydroponics had formed nodules 16 and 10 wk after inoculation, respectively. All positive controls of A. rubra were nodulated after 10 wk. In the second test, with plants growing in soil cores in glass tubes, none of the A. incana seedlings had formed nodules, and this was the case still when the seedlings had been kept for half a year.

Siberia

After 15 wk there were nodules only in three tubes with A. incana growing in soil no. 2. These plants had green leaves. No other soils gave nodules on A. incana and no soils gave nodules on M. gale or T. pratense. All positive controls of all species were nodulated.

Alaska and the Northwest Territories

Two of the five Alaska soils gave nodules. Soil no. 21 gave one single nodulated seedling of A. incana; soil no. 23 gave two nodulated seedlings of A. incana (in different tubes) and one nodulated seedling of T. pratense. All of the positive controls formed nodules. With the exception of one single plant of T.

K. HUSS-DANELL ET AL. / 401



TABLE 1

Description of sites and collection of soil samples. Site numbers in Siberia refer to sites used by the Russian-Swedish Tundra Ecology Expedition 1994 (Goryachkin et al., 1994). Any known land use and any noticed actinorhizal plants or legumes are

indicated. Dates are given as year-month-day

Sample Location, site characteristics, sampling date, collector

Spitsbergen:

1 5 km SE of Longyearbyn, Svalbard, Norway, 78?2'N, 15?9'E, <25 m a.s.l. No trees or shrubs. Carex sp., Alopecurus arcticus, Poa

alpigena, Oxyria digyna, Cerastium sp., Papaver dahlianum, Ra-

nunculus sp., Petasites frigidus. Relatively wet. Composite sample from several spots within 4 m2 at 0-0.15 m depth. Permafrost below this depth. 92-07-25, S Blixt

Siberia:

2 Site 1. Kachkovsky Bay, Kola Peninsula, 67?08'N, 40?55'E, 180 m

a.s.l. Sparsely spaced Betula pubescens and Juniperus shrubs, dwarf shrubs, patches of lichens. Composite sample from six

spots within 0.5 X 0.5 m at 0-0.15 m depth. 94-06-10, K Danell, E-B Olofsson

3 Site 2. NE Kanin Peninsula, 68?20'N, 45?14'E, 60 m a.s.l. South-

facing slope, Salix spp., Verratum album, Solidago sp., Pyrola sp., Deschampsia flexuosa, grasses, Vaccinium myrtillus, Polytri- chum. Composite sample from three spots within 1 x 1 m at 0- 0.2 m depth. 94-06-12, KD, EBO

4 Site 3. Kolguyev Island, 69?08'N, 49?23'E, 60 m a.s.l. Slope facing river, at small creek. Grasses (Festuca and others), Empetrum sp., mosses. Dryas sp. within 500 m distance. Composite sample from

four spots within 1 x I m at 0.02-0.2 m depth. 94-06-14, KD, EBO

5 Site 4. Pechora Bay, 68?46'N, 53?04'E, 5 m a.s.l. Wet tundra on sand bar near the sea. Empetrum sp., Eriophorum sp., Rubus cha-

maemorus, mosses, Cladonia sp. Composite sample from three

spots within 1 x 1 m at 0.02-0.15 m depth. 94-06-16, KD, EBO

6 Site 5. W Yamal Peninsula, 70?25'N, 68?02'E, 60 m a.s.l. Grass tun-

dra near lake. Salix lanata, grasses. Composite sample from three

spots within 1 x 1 m at 0.02-0.2 m depth. 94-06-18, KD, EBO 7 Site 7. Arctic Institute Island, 75?21'N, 82?01'E, 15 m a.s.l. Tundra,

close to the sea. Composite sample from three spots within 1 x 1 m at 0-0.2 m depth. 94-06-23, KD

8 Site 8. NW Taymyr Peninsula, 76?00'N, 93?E, 300 m a.s.l. Slope to river. Sedge tundra with mosses. Composite sample from three

spots within 1 x 1 m at 0.02-0.15 m depth. 94-06-24, KD, EBO 9 Site 9. Chelyuskin Peninsula, 77?05'N, 102?13'E, 90 m a.s.l. Polar

desert. Slight slope to river. Carex eusifolia, Polytrichum sp., Ra- comitrium sp., Cladina sp., Cetraria sp. Composite sample from three spots within 1 x 1 m at 0.02-0.15 m depth. 94-06-28, KD, EBO

10 Site 10. NE Taymyr Peninsula, 76?26'N, 111?16'E, 150 m a.s.l.

Sedge tundra with mosses. About 100 m from Dryas sp. Compos- ite sample from three spots within 1 x 1 m at 0.02-0.2 m depth. 94-06-30, KD, EBO

11 Site 11. Olenekskiy Bay, 73?19'N, 117?00'E, 20 m a.s.l. Wet grass tundra with Carex spp., Eriophorum sp., grasses and mosses. Nearest Dryas sp. at 15 m distance. Oxytropis nigrescens the only legume noticed. Composite sample from three spots within 3 x 3 m at 0-0.2 m depth. 94-07-06, EBO

12 Site 13A. New Siberian Islands: Belkovsky, 75?34'N, 135?38'E, 400 m a.s.l. Poorly vegetated patch, close to snowfield. Composite sample from three spots within 1 x 1 m at 0-0.1 m depth. 94-07-

09, EBO 13 Site 13B. New Siberian Island, 74?49'N, 138?42'E. Tundra. Salix po-

laris, mosses, lichens. Composite sample from three spots within I x I m at 0-0.1 m depth. 94-08-02, EBO

14 Site 12. Yana Delta, 72?25'N, 139?33'E. Tussock tundra. Salix po-

laris, grasses, Carex spp., Eriophorum sp. Nearest Dryas sp. at 500 m distance. Composite sample from three spots within 1 x 1 m at 0-0.12 m depth. 94-08-06, EBO

TABLE 1

(Cont.)


15 Site 13. New Siberian Islands: Faddeevsky, 75?29'N, 143?14'E, 200 m a.s.l. Site extremely influenced by frost upheaval. Grasses, mosses and lichens. Composite sample from three spots within I x 1 m at 0-0.15 m depth. 94-07-11, EBO

16 Site 14. Lopatka Peninsula, NW Indigirka, 72?1 1'N, 148?26'E, 20 m a.s.l. Tundra. Carex spp., Eriophorum sp., mosses. Nearest Dryas sp. at 500 m distance. Composite sample from three spots within 1 x 1 m at 0-0.12 m depth. 94-07-15, EBO

17 Site 15. NE Kolyma Delta, 69?32'N, 160?43'E, 60 m a.s.l. Tundra with mosses. Nearest Dryas sp. at 150 m distance. Nearest le-

gume at 1 km distance. Composite sample from three spots within 1 x 1 m at 0-0.1 m depth. Permafrost below this depth. 94-07-

19, EBO 18 Site 16. Ayon Island, 69?48'N, 168?37'E, 50 m a.s.l. Tundra. Salix

reptans, Dryas sp., grasses. Nearest legume at 200 m distance.

Heavily grazed by reindeer. Composite sample from three spots within 1 x 1 m at 0-0.1 m depth. 94-07-20, EBO

19 Site 17. SW Wrangel Island, 71?02'N, 179?11'E, 80 m a.s.l. Tundra. Carex spp., mosses, lichens. Nearest Dryas sp. at 500 m distance.

Composite sample from three spots within 1 x 1 m at 0-0.15 m

depth. 94-07-25, EBO

Alaska:

20 Coldfoot-Yukon River, 66?37'N, 150?40'W. Coniferous forest. Picea

mariana, Alnus crispa, Ledum sp., Rubus chamaemorus, Betula

glandulosa, thick moss layer, Sphagnum, Peltigera, Cladina. Al- nus crispa at sampling points. Composite sample from four points within 5 x 5 m at 0.01-0.1 m depth. 95-08-04, KD

21 N of Coldfoot, 67?31'N, 149?50'W. Thin forest stand of Picea mariana. Betula glandifolia, Salix spp., Potentilla spp., Shepherdia canadensis, Ledum sp., Dryas sp., Vaccinium spp., mosses. Near- est Alnus at about 1 km distance. Composite sample from four

spots within 3 x 3 m at 0.01-0.1 m depth. 95-08-04, KD 22 Toolik-Atigun Pass, 68?24'N, 149?19'W, 780 m a.s.l. Shore of creek.

Salix spp., Dryas sp., Shepherdia canadensis, Epilobium latifol- ium. Several nonidentified legumes. Composite sample from four

spots within 2 x 2 m at 0.01-0.1 m depth. 95-08-03, KD 23 Deadhorse-Toolik, 69?15'N, 148?45'W. Herb-rich tundra. Salix spp.,

Lupinus sp., Dryas sp., Carex sp., mosses. Collection site 50 m from Dalton Highway. Composite sample from four spots within 2 x 2 m at 0.01-0.1 m depth. 95-08-03, KD

24 Deadhorse at Prudhoe Bay, 70?10'N, 148?26'W, 10 m a.s.l. Sedge- tundra with Dryas sp. Several nonidentified legumes. Composite sample from five spots within 2 x 2 m at 0.01-0.15 m depth. 95-

08-03, KD

Northwest Territories:

25 Kent Peninsula, Cockbum Island, 68?05'N, 108?18'W, 60 m a.s.l.

Sedge-tundra with Salix lanata, Dryas integrifolia, Pedicularis lanata. Composite sample from four spots within 5 x 5 m at 0.01-0.1 m depth. 95-06-17, KD

26 Kent Peninsula, Walker Bay Camp, 68?21'N, 108?05'W, 10 m a.s.l. Tundra. At margin of palsa. Salix arctica, Dryas integrifolia, Car- ex spp., Eriophorum sp., grasses, Cetraria spp. Composite sample from three spots within 2 x 2 m at 0.01-0.08 m depth. 95-06-19, KD

27 Cambridge Bay, 69?06'N, 105?10'W, 10 m a.s.l. Wet sedge-tundra with Salix lanata, Dryas integrifolia, Saxifraga oppositifolia. Gen- tle slope to sea. Composite sample from four spots within 2 x 2 m at 0.01-0.05 m depth. 95-06-20, KD

Iceland:

28 Mogilsa, 13 km NE Reykjavik, 64?8'N, 21?27'W. South-facing slope of Esja Mountain above planted forest, close to small creek. An- thoxanthum odoratum, Equisetum spp., Galium boreale, G. nor- manii, Festuca sp., mosses. Composite sample from three spots within 2 X 2 m at 0-0.1 m depth. 91-06-27, H Sverrisson, K Huss-Danell




TABLE 1

(Cont.)


29 Mogilsa, 13 km NE Reykjavik, 64?8'N, 21?27'W, below no. 28.

South-facing slope above birch plantations. Vegetation and sample as for sample 28. 91-06-27, HS, KHD

30 Mogilsa, 13 km NE Reykjavik, 64?8'N, 21?27'W. SE of plantations.

Vegetation and sample as for sample 28. 91-06-27, HS, KHD

31 Hafnarfjor6our, Hvaleyrarholt, 64?05'N, 21?59'W, 20 m a.s.l. Grass-

land with Armeria maritima, Taraxacum sp., Rubus saxatilis, Al-

chemilla alpina, Polygonum sp., Equisetum arvense, Oxyria sp.,

Thymus sp., Galium sp., Festuca ovina. Trifolium repens and the

non-nodulated Dryas octopetala are common in this part of Ice-

land. Horse grazing, close to gravel road. Earthworms noticed.

Single sample from 0-0.15 m depth. 95-07-01, KHD

32 3 km NE of Geysir, Haukadalur, 64?20'N, 20?15'W, about 300 m

a.s.l. Hill slope with no sheep grazing. Eroded soil with stones,

Empetrum sp., grasses, Alchemilla alpina, mosses. Gravelly loam

soil. Example of land where plantation of Alnus is desirable.

Composite sample from four spots within 3 x 3 m at 0-0.1 m

depth. 91-10-24, HS

33 As for sample 32. 92-05-24, HS

34 As for sample 32. 92-10-28, HS

35 3 km NE of Geysir, Haukadalur, 64?20'N, 20?15'W, about 200 m

a.s.l. Hill slope with no sheep grazing. Betula pubescens and Salix

phylicifolia shrubs, sward with grasses and mosses. Example of

land where plantation of Alnus is desirable. Composite sample from three spots within 2 X 2 m at 0-0.2 m depth. 91-10-24, HS

36 As for sample 35. 92-05-24, HS

37 As for sample 35. 92-10-28, HS

Faeroe Islands:

38 Torshavn, 62?N, 6?47'W, 40 m a.s.l. Section 12d in the planted Larix

forest in Torshavn. Agrostis sp., Festuca sp., Deschampsia sp., Holcus sp., Alnus, and Vicia sp. within 100 m from sampling

points. Composite sample from five spots. 92-10-27, T Leivsson

39 Rangabotnur, Su6uroy, 61?33'N, 6?53'W, 170 m a.s.l. Far from set-

tlements. Grass heath (Tukhanen 1987 p. 114). Sheep grazed.

Composite sample from six spots 10 m apart, three of them from

eroded soil and three from under vegetation. 92-10-22, TL

pratense (soil number 27), none of the three soils from the Northwest Territories gave nodules on test plants.

Iceland

All three soils from Mogilsai induced nodules on A. incana. In sample 28 nodulation frequency increased from 17 to 37% and in sample 29 it increased from 0 to 16% during the period 8 to 18 weeks of plant growth. In sample 30 only a single plant was nodulated. Nearly all positive controls formed nodules in all three soils.

No nodules were obtained on A. incana whereas seedlings of T. pratense (in one tube) formed nodules in the Hafnarfjor6ur soil. Positive controls of both species were all nodulated.

After 10 wk 13% of A. glutinosa and 2% of A. incana had nodules but none of the A. sinuata were nodulated in pots with

Geysir soil (no. 32). Positive controls comprised A. incana and

nearly all of them were nodulated. A similar result was obtained for soil no. 35 with 17% of A. glutinosa and 5% of A. incana

being nodulated. None of the A. sinuata seedlings were nodulated. Almost all positive controls (A. incana) were nodulated.

When tested in hydroponics none of the three Alnus species formed nodules after inoculation with the soils 33 and 36. All

positive controls were nodulated. In the second test of soil no.

33 one single plant of A. rubra, receiving the heaviest inoculum, formed nodules. None of A. incana became nodulated in spite of the very long duration of the test. All of the positive controls

were nodulated. The third test of soils 33 and 36 was done in

soil cores in glass tubes. None of the seedlings formed nodules still after 6 mo.

The soils 34 and 37 from Geysir were kept for 6 mo in

glass tubes and gave rise to a few nodulated seedlings. Soil 37 formed nodules on 10% of the seedlings and soil 34 gave nodules on two seedlings. In spite of the very long cultivation time, nodules were small, less than 1 mm, and the seedlings remained small and had yellow to pale green leaves.

Faeroe Islands

In the first test, in hydroponics, all seedlings of A. incana and all seedlings of A. rubra were nodulated by Torshavn soil. No seedlings were nodulated by Su6uroy soil. In the second test, in soil cores in glass tubes, all seedlings in soil from Torshavn were nodulated. After 6 mo 25% of seedlings in Su6uroy soil were nodulated. All seedlings in soil from Torshavn were green and on average more than 10 cm high. These plants had 4 to 9 nodules each and nodules were 1 to 3 mm in size. Seedlings in soil from Su6uroy were yellow to pale green and their height was on average only 2 cm. They had 1 to 4 nodules per plant with a size of about 1. mm.

Discussion The presence of Frankia in a soil can be demonstrated by

two principally different methods: infection of host plants or studies of Frankia-DNA extracted from the soil (Picard et al., 1992; Huss-Danell and Myrold, 1994; Myrold and Huss-Danell, 1994; Myrold et al., 1994). Methods for enumerating Frankia in soil by quantification of Frankia-DNA do not necessarily give information on infective Frankia (Myrold and Huss-Danell, 1994). In the few comparisons available, the number of genomic units of Frankia in soil was found to be higher than the number of infective units of Frankia (Myrold and Huss-Danell, 1994;

Myrold et al., 1994). On the other hand, an infectivity test may overlook presence of infective Frankia if the growth conditions are unfavourable for root infection to occur. The present study focussed on occcurrence of infective Frankia and infective Rhi- zobium leguminosarum bv. trifolii, and therefore infectivity tests were used. Tests in the hydroponics system were designed for

quantitative measurements whereas tests in soil cores (glass tubes, pots) served as qualitative tests. Soil without any dilution, as used in the glass tubes, sometimes gave the problem that seeds and spores germinated and the resulting plants and mosses com-

peted with the test seedlings in the tubes. Soil suspensions in

hydroponics eliminated this problem, but dilute suspensions increased the risk of falling below the detection limit when the Frankia populations in the soil were small.

Alnus sp. was used in all soils and T. pratense in some of the soils. For additional information other actinorhizal plants (D. drummondii, Elaeagnus angustifolia, M. gale) were tried but

poor germination and seedling growth prevented use of these

species, except for a few plants of M. gale with Siberia soils. The present results on Frankia are therefore only valid for Alnus- infective Frankia and conclusions about Frankia belonging to other host-specificity groups (Huss-Danell, 1997) cannot be made (Zimpfer et al., 1997). Soils were from sites outside the natural distribution of Alnus (Hulten, 1990). Alnus incana was chosen as the main test species because of great experience of




TABLE 2

Growth conditions and occurrence of nodules on seedlings inoculated with soil. Soil samples are explained in Table 1. Species of test plants are abbreviated: Ag, Alnus glutinosa; Ai, A. incana; Ar, A. rubra; As, A. sinuata; Mg, Myrica gale; Tp, Trifo- lium pratense. Growth systems are abbreviated: H, hydroponics; P, pots; T, glass tubes with soil cores. Time refers to weeks following inoculation. Data on nodulation are expressed as number of nodulated seedlings/number of seedlings tested. NegC, negative controls; PosC, positive controls. - indicates that no

seedlings were available for examination.

Species, Nodulation

growth Time

system (weeks) Test NegC PosC

Ai, H 16 0/48

Ar, H 10 0/48

Ai, T 27 0/75

Ar, H 10

Ai, T 27

Ai, T 15 3/3a

Tp, T 15 0/2

Ai, T 15 0/7

Tp, T 15 0/1

Ai, T 15 0/5

Mg, T 15 -

Tp, T 15 0/3

Ai, T 15 0/7

Ai, T 15 0/5

Mg, T 15 -

Tp, T 15 0/2

Ai, T 15 0/12

Mg, T 15 -

Tp, T 15 0/1

Ai, T 15 0/8

Mg, T 15 0/1

Tp, T 15 0/1

Ai, T 15 0/5

Mg, T 15 -

Tp, T 15 0/2

Ai, T 15 0/7

Mg, T 15 0/1

Tp, T 15 0/2

Ai, T 15 0/4

Tp, T 15 0/2

Ai, T 15 0/2

Tp, T 15 0/2

Ai, T 15 0/3

Mg, T 15 0/2

Tp, T 15 0/2

Ai, T 15 0/8

Tp, T 15 -

Ai, T 15 0/5

Mg, T 15 -

Tp, T 15 0/2

Ai, T 15 0/5

Tp, T 15 0/1

Ai, T 15 0/7

Mg, T 15 -

Tp, T 15 0/2

Ai, T 15 0/10

Mg, T 15 -

Tp, T 15 0/1

Ai, T 15 0/5

Mg, T 15 -

Tp, T 15 -

Ai, T 15

6/6

0/10 0/29

1/1 1/1 1/1 1/1 1/1 1/1 1/1 1/1

2/2 1/1

1/1

2/2

1/1

1/1

2/2

TABLE 2

(Cont.)

Species, Nodulation

growth Time Soil sample system (weeks) Test NegC PosC

NegC

NegC

Alaska:

20 20

21

21 22

22

23

23 24

24

NegC NegC

Mg, T 15

Tp, T 15

Ai, T

Tp, T

Ai, T

Tp, T

Ai, T

Tp, T

Ai, T

Tp, T

Ai, T

Tp, T

Ai, T

Tp, T

0/3

0/3

16 0/37 16 0/35 16 1/35 16 0/59 16 0/45 16 0/44

16 2/12

16 1/51

16 0/22

16 0/30 16

16

2/2 8/8

3/3 2/2

3/3 1/1

2/2

3/3 2/2

1/1

0/16

0/16

Northwest Territories:

25

25

26 26 27

27

NegC

NegC

Iceland:

28

29

30

NegC

1/1 31 1/1 31 1/1 NegC 1/1 NegC 1/1 32 1/1 32

32 1/1 NegC

NegC 1/1 NegC 1/1 33, first test

33, first test 2/2 33, first test 1/1 NegC 1/1

NegC 1/1

NegC 1/1

1/1 1/1

1/1 1/1 2/2 1/1

1/1

1/1

1/1

1/1

0/12

33, second test

33, second test

NegC NegC 33, third test

NegC 34

NegC 35

Ai, T

Tp, T

Ai, T

Tp, T

Ai, T

Tp, T

Ai, T

Tp, T

16 0/19 16 0/46

16 0/51 16 0/66 16 0/11

16 1/25 16

16

Ai, P 8 7/42

18 15/41

Ai, P 8 0/44

18 7/44

Ai, P 8 1/47

18 1/47

Ai, P 8

18

Ai,T 16 0/11

Tp, T 16 7c/20

Ai, T 16

Tp, T 16

Ag, P 10 4/30

Ai, P 10 1/50

As, P 10 0/60

Ag, P 10

Ai, P 10

As, P 10

Ag, H 8 0/24

Ai, H 8 0/24

Ar, H 8 0/24

Ag, H 8

Ai, H 8

Ar, H 8

Ai, H 17 0/24

Ar, H 10 1/24

Ai, H 17

Ar, H 10

Ai, T 27 0/37 Ai

Ai, T Ai

Ag, P

27 2/67

10 5/30

0/2 9/9

3/3 10/10

0/3

12/12 see Alaska see Alaska

8/8

7/8

6/6

0/42 0/8b

3/3 7/7

see Alaska

see Alaska

6/7

0/30

0/60 0/60

5/5 8/8

6/6

0/14

0/16 0/11

6/6 5/5

0/10

0/9

see sample I

see sample I

aBoldface indicates tests with nodulated test plants. b Plants thinned after 7-9 wk. c All nodulated seedlings in the same tube.


Soil sample

Spitsbergen:

NegC

NegC

Siberia:

2 2

3 3 4 4 4

5

6 6 6 7 7 7

8 8 8 9 9 9 10 10 10 11 11

12 12

13 13 13 14 14

15 15 15 16 16 17 17 17 18 18 18 19 19 19

NegC



TABLE 2

(Cont.)

Species, Nodulation growth Time

Soil sample system (weeks) Test NegC PosC

35 Ai, P 10 2/43 7/8

35 As, P 10 0/74 -

NegC

NegC NegC

36, first test

36, first test

36, first test

NegC

NegC

NegC

36, second test

36, second test

NegC

NegC

36, third test

NegC 37

NegC

Faeroe Islands:

38, first test

38, first test

NegC

NegC 38, second test

NegC 39, first test

39, first test

NegC NegC 39, second test

NegC

Ag

Ai

As

Ag, H

Ai, H

Ar, H

Ag, H

8

8 8

0/24 0/24 0/24

Ai, H

Ar, H

Ai, H

Ar, H

Ai

Ar

Ai, T

Ai, T

Ai

Ai, H

Ar, H

Ai, H

Ar, H

Ai, T

Ai

Ai, H

Ar, H

Ai

Ar

Ai, T

Ai

see sample 32

see sample 32

see sample 32

see sample 33, first test



17 0/24

10 0/24

see sample 33, second test

see sample 33, second test

27 0/57

see sample 1 27 5/51

see sample 1

9

9

12

12

27

24/24

24/24

72/72

12 0/24

12 0/24

27 14/55

0/6

0/6

see sample 1

see sample 38 see sample 38

see sample 1

this species in infectivity tests (Arveby and Huss-Danell, 1988; Huss-Danell, 1991; Huss-Danell and Myrold, 1994; Huss-Danell et al., 1997). Within the genus differences among Alnus species have been found in inoculation tests with the extent of nodulation being in the order A. rubra > A. incana > A. glutinosa after 8 wk in hydroponics (Huss-Danell and Myrold, 1994). Out of these three species A. incana was geographically closest to a majority of the sites studied.

A consistent result throughout this study was the very high nodulation frequency of positive controls, i.e. soils with Frankia or Rhizobium added. This indicated that growth conditions during the experiments permitted nodulation to occur in all soils and growth systems and that absence of nodules on test seedlings was the result of low numbers of Frankia or Rhizobium in the soil. The always non-nodulated negative controls and the care taken to avoid contaminations suggest that nodulated test plants were due to infective Frankia (or Rhizobium) in the soil.

In most soils the population of infective Frankia (and Rhi- zobium in those soils that were tested) was very low or not detectable. Lack of detectable Frankia (or Rhizobium) in a soil can be explained by improper soil conditions such as organic

matter, nutrients, water, pH and temperature. Another possible explanation is that Frankia (or Rhizobium) has not been spread to the site. In the present study one of the samples from the Faeroe Islands, Su6uroy (no. 39), gave very poor nodulation whereas the other site, Torshavn (no. 38), always gave good nodulation. In Torshavn Frankia had been introduced when Al- nus was introduced there (Leivsson, pers. comm., 1992) and Frankia was apparently persisting at this site. The infective sample no. 23 from Alaska was collected 50 m from a highway, and spreading of soil microbes by road construction vehicles might be an explanation for presence of Frankia and Rhizobium. Be- sides of spreading by human activities, Frankia can be spread by water (Arveby and Huss-Danell, 1988; Huss-Danell et al., 1997) and by animals (Reddell and Spain, 1991; Paschke and Dawson, 1993; Burleigh and Dawson, 1995), but has not been shown to be wind-borne (Arveby and Huss-Danell, 1988). In many cases Frankia infective on Alnus is present in soils without hosts (see Huss-Danell (1997) for a discussion), but there are exceptions (Arveby and Huss-Danell, 1988). In Iceland, R. leg. trifolii is usually not present in the soil unless inoculated Tri- folium has been grown there (Hardarson and Jones, 1977; Sven- ning et al., 1998).

With the exceptions of the Torshavn sample and some Ice- landic samples, nodulation was sparse when it occurred on test seedlings. Sparse nodulation makes it difficult to distinguish with certainty between a very low population of soil-borne Frankia (or Rhizobium) and contaminations unless a very large number of test plants and negative controls are studied. A few soils were tested more than once and in different growth systems. Although the first tests gave negative results, nodules developed when a more heavy inoculum was used. (Iceland no. 33, second test; Faeroe Islands no. 39). When examinations of root systems were made more than once during a prolonged experiment (Iceland, no. 28-30) the nodulation frequency increased. Proliferation of Frankia in soil planted with host seedlings or a better penetration of the soil volume over time are possible explanations for the results from prolonged tests in soil.

N2-fixing root nodule symbioses have potential use in re- vegetation efforts. It is then crucial that plants are well nodulated and that the nodules are effective in N2 fixation. From the present results it is recommended that, unless the soil is known to have infective Frankia (or Rhizobium), any plantations should be performed with nodulated or inoculated plants to ensure that Fran- kia (or Rhizobium) is available in sufficient numbers in the soil. Inoculation admittedly adds one more step in nursery or planting work, but lack of infective root nodule bacteria in the soil gives the advantage that an introduced Frankia (or Rhizobium) will not have to compete with an indigeneous flora. Such a situation also offers an opportunity to introduce strain(s) with some pre- ferred characteristics.

Small nodules and small plants with pale leaves (Iceland 34 and 37, Faeroe Islands 39) can be a result of either a very recent nodulation so that N2 fixation has not yet begun or, per- haps more likely given the long experimental time, that the nodules were ineffective. No measures of nitrogenase activity were performed however. When a soil contains ineffective Frankia or Rhizobium it becomes even more important to inoculate any hosts to be grown in the soil so that the introduced strain(s) can outcompete the indigeneous ineffective bacteria population. In legumes such introductions are sometimes successful, sometimes not (Bottomley, 1992). Actinorhizal plants like Alnus need to be planted rather than sown directly in the field. This gives an opportunity to make sure that at least the introduced, perennial nodules contain a proper type of Frankia. In A. glutinosa (Hahn




et al., 1990) and in Elaeagnus (Simon et al., 1988) effective Frankia had higher compatibility than ineffective strains for nodulation.

Acknowledgments We are most grateful to Tr6ndur Leivsson at the Forestry

Service of the Faeroe Islands, Stefan Blixt at the Nordic Gene Bank, and Eva-Britt Olofsson at the Swedish University of Ag- ricultural Sciences who kindly collected soil samples and iden- tified plant species on site; Johan Grudemo who assisted with plant cultivations; Kristina Johansson who gave secretarial help; and Drs Monica Kahr, David Myrold, and Marijke Van Ghelue who provided seeds of A. incana, A. rubra, and A. glutinosa, respectively. Early parts of this study were performed at De- partment of Plant Physiology, Umefa University (KHD, ASH). This study was financially supported by the Swedish Council for Forestry and Agricultural Research and the Swedish Natural Sci- ence Research Council (KHD), the Nordic Gene Bank (HS) and the Swedish Polar Research Secretariat (KD).

References Cited

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Ms submitted December 1998




Documents

Occurrence of Alnus-Infective Frankia and Trifolium-Infective Rhizobium in Circumpolar Soils