Roots, Mycorrhizas and Rhizosphere Microbes NorFa - Workshop in Hyytiälä, Finland 20.-22.9.1994 Role of Roots, Mycorrhizas and Rhizosphere Microbes in Carbon Cycling in Forest Soil Extended abstracts of the NorFa - workshop Hyytiälä, Finland 20.-22.9.1994 Editors Heljä-Sisko Helmisaari Aino Smolander Arja Suokas Metsäntutkimuslaitoksen tiedonantoja 537 The Finnish Forest Research Institute. Research Papers 537 Helsinki 1995 Heljä-Sisko Helmisaari, Aino Smolander and Arja Suokas 1995. The role of roots, mycorrhizas and rhizosphere microbes in car bon cycling in forest soil. Metsäntutkimuslaitoksen tiedonantoja 537. The Finnish Forest Research Institute. Research Papers 537. Keywords: roots, mycorrhiza, rhizosphere, carbon cycling. Publisher: The Finnish Forest Research Institute; Accepted for publication by Research Director Eero Paavilainen, 22.12.1994. Correspondence: Heljä-Sisko Helmisaari and Aino Smolander, The Finnish Forest Research Institute, P.O. Box 18, FIN-01301 Vantaa, Finland. Distribution: The Finnish Forest Research Institute, P.O. Box 18, FIN-01301 Vantaa, Finland. Printed in: HAKAPAINO OY Helsinki, Finland, 1995 3 Roots, mycorrhizas and rhizosphere microbes... Contents FOREWORD 5 AUTHORS 7 EFFECTS OF NITROGEN AMENDMENTS ON THE GROWTH AND DISPERSAL OF SOME DIFFERENT ECTOMYCORRHIZAL FUNGI GROWN IN SYMBIOSIS WITH A HOST PLANT Kristina Amebrant 11 EFFECTS OF COLLEMBOLAN GRAZING ON THE ECTOMYCORRHIZAL SYMBIOSIS Kristina Amebrant, Hans Ek, Maria Sjögren and Bengt Söderström . 19 FINE ROOT DYNAMICS IN CANADIAN BOREAL FOREST STANDS AT DIFFERENT SUCCESSIONAL STAGES AFTER FIRE Leena Finer and Christian Messier 25 FINE-ROOT BIOMASS AND TURNOVER IN NORWAY SPRUCE AND SCOTS PINE STANDS Heljä-Sisko Helmisaari, Tarja Lehto and Aino Smolander 31 CELLULAR CHANGES AND ACCELERATED SENESCENCE OF CONIFER MYCORRHIZAS RELATED TO HIGH NITROGEN AVAILABILITY Toini Holopainen, Sari Janhunen and Helvi Heinonen-Tanski 37 THE EFFECTS OF SOIL MOISTURE AND TEMPERATURE ON CARBON ALLOCATION OF SCOTS PINE SEEDLINGS Hannu llvesniemi, Jyrki Haataja, Markus Huttunen and Eeva Korpilahti 43 UNBALANCED NUTRIENT STATUS AND MYCORRHIZAL ROOTS OF SCOTS PINE Sari Janhunen, Anneli Ylimartimo, Toini Holopainen 55 RHIZODEPOSITION IN ARABLE CROPS Bendt Jensen 59 TOMENTELLOID FUNGI (BASIDIOMYCETES, THELEPHORA CEAE S. STR.) - ARE THEY TRUE MYCORRHIZAL FUNGI? Urmas Köijalg 67 ECTOMYCORRHIZAL FUNGI AND FUNGICIDES Tarja Laatikainen and Helvi Heinonen-Tanski 71 VARIATION IN THE AMOUNT OF ORGANIC CARBON IN SOIL WITHIN A FOREST STAND: EFFECT OF TREES AND IMPLICATIONS FOR SAMPLING Jari Uski 77 4 DECOMPOSITION OF FINE ROOTS OF NORWAY SPRUCE (PICEA ABIES (L.) KARST.) AND SCOTS PINE (PINUS SYLVESTRIS L.) IN DIFFERENT SOILS Krista Löhmus, Mari Ivask and Ivika Ostonen 83 SEASONAL VARIATION OF FINE-ROOT BIOMASS IN PINUS SYLVESTRIS (L.) STAND Kirsi Makkonen 89 EFFECTS OF MYCORRHIZAS ON THE DEFENSIVE CHEMISTRY IN SCOTS PINE SEEDLINGS Anne Nerg, Toini Holopainen and Jarmo K. Holopainen 95 NITRITE REDUCTASE ACTIVITY IN THE MYCORRHIZAL ROOTS OF SCOTS PINE SEEDLINGS Karoliina Niemi, Helvi Heinonen-Tanski and Toini Holopainen 101 ECTOMYCORRHIZA DEVELOPMENT: 2-DIMENSIONAL ANALYSIS OF THE CYTOSKELETON Sara Niini, Mika Tarkka and Marjatta Raudaskoski 107 ECTOMYCORRHIZA: CARBOHYDRATES, MINERALS AND HORMONES Jan-Erik Nylund 111 THE ROLE OF ROOTS IN CARBON CYCLING IN FORESTS Hans Persson 119 EFFECT OF SHADELIGHT QUALITY ON DRY WEIGHT ALLOCATION AND MYCORRHIZAL DEVELOPMENT IN SCOTS PINE SEEDLINGS Tania M. de la Rosa, Tarja Lehto and Pedro J. Aphalo 127 BIOMASS AND TURNOVER OR ROOTS IN A MESOTROPHIC FEN Timo Saarinen 133 MICROBIAL BIOMASS IN THE RHIZOSPHERE OF TREES Aino Smolander, Ilari Lumme and Heljä-Sisko Helmisaari 137 A NEW HYPOTHESIS TO EXPLAIN THE NEGATIVE INFLUENCE OF NITROGEN ON ECTOMYCORRHIZAL DEVELOPMENT Hakan Wallander 141 MYCORRHIZAL ROOT COLONIZATION AND ERGOSTEROL CONTENT IN AN EXPERIMENTALLY POLLUTED SUBARCTIC BIRCH-PINE FOREST Martin Zobel, Olav Sarv and Martti Komulainen 147 5 Roots, mycorrhizas and rhizosphere microbes... Foreword This volume contains the extended abstracts of papers and post ers presented in the Nordic Workshop "The role of roots, mycor rhizas and rhizosphere microbes in carbon cycling in forest soil", which took place 20-22 September 1994 in Hyytiälä Forest Sta tion. The workshop was organised with the economic support kindly provided by Nor Fa. The aim of the workshop was to bring together Nordic scien tists working with the dynamics of belowground organisms in carbon cycling, to increase communication and understanding between researchers working in different disciplines and on dif ferent organisational levels (from cell to ecosystem), and to exchange information on new methods and research results deal ing with the belowground processes connected with roots in for est soil. We would like to thank the authors of papers and posters and all participants for contributing to a stimulating and successful meet ing. Vantaa 16th December 1994 On behalf of the organizers Heljä-Sisko Helmisaari Aino Smolander 6 7 Roots, mycorrhizas and rhizosphere microbes. Authors Arnebrant Kristina Department of Microbial Ecology, Lund University, Sölvegatan 37, S-223 62 Lund, Sweden. Aphalo Pedro J. Finnish Forest Research Institute, FIN-77600, Suonenjoki, Finland. Ek Hans Dept. of Chemical Ecology and Ecotoxicology, Lund University, Sölvegatan 37, S-223 62 Lund, Sweden. Finer Leena Finnish Forest Research Institute, Joensuu Research Station, P. 0. Box 68, SF-80101 Joensuu, Finland. Haataja Jyrki Department of Forest Ecology, P.O. Box 24, (Unioninkatu 40 B), FIN-00014 University of Helsinki, Finland. Heinonen-Tanski Helvi Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Helmisaari Heljä-Sisko Finnish Forest Research Institute, Department of Forest Ecology, P.O. Box 18, FIN-01 301 Vantaa, Finland. Holopainen Jarmo K. Ecological laboratory, Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Holopainen Toini Ecological Laboratory, Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Huttunen Markus Department of Forest Ecology, P.O. Box 24, (Unioninkatu 40 B), FIN-00014 University of Helsinki, Finland. Ilvesniemi Hannu Department of Forest Ecology, P.O. Box 24, (Unioninkatu 40 B), FIN-00014 University of Helsinki, Finland. Ivask Mari Institute of Environmental Protection, Estonian Agricultural University, Riia 12, EE-2400 Tartu, Estonia. Janhunen Sari Ecological Laboratory, Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Jensen Bendt Danish Institute of Plant and Soil Science, Department of Soil Science, Research Centre Foulum, P.O. Box 23, DK-8830 Tjele, Denmark. Kciljalg Urmas Institute of Zoology and Botany, 21 Vanemuise St. EE-2400 Tartu, Estonia. Komulainen Martti Laboratory of Ecological Zoology, Department of Biology, University of Turku, FIN-20500 Turku, Finland. 8 Korpilahti Eeva Department of Forest Ecology, P.O. Box 24, (Unioninkatu 40 B), FIN-00014 University of Helsinki, Finland. Laatikainen Tarja Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Lehto Tarja Finnish Forest Research Institute, FIN-77600, Suonenjoki, Finland. Liski Jari University of Helsinki, Department of Forest Ecology, P.O. Box 24, FIN-00014 University of Helsinki, Finland. Löhmus Krista Institute of Geography, Dept. of Nature Geography and Landscape Ecology, Tartu University, Ulikooli 18, EE-2400 Tartu, Estonia. Lumme Ilari Finnish Forest Research Institute, Department of Forest Ecology, P.O. Box 18, FIN-01301 Vantaa, Finland. Makkonen Kirsi Finnish Forest Research Institute, Department of Forest Ecology, P.O. Box 18, FIN-01301 Vantaa, Finland. Messier Christian Universite du Quebec ä Montreal, Gref, C.P. 8888 Succursalea, Montreal H3C 3PB, Canada. Nerg Anne Ecological laboratory, Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Niemi Karoliina Dept. of Plant Biology / Forest Pathology, P.O. Box 28, FIN-00014 University of Helsinki, Finland. Niini Sara Department of Botany, University of Helsinki, P.O. Box 7, FIN-00014 University of Helsinki, Finland. Nylund Jan-Erik Dept. of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S-750 07 Uppsala, Sweden. Ostonen Ivika Institute of Geography, Dept. of Nature Geography and Landscape Ecology, Tartu University, Ulikooli 18, EE-2400 Tartu, Estonia. Persson Hans Dept. of Ecology and Environmental Research, Swedish University of Agricultural Sciences, Box 7072, S-750 07 Uppsala, Sweden. Raudaskoski Marjatta Department of Botany, University of Helsinki, P.O. Box 7, FIN-00014 University of Helsinki, Finland. de la Rosa Tania M. 12 1 Finnish Forest Research Institute, FIN-77600, Suonenjoki, Finland. 2 Department of Botany, FIN-00014 University of Helsinki, Finland. Saarinen Timo Department of Botany, Ecological laboratories, P.O. Box 4, FIN-00014 University of Helsinki, Finland. 9 Roots, mycorrhizas and rhizosphere microbes.. Sarv Olav 12 department of Botany & Ecology, University of Tartu, Lai St 40, EE-2400 Tartu, Estonia, laboratory of Ecological Zoology, Department of Biology, University of Turku, FIN-20500 Turku, Finland. Sjögren Maria Department of Animal Ecology, Lund University, Sölvegatan 37, S-223 62 Lund, Sweden. Smolander Aino Finnish Forest Research Institute, Department of Forest Ecology, P.O. Box 18, FIN-01 301 Vantaa, Finland. Söderström Bengt Department of Microbial Ecology, Lund University, Sölvegatan 37, S-223 62 Lund, Sweden. Tarkka Mika, Department of Botany, University of Helsinki, P.O. Box 7, FIN-00014 University of Helsinki, Finland. Wallander Hakan Microbial Ecology, Department of Ecology, Lund University, Ecology Building, S-223 62 Lund, Sweden. Ylimartimo Anneli Department of Forest Ecology, P.O. Box 24, 00014 University of Helsinki, Finland. Zobel Martin 1 2 1 Department of Botany & Ecology, University of Tartu, Lai St 40, EE-2400 Tartu, Estonia, laboratory of Ecological Zoology, Department of Biology, University of Turku, FIN-20500 Turku, Finland. 10 11 Roots, mycorrhizas and rhizosphere microbes. Effects of nitrogen amend ments on the growth and dispersal of some different ectomycorrhizal fungi grown in symbiosis with a host plant Kristina Arnebrant Introduction It has often been reported that ectomycorrhizal fungi are nega tively affected by nitrogen fertilization. Generally these studies have been focussed on the effects found on fruitbodies (Menge and Grand 1978, Ohenoja 1978, Wästerlund 1982) and ectomyc orrhizal root tips (Menge et al. 1977, Alexander and Fairley 1983, Arnebrant and Söderström 1992). The effects of N amend ments on the mycelial part of the symbioses have thus far received much less interest. In many aspects, however, the extramatrical mycelium is the essential part of the symbiosis, since it, for an individual host plant, will substantially increase the surface area in contact with the substrate, while for the indi vidual fungus it is a prerequisite for dispersal and thus for the fit ness of the fungi. Furthermore, at the plant community level the mycelium is of importance since it connects different plants with each other, thereby creating the possibility of translocating car bon (Read et al. 1985, Finlay and Read 1986) and nitrogen (Arnebrant et al. 1993) from one host to another. Thus, possible Department of Microbial Ecology, Lund University, Sölvegatan 37, S-223 62 Lund, Sweden. 12 negative effects of nitrogen on the mycelial growth are of impor tance both to the host plant as well as to the fungus. In laboratory experiments, using a semi-hydroponic cultiva tion system, a decreased production of fungal biomass, estimated as ergosterol, has been reported both in roots (Wallander and Ny lund 1991, 1992) as well as extramatrical mycelium for three dif ferent ectomycorrhizal fungi (Wallander and Nylund 1992) as a result of increased nitrogen levels continously distributed in the nutrient solution. In the present study the effects of inorganic nitrogen on the production growth and dispersal of some ectomycorrhizal fungi grown in symbiosis with a host plant was studied. Material and methods Transparent perspex microcosms with non-sterile peat as sub strate were used in the experiments where the production of extramatrical mycelium was studied. 1, 2 or 4 mg of N g" 1 dry weight peat as (NH4)2SO 4, NaN03 or as a complete nutrient solution were used. Five different fungal isolates, grown in sym biosis with pine seedlings, were investigated. The effect of different N treatments on dispersal and compe tition between pairs of ectomycorrhizal fungi was investigated in pot experiments. Four pine seedlings, two colonized by one fun gus and two with the other,were diagonally planted in a pot. In the center of the pot, one bait-seedling, at the start of the experiment uncolonized, was planted. The pots were continously watered with different nitrogen solutions, 50 and 100 ppm N solutions of KN0 3, (NH4)2SO4 and a complete nutrient solution were used. Results The area covered by mycelium showed a tendency to be reduced for all fungi in all nitrogen treatments, although in most cases this reduction was not significant for the individual nitrogen treat ment (Table 1). However, no significant difference was shown neither between the different N-concentrations within each nitro gen treatment nor between the different nitrogen sources. Thus, the values obtained in all nitrogen treatments were pooled to obtain one value which showed the overall effect of nitrogen on mycelial growth. The overall effect of inorganic nitrogen on all the fungi was a significant reduction of the mycelial growth as 13 Roots, mycorrhizas and rhizosphere microbes. Table 1. Area (cm 2 ) covered by mycelium at harvest of the different ectomycorrhizal fungi in the different nitrogen treatments. Nitrogen was added in three different levels, 1, 2 and 4mg N g" 1 dry weight peat. The maximum area was 256 cm 2 . Pinus contorta was used as host plant for Paxillus involutus and P sylvestris to the other fungi. Values followed by different letters within a column indicate significant differences according to Duncan's multiple range test (p<0.05), N = 5. Percentages of control values are shown within parentheses. On the bottom line the table also includes values showing the effect of nitrogen on the area covered by mycelium. Percentages of control values are shown within brackets (from Arnebrant 1994). Fungus P. inv. 87.02 P. inv. 374 vgk 2 89.10 vg 1 87.10 S. bov. Experi- ment I II I II Control 234 a (100) 1 16 ab (100) 145 a (100) 206a (100) 121 a (100) 163 a (100) 96 a (100) pH-control 214 ab (92) 180 a (155) 124 a (86) 1 23 abc (60) 80 ab (66) 146a (90) 81 a (84) 1 164 ab (70) 93 ab (80) 44£l (30) 1 40 abc (68) 66 ab (55) 2.8 b (1.7) 12a (13) NaNOg 2 2i4 ab (92) 1 12 ab (97) 11 7 a (81) 1 35 abc (66) 70 ab (58) 8.7 b (5.3) 26a (27) 4 172 ab (74) n.d 1 73£i (50) 1 oo abc (49) n.d n.d n.d 1 182 ab (78) 102 ab (88) 26 a (18) 187 ab (91) 35 ab (29) 0.6 b (0.37) 43 a (45) (nh 4 ) 2 so 4 2 156 b (67) 68 ab (59) 49 a (34) 96 abc (47) 13 b (11) 8.5 b (5.2) 33 a (34) 4 172 ab (74) n.d 76 a (52) 36 c (17) n.d n.d n.d 1 211 ab (90) 64 b (55) n.d 1 38 abc (67) n.d n.d n.d Complete nutrient solution 2 209 ab (89) 46 b (40) n.d 82 bc (40) n.d n.d n.d 4 174 ab (74) n.d n.d 50 c (24) n.d n.d n.d F-ratio 2.175 3.952 2.08B 3.236 2.937 66.697 2.387 Sign, level 0.038 0.002 0.082 0.003 0.033 0.000 0.068 nitrogen effect 188* (80) 88* (76) 64* (44) 107* (52) ** 46 (38) *** 5.1 (3.1) 28** (29) 14 shown in Table 1. The mycelial growth of all fungi was found to be reduced by the nitrogen amendments, although the sensitivity to N varied between the isolates. One unidentified fungus was extremely sensitive and its growth was completely inhibited by all the N treatments. In contrast, the least sensitive fungus was one of the Paxillus involutus isolates which growth decreased by approximately 20 %. The mycelial growth of the other three iso lates was reduced by 50-70 %. In the experiments studying effects of nitrogen treatments on colonization potential a signifi cant reduction of ectomycorrhizal root tips was shown for all nitrogen treatments within one experiment including Hebeloma crustuliniforme and Piloderma croceum (Table 2). The distribu tion of the two fungal species on the bait seedling was, however, shown to be different depending on the nitrogen treatment (Table 2). In contrast, in another experiment that included P. involutus and Suillus bovinus all N treatments negatively affected S. bovi nus (Table 3). Discussion It was clearly demonstrated that nitrogen can affect the competi tive interactions between different ectomycorrhizal fungi. In the experiment that included H. crustuliniforme and P. croceum, it was indicated that different nitrogen sources can have different effects on the competitive success of different fungi. In this case, although both fungi were negatively affected of all nitrogen treat ments, P. croceum appeared to be relatively more sensitive to ammonium than to nitrate compared to H. crustuliniforme and thus lost in competitive success. The opposite was indicated in the treatment with a complete nutrient solution where the per centage of ectomycorrhizal root tips colonized with P. croceum tended to increase compared to all other treatments, and thus the relative success of this fungus was increased compared to H. crustuliniforme. In contrast, all nitrogen treatments including the complete nu trient solution, all affected the relative competitive success of Suillus bovinus and Paxillus involutus in the same way. Although the total percentage of ectomycorrhizal root tips only were signif icantly reduced in the nitrate compared to the ammonium treat ment, all significantly reduced the relative success of S. bovinus. Thus, not only the relative success of P. involutus increased in all nitrogen treatments, but also the total colonization level by this fungus tended to increase in the ammonium treatment. In contrast 15 Roots, mycorrhizas and rhizosphere microbes... Table 2. Data showing the colonization pattern of the bait-seedling from the pot experiment including Hebe loma crustuliniforme and Piloderma croceum. The pots were treated with 50 or 100 ppm N solutions of KN0 3 or (NH 4 ) 2 SO 4 or a 50 ppm Nof a complete nutrient mixture, and incubated for four months. One-way ANOVA was performed on either log- or arcsine square root-transformed values (percentages). Duncan's multiple range test p<0.05, N=s. Table 3. Data showing the colonization pattern of the bait-seedling from the pot experiment including Paxil lus involutus and Suillus bovinus. The pots were treated with 50 ppm solutions of KN03 , (NH4 ) 2SO4 or a complete nutrient mixture, and incubated for 6 months. One-way ANOVA was performed on either log- or arcsine square root-transformed (percentages) values. Duncan's multiple range test p<0.05, N=s. T reatment total no. of total no. of percentage total no. of percentage P. root tips ectomyc. ectomyc. P. croceum croceum of root tips root tips total ectom. Control 436 a 204 a 48. 1 a 15.2 a 7.88 a KN0 3 50 855 b 189a 24.2 b 13.4ab 7.56a 100 1097 bc 133ab 12.7bc 6 2ab 6.71 a (NH 4 ) 2S04 3.2 ab 50 766b L 00 0> 24.6b 2.58a 100 994 b 67b 7.5C 2.6 b 2.04a Complete nutrient solution 50 1746 c 103 ab 6.8C 16.5a 17.80a F-ratio 14.42 6.229 14.19 2.708 1.971 p (sign, level) < 0.001 <0.05 < 0.001 <0.05 0.121 Treatment total no. of root tips total no. ectom. root tips percentage ectom. root tips total no. S. bovinus percentage S. bovinus of total ectom. Control 496 ab 178a 36.0 ab 93.6a 56.3a KN0 3 50 1 087 bc 11 3a 1 0.9a 9.2b 10.5b (nh4 ) 2so4 50 343 a 178 a 46.8b 21 ,6b 13. 1 b Complete nutrient solution 50 1 155 c 260 a 22.7 ab 25.8b -Q CO F-ratio 11.785 0.909 2.844 4.622 10.937 p (sign, level) <0.001 0.459 0.071 <0.05 < 0.001 16 to the fungi used in the other pot experiment, both the fungi in this were included in the study concerning effects of different nitrogen sources on mycelial growth (Arnebrant, in press), and the differ ences in competitive ability could possibly be explained by the differences in nitrogen sensitivity. P. involutus was in that study shown to be the least affected fungus, although it's growth was significantly reduced with approximately 20 %. The mycelial growth of S. bovinus (although another isolate than was used in this study) was reduced with approximately 70 % compared to the growth in the control. This fungus was also the most sensitive one of the three used by Wallander and Nylund (1992). The fruitbody production by S. bovinus has been shown to decrease in ammoni um nitrate fertilized plots (Wasterlund 1982), while P. involutus is known to be one of those few that actually increases it's fruit body production as an effect of ammonium treatment (Hora 1959, Laiho 1979, Shubin 1988). The explanation to the reduced mycelial growth caused by in creased nitrogen level is not evident but has been substantially discussed by Nylund (1988) and Wallander (1994). What's even more intriguing to discuss is why different ectomycorrhizal fungi are differently sensitive to nitrogen. What mechanism makes P. involutus more successful than for example S. bovinus in nitrogen treated soils? References Alexander, I. J. & Fairley, R. I. 1983. Effects of fertilisation on popula tions of fine roots and mycorrhizas in spruce humus. Plant and Soil 71: 49-53. Arnebrant, K. 1994. Nitrogen amendments reduce the growth of extramatrical ectomycorrhizal mycelium. Mycorrhiza (in press). Arnebrant, K. & Söderström, B. 1992. Effects of different fertilizer treatments on ectomycorrhizal colonization potential in two Scots pine forests in Sweden. For. Ecol. Manag. 53: 77-89. Arnebrant, K., Ek, H., Finlay, R. D. & Söderström, B. 1993. Nitrogen translocation between Alnus glutinosa (L.) Gaertn. seedlings inoculated with Frankia sp. and Pinus contorta Doug, ex Loud seedlings connected by a common ectomycorrhizal mycelium. New Phytol. 124: 231-242. Finlay, R. D. & Read, D. J. 1986. The structure and function of the veg etative mycelium in ectomycorrhizal plants. I. Translocation of 14 C-labelled carbon between plants interconnected by a com mon mycelium. New Phytol. 103: 143-156. Laiho, O. 1979. Paxillus involutus as a mycorrhizal symbiont of forest trees. Acta For. Fenn. 106. 17 Roots, mycorrhizas and rhizosphere microbes. Menge, J. A. & Grand, L. F. 1978. Effect of fertilization on production of epigeous basidiocarps by mycorrhizal fungi in loblolly pine plantations. Can. J. Bot. 56: 2357-2362. Menge, J. A., Grand, L. F. &: Haines, L. W. 1977. The effect of fertiliza tion on growth arid mycorrhizae numbers in 11 -year-old Loblolly pine plantations. For. Sci. 23: 37-44. Nylund, J.-E. 1988. The regulation of mycorrhiza formation - carbohy drate and hormone theories reviewed. Scand. J. For. Res. 3: 465- 479. Read, D. J., Francis, R. & Finlay, R. D. 1985. Mycorrhizal mycelia and nutrient cycling in plant communities. In "Ecological interac tions in soil - plants, microbes and animals". Ed. by Fitter, A. H. with Atkinson, D. Read, D. J. & Usher, M. B. Special publica tions series of the British Ecological Society, no. 4: 193-217. Shubin, V. I. 1988. Influence of fertilizers on the fruiting of forest mushrooms. Acta Bet. Fenn. 135: 85-87. Wallander, H. 1994. A new hypothesis to explain allocation of dry mat ter between mycorrhizal fungi and pine seedlings in relation to nutrient supply. Plant and Soil (in press). Wallander, H. & Nylund, J.-E. 1991. Effects of nitrogen on carbohy drate concentrations and mycorrhizal development of Pinus syl vestris L. seedlings. New Phytol. 119: 405-411. Wallander, H. & Nylund, J.-E. 1992. Effects of excess nitrogen and phosphorus starvation on the extramatrical mycelium of ectomy corrhizas of Pinus sylvestris L. New Phytol. 120: 495-503. Wästerlund, I. 1982. Försvinner tallens mykorrhizasvampar vid göd sling? (Do pine mycorrhizae disappear following fertilization?) Svensk Botanisk Tidskrift 76: 411-417. 18 19 Roots, mycorrhizas and rtiizosphere microbes. Effects of collembolan graz ing on the ectomycorrhizal symbiosis Kristina Arnebrant 1 , Hans Ek 2 , Maria Sjögren 3 and Bengt Söderström 1 Introduction Little is known about effects of microarthropods on the function of the ectomycorrhizal symbiosis (Moore 1988). There are, how ever, a few studies focusing on the relationship between arbuscu lar mycorrhiza and their grazers and the effects on the host plant. Kaiser and Lussenhop (1991) demonstrated that collembolans can interfere with the establishment of arbuscular mycorrhiza. Warnock et al. (1982) found that the positive effect of arbuscular mycorrhizal colonization on the growth of leek disappeared in the presence of collembolans. This was explained as an effect of the collembolans grazing on the external hyphae. Finlay (1985) studied interactions between the collembola Onychiurus ambu lans and arbuscular mycorrhizal Allium porrum and observed that at low grazing intensities, mycorrhizal plant phosphorus uptake was stimulated, but at higher grazing pressure P uptake was reduced. Similar non-linear effects of collembolans on plant growth and P uptake have also been shown in arbuscular mycor rhizal Geranium robertianum (Harris and Boerner 1990). The Collembola-ectomycorrhiza interaction involves a host plant which may be indirectly affected by the grazing activities. In our study we have therefore tried to evaluate the effects of grazing 1 Department of Microbial Ecology, Lund University, Sölvegatan 37, S-223 62 Lund, Sweden. 2 Department of Chemical Ecology and Ecotoxicology, Lund University, Sölvegatan 37, S-223 62 Lund, Sweden. 3 Department of Animal Ecology, Lund University, Sölvegatan 37, S-223 62 Lund, Sweden. 20 not only on the two main components directly affected, the ecto mycorrhizal fungus and the Collembola, but also on the plant. Material and methods A microcosm system consisting of transparent polystyrene cham bers 245 mm x 245 mm x 25mm (so called screening plates) was used. In these systems three Pinus contorta seedlings were planted, one colonized by the ectomycorrhizal fungus Paxillus involutus planted in the center and on each side of this, two at the start of the experiment noncolonized seedlings. A sandy soil, with apH (H 2O) of 4.6 was used as substrate. The soil was microwaved prior to the experiment to obtain a sterile soil. To the microcosms 12 small cups (1.5 cm in diameter), each containing soil with 100 pg Nas NH4CI, covered with a net that allowed the fungal hyphae to penetrate but neither Collembola nor plant roots, were added. The microcosms were incubated for 88 days in growth cabinets. There were four different treatments, mycorrhizal or non-my corrhizal, with 0 or 50 collembolans of the species Onychiurus ar matus added per microcosm. Ten microcosms of each treatment were used. In a pilot experiment the effect of different densities of O. ar matus on mycelial growth was studied. In this experiment we add ed 0, 20, 75 or 200 animals per microcosm. Mycelial growth was measured continously. At harvest we measured plant biomass (as dry weight), 15 N-uptake, fungal bio mass (as ergosterol) on roots and in the soil, microbial biomass (as ATP content) in the soil and numbers of Collembola and nema todes. Results and discussion The mycelial growth rate was significantly stimulated by the presence of a low density (50) of Collembola, the fungus grew 60 % faster in the grazed treatment, 13.6 cm 2 day" 1 compared to 8.4 cm 2 day" 1 in the control. The colonization rate of the side plants was also higher in the microcosms with animals (data not shown). The production of extramatrical mycelium was increased in the grazed treatment, while the amount of fungal biomass found in the roots was unaffected (Table 1). Thus, in the grazed microcosms 62 % of the P. involutus grew as extramatri cal mycelium compared to 42 % in the nongrazed. Furthermore, 21 Roots, mycorrhizas and rhizosphere microbes... Table 1. Biomass of Paxillus involutus in plant roots and soil. A conversion fac tor of 9.6 pg ergosterol mg' 1 fungal dry weight was used to calculate biomass from ergosterol values. The amount of ergosterol found in soil from non-mycor rhizal microcosms was subtracted from the corresponding mycorrhizal treat ment (modified from Ek et al., 1994). Table 2. Number of Collembola at start and at harvest of the two experiments (modified from Ek et al., 1994) Table 3. Seedling biomass and proportion of the plant allocated to the shoot (modified from Ek et al., 1994). Table 4. Amount of 15 N taken up by the plants. In total 1.2 mg of 15 N, available only to the fungus, was added to each microcosm (from Ek et al., 1994). Grazing Amount of P. involutus Amount of P. involutus in root in soil (mg) (mg) + "I2.8 a 20.4a - "I3.2 a 11.5 b Mycorrhiza No. of collem- No. of collem- Percentage bola at start bola at harvest increase Main + 50 63 25 - 50 57 14 + 20 126 630 + 75 459 610 Pilot + 200 384 190 - 75 332 440 Mycorrhiza Grazing Plant biomass Biomass allocated (mg) to shoot + + 394a 41 %a + 344 a 36%a - + 156 b 30% b - 175 b 24%b Mycorrhiza Grazing Mid-plant (M9 15 N) Side-plant (M9 5 N) + + 232 a 21 5 a + 132 b 177a + 5.6 C 12 b - 4.3 C 12 b 22 the proportion of fungal biomass of the total plant and fungal bio mass increased in the grazed system to 5.3 % compared to being 3.7 % in the ungrazed. The Collembola thus induced a change in the carbon allocation pattern and increased the amount allocated to the soil. In the pilot experiment the collembolan population increased dramatically during the experiment, and even the low est density was higher than in the main experiment (Table 2). The mycelial growth was reduced in all treatments with collembolans and was only 66 % of the growth rate in the control in the treat ments with the highest density of O. armatus. The plant biomass was significantly higher in the mycorrhizal treatment, while grazing had no apparent effect (Table 3). Fur thermore, the proportion of biomass allocated to the shoot was significantly increased by the mycorrhizal treatment. The nitrogen content (both 14 N and ,S N) was consistently higher in mycorrhiz al compared to non-mycorrhizal plants. There was no effect of O. armatus on the total nitrogen content but the uptake of 15 N was significantly higher in the mycorrhizal and grazed treatment, thus the mycorrhizal midplants in the grazed microcosms had 76 % higher 15 N content than the corresponding plants with mycorrhiza but without grazing pressure (Table 4). There was, however, no linear relationship neither between the amount of ergosterol in the substrate and total nitrogen content in the plants nor between fun gal biomass in the cups and 15 N content in the plants. In the first case the grazed microcosms had higher amount of fungal biomass but no difference in total N content in the host plant was found, while in the second there was no difference in fungal biomass in the cups but significantly higher amount of 15 N in the plants. We thus found a similar effect of collembolan grazing as has been reported for arbuscular mycorrhizal systems, i.e. that a low density increase growth and nutrient uptake, while higher densi ties reduce growth. There are a few possible mechanisms for the stimulatory effect. The first is that the fungus reacts with over compensatory growth at a low grazing pressure. The increased amount of fungal biomass in combination with that younger hy phae might be more efficient in nutrient uptake explains the in creased nutrient uptake. A prerequisite for this explanation is that the collembolans are grazing directly of the ectomycorrhizal fun gus. We have, however, no such evidence. In a third experiment we collected the Collembola and analyzed their content of 15 N. No difference was found between animals from mycorrhizal or nonmycorrhizal microcosms but since the biomass of the collem- 23 Roots, mycorrtiizas and rhizosphere microbes... bolans was low the measurement was close to the detection limit. Furthermore, in the pilot experiment the population of O. armatus increased up to sixfold, and the Collembola were seen to be gath ered at the top of the chambers were a band of green algae grew. In the main experiment where we had taken precautions to avoid algal growth there was only a slight increase of the collembolan population and there was no difference between the mycorrhizal and nonmycorrhizal treatments. Thus, P. involutus does not ap pear to be a good food source for O. armatus. However, the re duced mycelial growth as an effect of higher densities of O. armatus indicate a direct grazing effect. Another explanation involves selective grazing by the collem bolans on other microorganisms, in this way reducing the compe tition. This hypothesis has been discussed by Newell (1984 a and b) and Parkinson et al. (1979). However, we could not find any clear evidence of this either since the ATP content was not re duced and in a later experiment, where we studied the microfugal community structure, no obvious differences were noted between treatments (data not shown). A third explanation could be that the Collembola makes nutri ents more easy available to the fungus. Referenses Ek, H., Sjögren, M., Arnebrant, K. and Söderström, B. 1994. Extramat rical mycelial growth, biomass allocation and nitrogen uptake in ectomycorrhizal systems in response to collembolan grazing. Appl. Soil Ecol. (in press). Finlay, R.D. 1985. Interactions between soil micro-arthropods and endomycorrhizal associations of higher plants. In: Fitter, A.H., Atkinson, D., Read, D.J. and Usher, M.B. (eds.), Ecological Interactions in Soil. Blackwell, Oxford, pp. 319-331. Harris, K. K. and Boerner, R. E. J. 1990. Effects of belowground graz ing by collembola on growth, mycorrhizal infection and P uptake of Geranium robertianum. Plant and Soil 129: 203-210. Kaiser, P. A. and Lussenhop, J. 1991. Collembolan effects on establish ment of vesicular-arbuscular mycorrhiza in soybean (Glycine max). Soil Biol. Biochem. 23: 307-308. Moore, J. C. 1988. The influence of microarthropods on symbiotic and non-symbiotic mutualism in detrital-based below-ground food webs. Agriculture, Ecosystems and Environment 24: 147-159. Newell, K. 1984 a. Interaction between two decomposer basidiomycetes and a collembolan under Sitka spruce: Distribution, abundance and selective grazing. Soil Biol. Biochem. 16: 227-233. 24 Newell, K. 1984b. Interaction between two decomposer basidiomycetes and a collembolan under Sitka spruce: Grazing and its potential effects on fungal distribution and litter decomposition. Soil Biol. Biochem. 16: 235-239. Parkinson, D. Visser, S. and Whittaker, J. B. 1979. Effects of Collembo lan grazing on fungal colonization of leaf litter. Soil Biol. Bio chem. 11: 529-535. Warnock, A. J., Fitter, A. H. and Usher, M. B. 1982. The influence of a springtail Folsomia Candida (Insecta, Collembola) on the myc orrhizal associations of leek Allium porrum and the vesicular arbuscular mycorrhizal endophyte Glomus fasciculatus. New Phytol. 90: 285-292. 25 Roots, mycorrtiizas and rhizosphere microbes. Fine root dynamics in Cana dian boreal forest stands at different successional stages after fire Leena Finér 1 and Christian Messier 2 Background Vegetation types vary in southern boreal forests in the Northern Clay Belt of Quebec and Ontario in relation to the surficial deposits and successional stages. Clay deposits are typical on lowlands where early successional stands are characterized by an abundance of deciduous species, primarily trembling aspen (Pop ulus tremuloides) and paper birch (Betula papyrifera), which are replaced later in succession by the coniferous species white spruce (Picea glauca), balsam fir (Abies balsamea) and eastern red cedar (Thuja occidentalis) (Bergeron and Dubuc 1989). The growth and structure of the forests are controlled by three distur bances: fire, spruce budworm (Choristoneura fumiferana) and man. The fire cycle is around 100 years, and the forests in the area seldom reach the climax stage of succession. Spruce bud worm outbreaks occur at regular intervals. The forest is vulnera ble to attack in the late successional stages dominated by white spruce and balsam fir. Conifers grow in mixed stands, and deaths caused by spruce budworm create gaps. The human impact has been minor so far. The above-ground plant succession is relatively well docu mented in forests of the Clay Belt region, but hardly anything is known about the below-ground dynamics. The aim of the study 1 Finnish Forest Research Institutes, Joensuu Research Station, P. O. Box 68, SF-80101 Joensuu, Finland. 2 Universite du Quebec ä Montreal, Gref, CP. 8888 Succursalea, Montreal H3C 3PB, Canada. 26 was to investigate how the fine root distribution and production of trees and ground vegetation vary in forest stands at different suc cessional stages after fire. This was one of the many projects be ing carried out in the boreal forests of the area, and was conducted by the forest research group of the University of Quebec in Mon treal. Material and methods Study sites The material was collected from three southern boreal forest stands at different successional stages after fire around lake Duparquet in northeastern Quebec, Canada. The youngest stand, which had developed since the last fire 48 years ago, was domi nated by paper birch and trembling aspen. The ground layer was dominated by herbs and mountain maple (Acer spicatum). Trem bling aspen, white spruce and balsam fir were the main tree spe cies in the mid successional stand, which had been burnt 122 years ago. The ground layer was formed by herbs, grasses and shrubs; the mountain maple and American yew (Taxus canaden sis) especially were important species. The late successional, 232-year-old stand was characterized by eastern red cedar, bal sam fir and aspen. The ground layer was dominated by mountain maple and American yew. The thickness of the organic layer had increased as a result of ageing of the stand from 5.3 cm to 8.0 cm. Sampling The study was carried out in each of the tree stands on three rep licates of 10 x 10 m plots for each of the two treatments: 1) gap 2) control. Fine and small roots (0 < 10 mm) were sampled on the control plots by the core method at the beginning of June 1993. In the laboratory the roots were separated from eight soil cores per plot (area 38 cm 2 ) and divided into diameter classes. The small roots (0 2-10 mm) were further separated by species. The length and biomass of fine roots were measured. The fine root production was measured by the ingrowth bag method on both the control plots and gaps. Six 30 cm long bags, filled with rootfree clay from the site, were installed on each plot in August 1992. The ingrowth bags were dug up in September 1993. Roots, mycorrhizas and rhizosphere microbes. 27 Table 1. Total fine and small root (0 < 10 mm) biomass, length and biomass production in stands at different successional stages after fire. Standard devia tion in parentheses. Preliminary results and discussion Root biomass The youngest of our stands had not yet reached its maximum above-ground biomass, whereas the 122- and 232-year-old stands had passed the stage of maximum biomass, partly due to spruce budworm outbreaks in the late 1980's. The fine and small root biomass did not vary between the sites (Table 1). The proportion of fine and small root biomass out of the total biomass corre spondingly increased with the aging of the stand. However, total root length was longer in the youngest stand than in the older stands, which could indicate a better nutrient uptake ability in the early successional stage compared to later stages. As in many studied forest ecosystems, the root systems were superficial in all stands (Fig. 1) (Persson 1980 a, 1983, Gale and Grigal 1987). The results did not indicate any changes in total rooting depth along with succession. The roots of tree species accounted for 80, 66 and 67 % of the small root biomass, respectively (Fig. 2). The tree density did not vary between the sites, which may explain why the proportion did not correlate with stand age as has been reported in some other stands (Perssonl979, Vogt et al. 1983). Aspen dominated the ear ly and mid successional stands, but was replaced by cedar in the old stand. The abundance of birch decreased along with the in crease in the abundance of spruce and fir. American yew became more dominant than the other shrubs at mid succession. Root production The fine root production was studied with the ingrowth bag method, which is known to give results comparable to the Site Root biomass g/m 2 Root length m/m 2 Root biomass pro- duction g/m 2 /yr 48 yrs 1056 (289) 12857 (3667) 130 (75) 122 yrs 827(193) 8200(1107) 61 (36) 232 yrs 952 (170) 7181 (404) 47(18) F-value 0.80 F-value 5.55 F-value 2.43 p-value 0.49 p-value 0.04 p-value 0.17 28 Figure 1. Fine and small root (0 3, NH4-acetate and ureaformaldehyde each in four levels corresponding to 0, 50, 150 and 300 kg/ha N. In the second exper iment (Kottke et al. 1994) two ammonium sources, NH4CI and (NH4)2SO 4, at total doses of 50 and 100 kg/ha N were used. Mycorrhizas in industrial environment: Samples of several mycorrhizal types of Scots pine were collected from the environ ment of a pulp mill near the city of Kuopio. During the study pe riod, the annual S02 emissions were about 10 000 t/y, NO x emissions 400 t/y and NH 4 emissions 16 t/y. Elevated nitrogen concentrations of the humus layer have been measured in this en vironment (Holopainen et al. 1994). Pure cultures of mycorrhizal fungi: Experiments for com parative purposes were carried out with pure cultures of Cenococ cum geophilum and Paxillus involutus. The fungi were grown on Petri dishes on MMN medium where the levels of ammonium chloride (NH4CI) added were 62.5, 125, 250 (optimum), 500 and 750 mg/L. Methods: The different mycorrhizal types were identified and counted under a dissecting microscope (Holopainen and Heino nen-Tanski 1993). For electron microscopy mycorrhizas and piec es of pure cultures were fixed in glutaraldehyde and os miumtetroxide and thin sections stained on grids with uranylacetate and lead citrate (Holopainen and Heinonen-Tanski 1993). Electron energy loss spectroscopy (EELS/ESI) was ap plied at the University of Tubingen, Germany (Kottke et al. 1994). Results and discussion Our results on shoot and root growth and mycorrhiza develop ment in relation to nitrogen availability in experimental expo sures were in agreement with earlier observations (Alexander and Fairley 1983, Laiho et al. 1987, Gagnon et al. 1988, Högberg 1989). In our experiments nitrate inhibited more clearly the myc orrhiza development than ammonia (Holopainen and Heinonen- Tanski 1993). At the cellular level all the used nitrogen sources induced a similar developing pattern of changes (Figs. 1 and 2). In the first phase, development of dark vacuolar accumulations accompanied with a decline of glycogen granules and lipid bod ies, were evident. A dark staining and gradual disintegration of fungal cytoplasm were observed after exposure to higher nitro- 39 Roots, mycorrhizas and rhizosphere microbes. Figures 1 -4. Electron micrographs of Scots pine mycorrhizas. 1) Normal structure of C. geophilum in sheath (low nitrogen availability). 2) Fungal cells of C. geophilum in sheath showing cytoplasm disintegration (ar rows) and large electron dense vacuolar accumulations (a) (NH 4 -N, 300 kg/ha). 3) Electron dense vacuolar bodies in a brown dichotomous mycorrhiza in an industrial environment (pulp mill, 600 m N). 4) EELS/ESl image showing nitrogen deposition (light area) in a vacuolar body in C. geophilum under high nitrogen availability. 40 gen levels (Fig. 2). Intracellular penetrations of fungi were often observed in senescing host cells (Holopainen and Heinonen-Tan ski 1993). Very similar cellular changes and limited infection lev els were observed in mycorrhizas collected from the industrial environment (Fig. 3) (Holopainen et ai. 1994). The EELS/ESI-analysis revealed that the vacuolar bodies in C. geophilum contained high levels of nitrogen (Fig. 4) accompa nied with accumulation of phosphorus (Kottke et al. 1994). This observation suggests that vacuolar bodies may consist of protein aceous material (Turnau et al. 1994) or stored aminoacids (Plas sard et al. 1991), although other elements seem to deposit into the vacuoles together with stored nitrogen, as well (Turnau et al. 1994). The structural observations from the pure cultures of C. geophilum and P. involutus agreed very well to the observations from mycorrhizas. This indicates that mycorrhizal state is not nec essary for the formation of vacuolar storage bodies and accelerat ed senescence of fungi, which suggests that nitrogen has also direct degenerating effects on mycorrhizal fungi. Conclusions Under high nitrogen availability mycorrhizal fungi, both in sym biosis and pure cultures, can store nitrogen as nitrogen-rich vacu olar deposits. Sooner or later, depending on species, the storage capacity is saturated, leading to toxic effects of excess nitrogen. At ultrastructural level this is observable as accelerated senes cence and degeneration of fungal cytoplasm. Since the carbon availability was not a limiting factor in pure cultures, these obser vations suggest that mycorrhizal fungi can be directly affected by soil nitrogen, possibly leading to declined infection level and root vitality. Further studies are needed to identify the stored nitrogen rich compounds and mechanisms involved in cytoplasmic degen eration. References Alexander, I.J. & Fairley, R.L. 1983. Effects ofN fertilization on popu lations of fine roots and mycorrhizas in spruce humus. Plant and Soil 71: 49-53. Högberg, P. 1989. Growth and nitrogen inflow rates in mycorrhizal and non-mycorrhizal seedlings of Pinus sylvestris. For. Ecol. Manag. 28: 7-17. 41 Roots, mycorrhizas and rhizosphere microbes. Holopainen, T., Halonen, A. & Heinonen-Tanski, H. 1994. Injuries to Scots pine mycorrhizas and chemical changes in forest soil in the environment of a pulp mill in Central Finland. Water Air Soil Pollution (in press). Holopainen, T. & Heinonen-Tanski, H. 1993. Effects of different nitro gen sources on the growth of Scots pine seedlings and the ultrastructure and development of their mycorrhizae. Can. J. For. Res. 23: 362-372 Gagnon, J., Langlois, C.G. & Fortin, J.A. 1988. Growth and ectomycor rhiza formation of containerized black spruce seedlings as affected by nitrogen fertilization, inoculum type, and symbiont. Can. J. For. Res. 18: 922-929. Kottke, 1., Holopainen, T., Alanen, E. & Turnau, K. 1994. Deposition of nitrogen in vacuolar bodies of Cenococcum geophilum Fr. myc orrhizas as detected by electron energy loss spectroscopy. Manu script. Laiho, 0., Sarjala, T., Hyvärinen, R., & Rautiainen, L. 1987. Lan noituksen vaikutus männikön mykoritsoihin. Effects of fertiliza tion on mycorrhizae in pine stands. Folia Forestalia (Helsinki) 699: 1-22. Nihlgärd, B. 1985. The ammonium hypothesis - an additional explana tion to the forest dieback in Europe, Ambio 14: 2-8. Plassard, C., Scheromm, P., Mousain, D., & Salsac, L. 1991. Assimila tion of mineral nitrogen and ion balance in the two partners of ectomycorrhizal symbiosis: data and hypothesis. Experientia47: 340-349. Turnau, K„ Kottke, I. & Dexheimer, J. 1994. Paxillus involutus/Pinus sylvestris mycorrhizae from heavily polluted forest 11. Ultrastructural and cytochemical observations. Botanica Acta 107: 73-80. Wallander, H. & Nylund, J-E. 1991. Effects of excess nitrogen on car bohydrate concentration and mycorrhizal development of Pinus sylvestris seedlings. New Phytol. 119: 405-411. 42 43 Roots, mycorrhizas and rhizosphere microbes. The effects of soil moisture and temperature on carbon allocation of Scots pine seedlings Hannu Ilvesniemi, Jyrki Haataja, Markus Huttunen and Eeva Korpilahti Introduction The growth and the partition of carbohydrates to different plant organs of the seedlings is a dynamic function of light, tempera ture, moisture and nutritional conditions of the site, and the allo cation pattern has an effect on the growth of trees (Linder and Rook 1984, Axelsson and Axelsson 1986, Mäkelä 1988, Nikin maa 1993). The effects of these environmental factors can be seen e.g. on the root to shoot ratio of plants. The aim of this study was to analyze the effects of soil moisture and temperature on the growth and growth allocation of Scots pine seedlings. Material and methods The experiment was carried out in the field laboratory of the Hyytiälä experimental station (61° 48' N, 24°19'E). 112 two year old seedlings were grown in 8 liter draining pots, the temperature and moisture of which were adjusted. There were two tempera ture and two moisture treatments and the treatments were named as moist-warm (MW), moist-cold (MC), dry-warm (DW) and dry-cold (DC). The experiment consisted of two growing sea sons. 56 of the seedlings were over wintered outdoors, and dur ing this period all the seedlings, also those of the dry treatment Department of Forest Ecology, P.O. Box 24 (Unioninkatu 40 B), FIN-00014 University of Helsinki, Finland. 44 Figures 1a,b. The soil water potential of the growth media in different treatments during the growth period 1992 (a) and 1993(b). were watered. The growth media was homogenized and sieved (< 0.5 cm) forest soil consisting of eluvial and upper part of illuvial layers mixed together. The same amount of soil (7.85 kg) was weighed to all pots to obtain soil bulk density of 1.2 kg dm 3 . When the seedlings were planted (1.6. 1992) all seedlings were watered to the -10 kPa soil water potential. During the fol lowing month the soil water potential was allowed to decrease to -60 kPa. Some water was given to these seedlings during the dry 45 Roots, mycorrhizas and rhizosphere microbes... ing period to avoid strong moisture gradient along the depth of the pot. Meanwhile the seedlings growing in moist treatment were watered with successive water additions so that the treatment con ditions from -1 to -2 kPa was reached (Fig. la). In the beginning of the growth period 199 5 the soil water potential in all pots was around -10 kPa which level was kept over the growth period for the moist treatment. The treatment level of the dry soil -40-50 kPa was achieved in the beginning of June (Fig. 1 b). The temperature treat ments were arranged so that a reference electrode was installed to the depth of 7 cm in soil in an open spot, resembling the soil temperatures of a clear-cut area. In 1992 the warm treatment was adjusted to be 4 to 1 °C higher than the ref erence temperature the difference decreasing along the decrease in soil temperature. The cold treatment was around 5 °C colder than the soil temperature in an open area. Due to the breakdown of the thermostat, the ten: peratures in treatments were higher and the difference between treatments was smaller as planned in the beginning of the period (Fig. 2a). The effective temperature sum in soil (+5 °C threshold value) between 10.6 and 14.9 was 1133 d.d. in warm treatment and the corresponding value in cold soil was 721 d.d. During the growth period 1993 the temperature of the warm soil treatment followed that of the reference soil and the cold treatment was 4 °C colder than that (Fig. 2b). The effective temperature sum between 13.5 and 11.10 was 977 d.d. in warm soil and 525 d.d. in cold soil. In summer 1992 eight and in 1993 ten measurement periods of gas exchange were performed covering evenly the whole growth period. At the same occasion one or two seedlings from each treatment was randomly chosen to the measurement. In 1992 six gas exchange measurement cuvettes were available so that at every other measurement period two seedlings from two treat ment combinations was measured at the same time. In 1993 only four cuvettes were used, and one seedling from each treatment was measured at time. Tie measurement period was 5-6 days ex cept the last four measurements which lasted for 10 days. The amount of transpiration and net photosynthesis, as well as the photosynthetically active radiation and air temperature was meas ured. The cuvette was closed during the measurement period (around one minute), otherwise the cuvettes were open. The tech nical details of the measurement system has been presented in Hari et. ai. 1990. Each of the cuvettes was measured with the in terval of 22 minutes in 1992 and 16 minutes in 1993 day and night. 46 Figures 2a,b. The tern- perature of the growth media during the growth 1993 (b) I The thick line corresponds to the refer- ence value, and the thin lines are those meas- ured from two different temperature treatments. The growth and growth allocation was studied by measuring the increase in dry-weight and by using I4C labeled carbon dioxide as a tracer in the beginning of the experiment, before the potting, t^ie res'l we '§ht of the seedlings and the volume of the roots was measured as follows: After gently rinsing the roots the fresh weight of the seedlings was recorded. The root system was then immersed in a water filled container of known weight, placed on a weight-recorder. The weight of the container was then re wejgjie( j t0 determine the weight (volume) of water displaced 47 Roots, mycorrhizas and rhizosphere microbes. equivalent to the volume of the roots. The volume dry weight of the roots was determined of an independent material following the same procedure to determine root volume. The dry-weight of the experimental seedlings root biomass was then calculated using the recorded water displacement weight (vol) and volume dry weight value. Shoot dry weight was calculated by subtraction and corre sponding correction for moisture. By subtracting the dry weight in the beginning of the period from the dry-weight at harvesting, the increase in dry weight could be calculated. At the end of the treatment period the seedlings measured were lifted, washed free from sand, and separated into stem, nee dles and roots. The needles were further divided by age and the roots to into fine (diameter < 1 mm) and coarse (diameter > 1 mm) roots. Sample needles were selected and the length and the width of the needles were measured for the surface-area determinations. A coefficient to transform dry-weight values for needle surface area was obtained. The d fferent plant parts were then oven dried at 105 °C for 24 h after which their dry weights were determined. The tissue N concentrations were analyzed with the CSN-analys er. For the analyses of other elements the samples were digested in the mixture of HN03 and HCI04 by heating the samples step wise up to 250 °C. The element concentrations were measured by the plasma emission spectrometry (ICP). For the tracer addition, seedlings in photosynthesis measure ment were enclosed to the measurement cuvette and the tracer brought into the cuvette as NaH 14C0 3 from which 14C0 2 was re leased by addition of H2S04. In 1992 the seedlings were enclosed to the cuvette for 20 minutes, and in 1993 for one hour. The tracer levels used in the successive years were 0.03 and 0.09 pCi. 14 C0 2 respiration of the roots was measured after this for the rest of the measurement period. Tht evolved 14C0 2 was trapped by sucking air from the pot through Lumasorb II solution. The disintegration of the I4C was determined from the trap solution using a scintilla tion counter. For the determination of 14 C assimilated into the biomass, sub samples were prepared from the different plant parts after the seedlings were lifted and dried. The samples were oxidized in a sample oxidizer (Junitek) and the released carbon was trapped in a solution, the activity of which was determined. From these measurements the total activities of different plant parts were cal culated. 48 Figures 3a,b: The tran spiration (a) and photo synthesis (b) rates of the seedlings grown on MW or DW conditions on 1.7.1993. The lower wa ter potential in dry soil has decreased both the transpiration and photo synthesis rates. Results Photosynthesis and transpiration The results of the photosynthesis and transpiration measurements are still to be analyzed. For the purpose of an example some peri ods of measurement were calculated. Results from the first of July 1993 are presented here. During the day air temperature increased from 10 °C to 23 °C, and the water content in the air 49 Roots, mycorrhizas and rhizosphere microbes. was between 7 and 8 gm 3 . The soil water potential in dry treat ments was -35 kPa (DC and-42 (DW), and -12 kPa in moist treatments. For the seed! ngs growing in dry soil, the maximum transpiration rates were 8 mgm 2 s ' (DC) and 7 mgnvV (DW). In moist soil the maximal transpiration rates on that day were 10 mgnrV. It can be seen that the dry soil decreased the maximal transpiration rate, but especially in moist soil the temperature did not have any effect on transpiration rate (Fig. 3a). The photosyn thetically active radiation varied occasionally between 200-1100 pmolm 2s'. The rate of photosynthesis followed closely the inten sity of the radiation. In both moist treatments the maximum rates of photosynthesis were 200 pgm 2 s '. In DW treatment the maxi mum was 150 (jgm 2 s _l and in DC treatment 130 pgnrV (Fig. 3b). As the transpiration, also the photosynthesis rate was affected by the soil water potential. Growth In the first growing season, the soil water potential had greater effect on soil growth thjin soil temperature, seedlings in moist soil growing more than those in dry soil (Fig. 4). In 1993 both the seedlings as well as roots separately, were growing faster throughout the growing period in warm soil. The soil water potential did not seem to have an effect on growth. The growth rate of the seedlings growing in DW soil was remarkably high. The difference to other t-eatments was largest in the growth of fine roots. This response on growth rate is not in good agreement with the results seen on th e photosynthesis and transpiration rates on the example day. Any good explanation for this is not availa ble before the photosynthesis measurements have been analyzed in more detail. The explanation for the smaller effect of tempera ture on the first growing season can be in the fact, that for the first month there was no tenperature difference between the treat ments and the temperatures were also remarkable high during that period (Fig. 2a, 4a, b). In the beginning of the experiment the seedlings were not of equ il size and the effect of the original bio mass can be seen especially in the material collected during 1992. This had also effect on the differences detected between different treatments. The growth of the ne idles was highest in the DW treatment, and that of fine roots in M W soil. In 1993 the amount of dead nee dles was highest in seedlings grown in cold soil, only some of the needles formed in 1991 still alive. 50 Figures 4a, b. The weights of the seedlings presented as a treat ment mean of the sam ples collected over the growing season, (a) 1992, (b) 1993. The dry weight in the beginning of the experiment was calculated from the fresh-weight by a coeffi cient obtained from an independent material. Biomass allocation jn beginning of the experiment the root to shoot ratio was 2/5 . . , , ■ , , a in next growth period the mean for the whole material was 2/3. This can be an acclimation reaction to the lower nutrient supply from the forest soil compared to the conditions in nursery. xhe root to shoot ratio showed similar trend as the growth, the rat jo being highest in the seedlings grown on moist soil. The .... .. .. .. . . . WTr hl§hest root t0 shoot ratl ° was found in MW treatment (Fig. 5). 51 Roots, mycorrhizas and rtiizosphere microbes. Figure 5. The dry-weight based root to shoot ratio in 1995 presented as a treatment mean over the whole growth period. Figure 6. The root to shoot ratios in treatments at differen harvesting periods over the growth period 1993 measured as 14C bound to biomass. 52 Figure 7. The ratio be- tween 14C02 released from the pot as respira- tion and 14C bound to the root biomass. The treatments did not seem to have dramatic effect on the seasonal 14C-allocation pattern of the seedlings during the growth period. In general, an early spring peak on proportional root growth was followed by an increase in the allocation to the nee dles in late June and July. In August, when the new needles were not growing any more, but were producing effectively carbohy drates, the proportion of the carbon allocated to the roots in creased again (Fig. 6). Root respiration In the rate of respiration from the pot, remarkable differences were found between different treatments. The respiration expressed as a ratio to the 14C bound into the root biomass was highest in DW treatment and smallest in MC treatment (Fig. 7) The increase in soil water deficit and also higher soil temperature seemed to increase respiration. The respiration rate here consists of root respiration and microbial respiration in rhizosphere. All the labeled 14C released has anyhow been synthesized, trans ported and allocated by the seedling, because no I4 C was given directly to the soil. 53 Roots, mycorrhizas and rhizosphere microbes... References Axelsson, E. & Axelsson, B. 1986. Changes in carbon allocation pat terns in spruce and pine trees following irrigation and fertiliza tion. In: Luxmoore, R. J., Landsberg, J. J. & Kaufman, M. R. (eds.) Coupling of C arbon, Water and Nutrient Interactions in Woody Plant System:. Tree Physiol. 2 1-3: 189-204. Hari, P., Korpilahti, E., Pohja, T. & Räsänen, P.K. 1990. A field system for measuring the exchange of forest trees. Silva Fenn. 24: 21- 27. Linder, S. & Rook, D.A. ISB4. Effects of mineral nutrition on carbon dioxide exchange anc partitioning of carbon in trees. In: Bowen, G.D., Nambiar, E.K S. (eds.) Nutrition of plantation forests. Academic Press, London, pp. 211-236. Mäkelä, A. 1988. Performance Analysis of a Process-based Stand Growth Model Using Monte Carlo Techniques. Scand. J. For. Res. 3: 315-331. Nikinmaa, E. 1993. Analysis of the Scots pine; matching structure with function. Acta For. Fenn. 235. 54 55 Roots, mycorrhizas and rhizosphere microbes. Unbalanced nutrient status and mycorrhizal roots of Scots pine Sari Janhunen 1 , Anneli Ylimartimo 2 and Toini Holopainen 1 Introduction Recently there has been a wide interest in effects of excessive nitrogen on mycorrhizal roots, and the majority of the reports indicate negative effects < in mycorrhiza development (Alexander and Fairley 1983, Wallander and Nylund 1991, Holopainen and Heinonen-Tanski 1993). However, research on the effects of excess nitrogen on tree nutrition in relation to other nutrients, and mycorrhiza development, is still limited. The aim of this study was to assess the effects of unbalanced nutrient status involving excessive N, and deficient K, Mg and Ca concentrations, alone and in combination, on the development of mycorrhizal roots of Scots pine. Materials and methods Scots pine (Pinus sylvestris L.) seedlings in this factorial experi ment, with 16 nutrient combinations, were 1.5-yr-old. The seed ling material and growing methods are described in more details in Ylimartimo et al. (1991-)- Nutrients were given along with the irrigation water once a week during the growing season. The mass ratios of N, K, P, Ca and Mg in the optimal nutrient solution were 100:45:14:8:8. The aim of the treatments was to induce 1 Ecological Laboratory, Departmt nt of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. 2 Department of Forest Ecology, P.O. Box 24,00014 University of Helsinki, Finland. 56 deficient (0), normal (1), or excess (2) foliar N, K, Mg and Ca concentrations as follows: Combined needle and root samples were taken from each treatment for nutrient analysis at the end of September 1992. Ni trogen was analysed by the micro Kjeldahl method. Concentra tions of P, K, Ca, Mg and B in the needles were analyzed by ICP and AAS (Williams 1984), Ca, Mg and K of the roots were deter mined by AAS and P by molybdenum blue method (Allen 1989). The mycorrhizal short root tips were identified after staining in Ponceau S (Daughtridge et al. 1986), and the total number of mycorrhizas and short roots / 1 m was determined. The calcula tion was made from the total samples of about 100 cm, consisting of 1-5 cm long root pieces collected randomly throughout the root system. Results and discussion The dry masses of roots were highest in the seedlings with exces sive N and mostly with optimum K nutrition (Fig.l). In these seedlings the N and K concentrations in the needles (Ylimartimo et al. 1994) and in the roots were higher than in the seedlings with optimum N and deficient K nutrition. Nitrogen also affected the number of mycorrhizal short roots. Approximately 57% of root tips in the seedlings with optimum N treatment were mycor rhizal (living mycorrhizas), but in excessive N treatments only 23%, respectively. This difference was very significant (pcO.OOl). With optimum N treatment only 6% of the root tips were non-mycorrhizal, but in excessive N treatments even 26% (difference was significant, p<0.001), respectively. At the same time, the total number of short roots was significantly (p<0.001) lower in the seedlings with excessive N treatment (Fig. 2). The reason for lower mycorrhization with excess N is possibly the statistically significant decrease in the numbers of monopodal 57 Roots, mycorrhizas and rhizosphere microbes. Figure 1. The dry mass (means ± S.D.) of Scots pine roots according to foliar NKMgCa status, I.e. status of N, K, Mg, and Ca concentration levels in current needles: 0, deficient; 1, normal; 2, excess level (e.g., 2101 indicates a combi nation of excess N, nor mal K and Ca, and defi cient Mg concentrations. One level marked with + is considered a border line case). Stars indicate means significantly (p<0.001) greater than in one or more of the other means marked without stars (according to Tuk ey's Multiple Range Test). Figure 2. The total number of short roots / 100 cm (including both the mycorrhizal and non mycorrhizal short roots, means ± S.D.) according to foliar NKMgCa status. Stars indicate means sig nificantly (p<0.001) greater than in one or more of the other means marked without stars (ac cording to LSD Multiple Range Test). (pcO.OOl), dichotomously branched (pcO.OOl) and dark stained and black mycorrhizas (p <0.010). Deficient K nutrition increased the total number of short roots (Fig. 2.), which appeared to be related to significant (p<0.01) increase of old mycorrhizas. The effects of N on mycorrhiza formation are well reported. How ever, the effects of K are not so well known. The other nutrients 58 did not have any drastic effects on the development of short roots, though Mg and Ca concentrations in the roots were lower in deficient treatments compared to optimum treatments. Conclusions Excessive N reduces very clearly the short root development and the numbers of mycorrhizal short roots, which is in agreement with several earlier observations (Alexander and Fairley 1983, Wallander and Nylund 1991, Holopainen and Heinonen-Tanski 1993). Besides nitrogen, potassium also has a role in controlling the development of mycorrhizas in Scots pine roots. Deficient K nutrition increases the total number of short roots by increasing the proportion of old mycorrhizas. References Alexander, I.J. & Fairley, R. I. 1983. Effects of N fertilization on popu lations of fine roots and mycorrhizas in spruce humus. Plant and Soil 71: 49-53. Allen, S.E. 1989. Chemical analysis of ecological materials. Blackwell Scientific Publications. Great-Britain. 369 p. Daughtridge, A.T., Boese, S.R., Pallardy, S.G. & Garrett, H.E. 1986. A rapid staining technique for assessment of ectomycorrhizal infection of oak roots. Can. J. Bot. 64: 1101-1103. Holopainen, T. & Heinonen-Tanski, H. 1993. Effects of different nitro gen sources on the growth of Scots pine seedlings and the ul trastructure and development of their mycorrhizae. Can. J. For. Res. 23: 362-372. Wallander, H. & Nylund, J.-E. 1991. Effects of excess nitrogen on car bohydrate concentration and mycorrhizal development of Pinus sylvestris L. seedlings. New Phytol. 119: 405-411. Williams, S. (ed.) 1984. Official methods of analyses of the Association of Official Analytical Chemists. 14th edition. Virginia. 1141 p. Ylimartimo, A., Pääkkönen, E., Holopainen, T. & Rita, H. 1994. Unbal anced nutrient status and epicuticular wax of Scots pine needles. Can. J. For. Res. 24: 522-532. 59 Roots, mycorrhizas and rhizosphere microbes... Rhizodeposition in arable crops Bendt Jensen Introduction Quantification of the annual input of crop residues to arable soils is a key issue in the study of soil organic matter dynamics. Unlike inputs from above-grouid plant parts, quantification of the below-ground C input is much more complicated. Isolation of roots by soil washing ur derestimates the input to soil of root related C. A considerable portion of the fine roots and root-hairs is lost during the soil washing procedure. Root exudates, leakates, lysates and sloughed-off cells are not accounted for. Most of the C lost during the soil washing procedure is very labile and influences the soil microbial biomass and activity. In order to quantify and distinguish root-derived C from in digenous soil organic C, 14 C0 2-labelling of plant tops has been used. Most studies under controlled conditions in the laboratory are based on a continuous exposure of plant tops to 14 C0 2 . Under field conditions, pulse-labelling with 14 C0 2 is easier to handle compared to continuous 1 ibelling, and series of pulses at regular intervals during the growth period have been found to provide a reasonable estimate of the cumulative below-ground C input. Experiments under field conditions have been based on label ling periods ranging froir 15 min to 2 hours, and it has been as sumed that the time of labelling during the day was of minor importance for the distribution of assimilated C. However, this as sumption is questionable since temperature and light intensity are subject to diurnal and seasonal fluctuations. This project examine:; the rhizodeposition from cereal crops under field conditions. Effects of labelling time and light intensity on assimilate distribution in spring barley were also included. Dis tribution of assimilates in three cruciferous species was exa- Danish Institute of Plant and Soil £ cience, Department of Soil Science, Research Centre Foulum, P.O. Bo> 23, DK-8830 Tjele, Denmark. 60 Figure 1. Equipment used in the field for 14CO2-gen eration, temperature con trol and photosynthesis chamber. mined under different autumnal conditions (low light and temper ature regimes). Materials and methods Figure 1 shows the mobile system developed for 14 C-pulse label ling plants under field conditions (Jensen 1993). The system allows the rhizosphere respiration (root and microbial respira tion) to be determined during and after labelling by placing a seal between above- and below-ground compartments. The labelling system was used during the growing seasons 1990 and 1991 to determine the rhizodeposition of spring- and winter barley, respectively (Jensen 1993, 1994 a). Crops were la belled at four growth stages, each labelling lasting 8 h. The barley plants were grown in stainless steel cylinders (20 cm diam., 50 cm deep) that were pushed into the soil. Decomposition of labelled C left in the root-soil system after labelling was followed for 3 to 5 months in the winter barley experiment. 61 Roots, mycorrhizas arid rhizosphere microbes. In two additional experiments the below- and aboveground plant parts were not separated and consequently the rhizosphere respiration was not measured. The effect of labelling time (IV2, 4 or BV2 h after onset of light) and of light intensity (75 and 160 W m" 2 ) on the distribution of 14 C in spring barley was examined in a green house experiment (Jensen 1994b). The effect of temperature (5 and 10° C) on assimilate distribu tion in three cruciferous species (winter rape, turnip and white mustard) was examined in temperature regulated rooms (Jensen 1995). The effects of light intensity (75 and 160 W m" 2 ) and tem perature (5 and 10 and 20''C) were examined for white mustard. Results and discussion Distribution of labelled C Table 1 shows the distribution of labelled C in spring barley six days after labelling. During early elongation (29 May), 36.7% of the recovered C was translocated below-ground. Equal amounts of 14 C are generally translocated below-ground and retained in the shoots at earlier growth stages and thus 43.2% of 14 C recovered was translocated below-ground during tillering of winter barley. The proportion of 14 C recovered below-ground decreased with plant age. Rhizosphere respiration accounted for 1.5-8.3% and 2.5-32.5 of the 14 C recovered in spi ing (Table 1) and winter barley, respec tively. This corresponds t3 20-67% and 18-69%, respectively, of 14 C translocated below-giound. Estimated C input to the soil during the growing season When estimating the tota] C input to the soil during the growing season of spring barley, it is assumed that the instantaneous total below-ground C-production (Table 1, lower part) can be applied to periods before and after each labelling date. The growing sea son was set to 95 days (22 days after sowing to 10 days before maturity). Values from the first, second, third and fourth labelling were taken to represent 45, 20, 15 and 15 days periods, respec tively. Furthermore, it was assumed that two times more 14 C would have been transl seated below-ground if the labelling period had been 24 hours. Consequently, the amount of labelled C retained in the macro-rool free soil for more than 6 days was esti mated to 95.5 g C m~ 2 yr" 1 . As macro-root C (30.3 g C m" 2 yr" 1 ) is 62 Table 1. Distribution of 14 C recovered and estimated C-production (g C m- 2 labelling" 1 ) as measured 6 days after labelling spring barley. Values are given for macro-roots, soil plus micro-roots, rhizosphere respiration, total below-ground and shoot as mean of 3 replicates (standard deviation in parenthesis). eventually left in the soil as dead root biomass, this contribution 9 1 was included too. Rhizosphere respiration (39.4 gC m yr") made up 23.3% of the total amount of 14 C translocated below ground during the growth period, the total amount of C translo cated below-ground being 165.2 g C m~ 2 yr" 1 . Figure 2 shows the amount of spring barley macro-root C and the cumulated below-ground labelled C. At maturity (7 August) only 45.0 g C m" 2 could be isolated in macro-roots. Thus macro roots isolated at harvest made up only 36% of labelled C retained in the soil for more than 6 days corresponding to 27% of the C translocated below-ground. Total below-ground input correspon ded to one-third of above-ground C harvested at maturity. In the winter barley experiment the growing season was set to 120 days that is from 13 March, when the soil temperature ex ceeded 5° C in a depth of 30 cm, to 10 days before maturity (2 August). The amount of labelled C retained in the macro-root free soil, in macro-root C and in rhizosphere respiration was 96.4, 47.2 and 93.6 g C m" 2 yr" 1 , respectively. The total amount of C translocated below-ground was 237.2 g C m" 2 yr" 1 , of which 45% was rhizosphere respiration. At maturity (2 August), only 79.7 g C m" 2 could be isolated in macro-roots. For both crops, the cumulated amounts of labelled C retained in macro-roots was much smaller than the amounts of macro-root- C isolated at maturity by soil washing. Labelling Labelled Whole soil minus Rhizosphere Total below Shoot date macro-roots macro-roots respiration ground % of 14 C recovered 29 May 7.6 (2.1) 20.7 (3.0) 8.3 (2.2) 36.7 (3.5) 63.3 (3.5) 21 June 2.0 (0.1) 7.8 (6.5) 4.4 (0.7) 14.3 (6.3) 85.7 (6.3) 4 July 1.0 (0.4) 5.8 (7.1) 1.5 (0.5) 8.3 (6.7) 91.7 (6.7) 18 July 0.7 (0.4) 1.1 (0.6) 3.0 (0.9) 4.7 (0.7) 95.3 (0.7) g labelled C m" 2 29 May 0.30 (0.3) 0.87 (2.5) 0.33(1.9) 1.50 (0.4) 2.55(1.4) 21 June 0.07 (0.1) 0.31 (1.6) 0.16(0.1) 0.54 (0.3) 3.13(4.6) 4 July 0.03 (0.1) 0.17(1.2) 0.05 (0.1) 0.24 (0.2) 2.71 (4.6) 18 July 0.01 (0.0) 0.01 (0.0) 0.04 (0.2) 0.06 (0.0) 1.15(3.0) 63 Roots, mycorrtiizas and rhizosphere microbes. Figure 2. Spring barley experiment. Comparison of macro-root biomass from root wash (g C m" 2 ) and cumulated total amount of C translocated below-ground. Other studies have estimated the total below-ground input from wheat to 900-2950 kg C ha" 1 yr" 1 . Lower values of 475 and 583 kg C ha" 1 yr" 1 in whe it and barley, respectively have been re ported in experiments, where the 14 C0 2 labelling was carried out between 8.45 and 10.15 am and the plant-soil-systems harvested after 24 h. The 14 C movement between above- and below-ground plant parts has been shown to occur mainly between labelling and day 5, and the time of labelling during the day may affect the as similate distribution when short pulses are applied. Exudation from roots may be overestimated if roots are not re moved from the soil soon after shoot detatchment. Part of the 14 C in the soil plus root may tl lerefore be material released from dying and lysing root cells. This if the plants were allowed to grow on beyond 6 days after labelling, part of the 14 C found in the soil in this study would not have been released to the soil but retranslo cated to the shoot later in the growth period. The estimated total input of C into the soil may therefore overestimate the "true" rhizodeposition. Based on similar assumptions as applied to the below-ground production of spring barley (Table 1), the total shoot production can be calculated to 470.3 g C m~ 2 . At maturity the yield of straw plus grain was 534.9 g C m" 2 in adjacent reference plots not ex- 64 Figure 3. Winter barley experiment. The relative amounts of labelled C left in the root-soil sys tem 6 days after each la belling, at maturity and on 11 October. The amount of labelled C translocated below ground after labelling is set to 100%. posed to the labelling procedure. Corresponding harvest yield in plots passing the labelling procedure on 18 July was 467.9 g C m" 2 . Decomposition of below-ground labelled C (winter barley) In the winter barley experiment, decomposition of labelled C left in the root-soil system was followed for 3-5 months (Fig. 3). The amount of labelled C left in the root-soil system 6 days after labelling plus labelled C in rhizosphere respiration (root- and microbial respiration) during these 6 days was set to 100%. The decomposition of labelled C following day 6 was slower than in day 1-6. At maturity (2 August), 24-82% of total below-ground translocated C was left. On 11 October the corresponding figures were 41-50%. Of the labelled C left in the root-soil system 6 days after label ling, 65-98% was still retained at maturity and 69-73% on 11 Oc tober. Relating these percentages to the estimated total cumulated C left in soil 6 days after labelling (143.6 g C m" 2 ), about 117 g C m" 2 was still in soil at plant maturity. Effect of labelling time, light intensity and temperature The green house experiment showed, that neither the time of labelling nor the light intensity significantly influenced the 14 C distribution four hours after labelling start (Table 2). However, 65 Roots, mycorrhizas and rhizosphere microbes. Table 2. The proportion of 14 C ecovered below-ground in percent of total 14 C recovery. Mean of three pots. Values in each column followed by the same letter are not significantly different at P=0.05 (standard deviation in brackets). when exposed to low light intensity the percentage of 14 C in the soil-root system tended tc be higher (15.4% and 6.0% at late till ering and late elongation, respectively) than when exposed to high light intensity (12.1% and 5.2%, respectively). Also, the percentage of 14 C in the soil-root system tended to be higher early in the morning (1.5 h after onset of light) than later during the day. Under field conditions, the light intensity is generally higher in the middle of the day ttian in the morning. In that respect it is interesting to see, that the distribution of 14 C in the morning (low light) is significantly different from the distribution at higher light intensities in the middle of the day. The present results suggest that the total C-input to the soil may be overestimated, when the distribution and the total below-ground C translocation of photo syntate is based on a short pulse-labelling very early in the morn ing of a predominant sunny day. The temperature regulated room experiment demonstrated that the temperature effec r. on assimilate distribution in three cru ciferous species was small (Table 3). 7 weeks after planting all three species showed an increasing below-ground -to- shoot ratio of labelled C by increasing temperature, whereas the ratio de creased by temperature 11 weeks after planting. The only signifi cant effect of temperature was found for winter rape 11 weeks after sowing. The effect of temperature may depend on plant de velopment and the effect may be greater in winter rape which is physiologically able to adapt to low temperatures with the pur pose of living through the winter. In a second experiment with white mustard, the effect of tem perature and light intensity was examined in more detail. Again there was no significant effect of temperature. As in the green Time after onset Light intensity Late tillering Late elongation of light (h) 1.5 low A 19.8 (5.2) A 7.6 (0.9) 4.0 low ab15.4 (1.5) ab 6.0 (1.2) 4.0 high B 1 2.1 (3.0) b 5.2 (0.8) 8.5 low ab 16.0 (1.8) ab 6.4 (1.1) LSD 95% 6.1 1.9 66 Table 3. Total below-ground -to- shoot ratio of labelled C determined 6 days after labelling of plants labelled at different temperature and plant age. Mean of three pots. Values in each column followed by the same let ter are not significantly different at P=0.05 (standatd deviation in brackets). house experiment the below-ground -to- shoot ratio of labelled C tended to increase by decreasing light intensity. Refrences Jensen, B. 1993. Rhizodeposition by 14 C-pulse-labelled spring barley grown in small field plots on sandy loam. Soil Biol. Biochem. 25: 1553-1559. Jensen, B. 1994 a. Rhizodeposition by field-grown winter barley ex posed to 14 C0 2 pulse-labelling. Appl. Soil Ecol. 1: 65-74. Jensen, B. 1994b. Distribution of 14 C in pulse-labelled spring barley: Effect of light intensity and length and photoperiod before label ling. Acta Agricultural Scandinavica. Sect. B, 43 (in press). Jensen, B. Effect of autumnal conditions (low temperature and light intensity) on assimilate distribution in three cruciferous species with different cold tolerance (in preparation). Species Weeks after Temperature Winter rape White mustard Tyfon sowing (°C) 7 5 b 0.28 (0.06) A 0.40 (0.11) AB 0.37 (0.15) 7 10 ab0.34 (0.04) A 0.61 (0.26) A 0.51 (0.09) 11 5 A 0.38 (0.03) A 0.68 (0.15) AB 0.37 (0.07) 11 10 B0.27 (0.05) A 0.56 (0.02) B 0.32 (0.05) LSD 95% 0.082 0.293 0.175 67 Roots, mycorrhizas and rhizosphere microbes. Tomentelloid fungi (Basidio mycetes, Thelephoraceae s. str.) - are they true mycor rhizal fungi? Urmas Kõljalg The fungal family Thelephoraceae sensu strictu contains well known (generally accepted) mycorrhizal genera as Bankera Coker and Beers ex Pouzar, Boletopsis Fayod, Hydnellum P. Karst., Phellodon P. Karst., Sarcodon Quelet ex P. Karst. and Thelephora Ehrh.: Fr. There are also six resupinate genera (viz. Pseudotomentella Svrcek, Tomentella (Pers) Pat., Tomentellago Hjortstam & Ryvarden, Tomentellastrum (Bourd. and Galz.) Svrcek, Tomentellina Höhnel and Litsch. and Tomentellopsis Hjortstam) with over 100 species in use (Hjortstam 1974, Hjorts tam and Ryvarden 1988, Larsen 1971, 1974, 1981) belonging to this family. The term tomentelloid fungi has been used for all of them. Their fruitbodies occur mainly on the underside of well decayed wood and inside litter in forest ecosystems. It has been asserted that tomentelloid fungi can form ectomyccorhiza (Miller 1982), and the term Torr entella-like. has been used for several unidentified ectomycorrh iza (Danielson and Pruden 1989). The basidiospores of Tomentella crinalis (Fr.) M. J. Larsen have been used for synthesizing mycorrhiza with Pinus sylvestris L. seed lings under sterile conditions (Köljalg 1992). Up to present time the tomentelloid fungi have been investi gated mainly by taxonomists or so called organism-mycologists. The main reason for this is that the germination of spores and iso lation from fruitbody tissue on artificial media as rule have failed. Only known species of these fungi in culture is T. crinalis. The Institute of Zoology and Botany, 21 Vanemuise St, EE-2400 Tartu, Estonia. 68 aqueous suspension of basidiospores of this fungus and one week old seedlings of P. sylvestris were introduced simultaneously into 500 ml flasks, containing vermiculite and ground peat moss mois tened with modified Melin-Norkrans nutrient solution, so that the spores fell close to and on the radicle. The basidiospores germi nated in two flasks of four and mycorrhiza developed which re sembled, morphologically as well as anatomically, ectendomyc orrhiza. The negatively geotropic fruitbodies, with toothed hy menophore typical of T. crinalis, also formed on the substrate (Köljalg 1992). Several species like Pseudotomentella tristis (P. Karst.) M. J. Larsen and Tomentella punicea (Alb. and Schw.: Fr.) Schröter can form their fruitbodies under forest litter near tree roots, and these species have rhizomorphs connected with roots (Köljalg 1992). Our knowledge is very limited on the ecology of tomentelloid fungi. We cannot answer exhaustively for the questions like: Are tomentelloid fungi mycorrhizal, weakly pathogenic or sapro trophic fungi? What is the task of these fungi in carbon cycling in forest belowground ecosystems? The sum of authors observations on the ecology of tomentel loid fungi are briefly as follows: 1. The fruitbodies of tomentelloid fungi come out mainly in old forests and usually on the underside of well-decayed logs or inside litter. Both of them are as rule full of living roots of trees. For example the fruitbodies of most species of Pseu dotomentella are collected from old mixed coniferous forests in Europe, Asia and North-America. The P. tristis is almost only species of Pseudotomentella which can fruit (we do not know anything about mycelial) in comparatively young or managed forests. 2. Also, the fruitbodies can develop on recently fallen trees but in this case the mycelia is distributed (if it is possible to follow it) in the litter. It means that nutrients can arrive from other source. 3. Sometimes we can follow mycelia or rhizomorphs emanating from the fruitbodies up to the living roots of trees. 4. We do not know how long the individual mycelia have been growing before fruitbody will develop and what is the size of area the individual mycelia can colonize. From all what has been said above we can conclude that the fruitbodies of tomentelloid fungi come out first of all in the late successional stages of plant communities (old coniferous, mixed or broad-leaved forests, old wood-meadows) and the mycelia 69 Roots, mycorrtiizas and rhizosphere microbes. (when it comes we do not know) colonize mainly litter and well decayed wood. There are probably different successors of mycor rhizal fungi in different succession stages of forest. If we accept that tomentelloid fungi co ild be weakly pathogenic or mycorrhiz al then they are probably late successors which colonize special substrate - old logs and litter. As we know the well-decayed logs are like seed panks and often there are numerous seedlings grow ing on these logs. In this case it is possible that tomentelloid fungi have, among others (viz. Amphinema byssoides (Fr.) J. Eriksson), weakly pathogenic or mutually beneficial relationships with those seedlings. Finally, in this stage of knowledge we can just suppose that to mentelloid fungi are true roycorrhizal fungi and call them non-tra ditional mycorrhizal fungi. References Danielson, R. M. & Pruden, M. 1989. The ectomycorrhizal status of urban spruce. Mycologia 81: 335-341. Hjortstam, K. 1974. Studies in the Swedish species of the genus Tomen tella (Thelephoraceai). 111. The genus Tomentellopsis. Svensk Botanisk Tidskrift 68: 51-56. - & Ryvarden, L. 198fc. Tomentellago gen. Nov. (Thelephoraceae, Basidiomycetes). M>cotaxon 31: 39-43. Köljalg, U. 1992. Mycorrhiza formed by basidiospores of Tomentella crinalis on Pinus syhestris. Mycol. Res. 96: 215-220. Larsen, M. J. 1971. The genus Pseudotomentella (Basidiomycetes, Thelephoraceae s. str ). Nova Hedwigia 22: 599-619. - 1974. A contribution to the taxonomy of the genus Tomentella. Mycologia Memoirs i, 145 p. - 1981. The genus Tonentellastrum (Aphyllophorales, Thelepho raceae s. str.). Nova Hedwigia 35: 1-16. Miller, O. K. 1982. Taxono ny of ecto- and ectendomycorrhizal fungi. In: Schenck, N. C. (ed.). Methods and Principles of Mycorrhizal Research. The American Phytopathological Society, St. Paul, Minn. pp. 91-101. 70 71 Roots, mycorrhizas and rhizosphere microbes. Ectomycorrhizal fungi and fungicides Tarja Laatikainen and He lvi Heinonen-Tanski Introduction This study deals with the fungicide effects on ectomycorrhizal fungi of coniferous trees in Finland. The effects on the rhizo sphere microbial population on plant roots are also studied. Addi tionally, the capability of ectomycorrhizal fungi to degrade those fungicides is determined. Microorganisms in the rhizosphere have a marked influence on the growth of plants. Microbial population benefits the plant in many ways: by increasing, recycling and solubilization of mineral nutrients; by synthesizing vitamins, amino acids, auxins and gib berellins, which stimulate plant growth; and by producing antibi otics against potential plant pathogens (Atlas and Bartha 1992). Ectomycorrhizal fungi ha ve the most important role in this asso ciation because of their close relationship to plant roots. The al location and partitioning of carbon provide resources to plants for acclimation to environmental stress (Geiger and Servaites 1991). Ectomycorrhizal fungi tat e part in that allocation, for example, by transporting carbon betwe en plants. Pesticides have widely been used in the forests and nurseries of forest trees against fungal diseases, weeds and herbivores. However, only little is known about the effects of those pesticides on the ectomycorrhizal fungi and other microorganisms. Pesticide use has been more common in agriculture fields, especially in in dustrial countries. Many of these pesticides are persistent in soil and can, therefore, effect seedling growth when those fields will be afforested. This can be a greater problem in Finland than in lower latitudes because of cold climate: the half life of pesticides can be much longer here (Heinonen-Tanski 1989). Department of Environmental Scie ices, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. 72 In an earlier study the fungicides have proved to be the most toxic to ectomycorrhizal fungi of Scots pine and Norway spruce (Laatikainen and Heinonen-Tanski 1994). Three herbicides, hex azinone, chlorthiamid and linuron, two fungicides, benomyl and propiconazole, and an insecticide cypermethrin were tested with 56 different mycorrhizal fungi taken from our culture collection. Concentrations were 1 ppm and 10 ppm for fungicides and cyper methrin and 1 ppm for herbicides. The fungicides tested in this study proved to be more toxic to mycorrhizal fungi than herbi cides and insecticide cypermethrin. Fungicide propiconazole had the clearest inhibitory effect on mycorrhizal growth. Material and methods In the summer 1994 a research was started to find out if fungi cides copper oxychloride, propiconazole and chlorothalonil have any effect on ectomycorrhizal fungi of pine seedlings. These fun gicides are some of the commonly used fungicides at forest nurs eries in Finland. Two-years-old Scots pine seedlings were grown in sand-peat filled pots during the summer. After the period of growth the seed lings were treated with one of these fungicides at the same con centrations and the same intervals as recommended at the nurseries of forest trees. The fungicide was given straight to the surface of the pot. There was a control group of seedlings which was not treated at all. The first sampling was performed two weeks after the first treatment. The seedlings were cut off and the needles were col lected and frozen for the later analysis of residues of the pesticides and their major metabolites. The surface soil (0-5 cm) of the pots was separately collected. The samples from the surface soil and the rest of the soil from the pots were dried at room temperature and frozen for the later residue analysis. The activity of soil microorganisms of the surface soil (0-5 cm) was examined as soil respiratory measurement, and by the ac tive bacteria counts, and measuring active hyphal lengths. Soil respiration was performed as where 0.1 M NaOH-solution was used to trap C02 (Paul and Clark 1989). The numbers of active bacteria and active hyphal lengths were esti mated by direct observation after FDA staining with epifluores cent microscopy. The toxic effects of fungicides on the ectomycorrhizal fungi will be obtained by ultrastructural observations with electron mi- 73 Roots, mycorrhizas and rtiizosphere microbes. croscopy. The roots of the seedlings were cleaned with tap water on sieves and the samples from the short-root tips infected by dif ferent type of mycorrhiza I fungi were taken and stained for later observation. The short roc ts of different type of mycorrhizal fungi present will be counted according to five classes (Holopainen and Heinonen-Tanski 1993). The fungicides and their major metabolites both from needle and soil samples will be analyzed by GLC. The temperature and the rainfall during the test period were measured and recorded by datalogger and computer. Discussion Healthy mycorrhizal fungi are important and even necessary for the proper growth of seedlings at the nurseries and for the suc cessful start for development of the seedlings after the plantation (Halonen and Laiho 1993)- The inoculation of ectomycorrhizal fungi to the soil of nurseries have proved to produce marked decrease in mortality of host trees (Atlas and Bartha 1992). Only little attention has been paid on the resistance of mycor rhizal fungi to the pesticides, despite of the extend usage of those pesticides. Some pesticides have been studied earlier (Laiho and Mikola 1964, Marx and Rowan 1981), but the use of the most of those pesticides has been rejected nowadays, except benomyl. Pesticides, specially fungicides which are used for fungal diseas es, seem to affect also the ectomycorrhical fungi of the plant pro tected. Cultivations on pt tri dishes have shown inhibitory effect of commonly used fungicides on the most of the ectomycorrhizal fungi of coniferous trees tested (Laatikainen and Heinonen-Tan ski 1994). In some studies, when some significant pesticide ef fects on soil microbial populations and activities have been indicated, recovery has generally been rapid (Tu 1978, Tu 1993). Surviving microorganism 5 had replaced the sensitive species and, thus, maintaining the metibolic integrity of the soil (Tu 1978). Information of fungal capability to degrade pesticides may help to decide which pesticides would be less harmful when using them at the nurseries of forest trees and in the forests (Glad et al. 1981, Tu 1993). Martin et al. (1991) discovered that rate of micro bial degradation of certain fungicides was faster in soil previously treated with these same fungicides than in untreated soils. The mi croflora, which is able to degrade those fungicides, increases as the treatments are repealed (Martin et al. 1991, Donelly et al. 1994). Both selective inl ibitory and stimulatory effects of pesti- 74 cides due to concentration used on microorganisms have been re corded. For instance, phenoxyacetic acid-containing herbicides had slightly stimulatory effect on the mycorrhizal fungi at low concentrations (Dasilva et al. 1977). Biodegradation can be a result of cooperation of different mi croorganisms. For example, combinations of various bacteria strains were shown to degrade the PCBs more effectively (Donel ly et al. 1994). Synergist effects between fungi and bacteria in degradation of pesticides have also been observed (Levanon et al. 1994). At the nurseries of forest trees simultaneous treatments with fungicides and herbicides are common. Furthermore, sometimes one fungicide during the summer and another at the end of the au tumn are used with the same seedlings at nurseries of forest trees. There is no information how all these pesticides will affect togeth er ectomycorrhizal fungi of seedlings. Coappearance of different pollutants and their metabolites has shown to reduce biodegrada tion by microorganisms in the same cases (Burback et al. 1994). Different fungicides can affect fungi in different ways. Cop per oxychloride, one of the fungicides tested in this study, is de graded to Cu 2+ -ions, which are very persistent is soil. Cu 2+ -ions are known to accumulate into cells, especially into spores, which causes the coagulation of cell proteins. Cu 2+ has shown to inhibit some and stimulate the other bacteria to degrade insecticide par athion (Tchelet et al. 1993). Propiconazole is the sterol biosynthesis inhibitor (SBI) fungi cide. In laboratory tests, low dosages of propiconazole stimulated the soil respiration (Elmholt 1992). The stimulation may have been due to the fungicide causing a sub-toxic stress effect, result ing in a diversion of carbon from biosynthesis to maintenance en ergy requirements. Chlorothalonil is a non-systemic foliar fungicide. Tests with chlorothalonil have shown significant in crease in oxygen consumption from the decomposition of organic matter indigenous to the soil. Suppression of invertase and amy lase enzymatic activities for one day was also observed. The in hibitory effect disappeared after two days (Tu 1993). Most of the side-effect studies of pesticides are performed as laboratory experiments and usually at higher temperatures than occurring here in Finland. Furthermore, the results from labora tory side-effect tests can not be extrapolated to the field situation. It has been proved that the side-effects in the field can be pro voked at dosages considerably lower and they may last longer than in the laboratory, because the treatments affect the soil mi croorganisms indirectly (Elmholt 1992). Therefore, field studies 75 Roots, mycorrtiizas and rhizosphere microbes... are needed for assessing which pesticides and in which concentra tions could safely be used in plant protection. The information how fungicides affect cell structures of ecto mycorrhizal fungi will help to analyze the mechanisms by which the fungal growth and, thereby, the growth of seedling is disturbed by fungicide. That may be important when considering, why so many seedlings will die when planted to forests or old agricultural fields. New information i bout pesticide effects on microbiologi cal activity and nutrient contents of soil will also be important. This knowledge can also be useful when developing new biotech niques for fungi. References Atlas, R.M. & Bartha, R. 1992. Microbial Ecology. Fundamentals and Applications. Third Edition. The Benjamin/Cummings Publish ing Company, Redwood City, CA. 563 p. Burback, 8.L., Perry, J.J. & Rudd L.E. 1994. Effect of environmental pollutants and metabi »lites on a soil mycobacterium. Appl.Micro biol. Biotechnol. 41: 134-136. Dasilva, E.J., Henriksson, L E. & Urdis, M. 1977. Growth responses of mycorrhizal Boletus and Rhizopogon species to pesticides. Trans. Br. Mycol. Soc. 68(3): 434-437. Donelly, P.K., Hedge, R.S. & Fletcher, J.S. 1994. Growth of PCB degrading bacteria on compounds from photosynthetic plants. Chemosphere Vol. 28(5): 981-988. Elmholt, S. 1992. Effect of propiconazole on substrate amended soil respiration following laboratory and field application. Pestic. Sci. 34: 139-146. Geiger, D.R. & Servaites, J.C. 1991. Carbon allocation and response to stress. In: Mooney, H., Winner, W.E. & Pell, E.J. (eds.). Response of Plants to Multible Stresses. Academic Press, San Diego, p. 103-127. Glad, G., Göransson, 8., Popoff, T., Theander, O. & Torstensson, N.T.L. 1981. Decomposition of linuron by fungi isolated from soil. Swedish J. Agrc. Res. 11: 127-134. Halonen, A. & Laiho, O. 1991. Mycorrhizae of afforested fields. Developing methods for afforestation of fields, Interim report (eds. Ferm, A. & Polet, K.), The Finnish Forest Research Insti tute, Kannus Research Station No 463. Heinonen-Tanski, H. 1989. The effect of temperature and liming on the degradation of glypliosate in two arctic forest soils. Soil Biol. Biochem. 21: 313-317. Heinonen-Tanski, H. & Hobpainen, T. 1991. Chapter 18. Maintenance of ectomycorrhizal ungi. Methods in Microbiology 23: 413- 422. 76 Holopainen, T. & Heinonen-Tanski, H. 1993. Effects of different nitro gen sources on the growth of Scots pine seedlings and the ultrastructure and the development oh their mycorrhizae. Can. J. For. Res. 23: 362-372. Juuti, S. & Ruuskanen. J. 1993. Trichloroacetic acid - a secondary organic air pollutant. Proceedings First Finnish Conference of Environmental Sciences pp. 109-112. Kott, J. A. 1993. Results of Rothamsted experiments on soil tillage, ero sion and leaching of nutrients and pesticides. NJF-seminarium 228: Soil tillage and environment pp. 6-27. Laatikainen, T. & Heinonen-Tanski, H. 1994. The effects of pesticides on the ectomycorrhizal fungi, Fourth European Symposium on Mycorrhizas, Granada 11-14 July, Abstracts p. 197. Laiho, O. & Mikola, R 1964. Studies on the effect of some eradients on mycorrhizal development in forest nurseries. Acta For. Fenn. 77: 3-34. Levanon, D., Meisinger, J.J., Dodling, E.E. & Starr, J.L. 1994. Impact of tillage on microbial activity and the fate of pesticides in the upper soil. Water, Air Soil Pollut. 72: 179-189. Marx, D.H. & Rowan, S.J. 1981. Fungicides influence growth and development of specific Ectomycorrhizae on Loblolly pine seed lings. For. Sci. 27: 167-176. Martin, C., Davet, P., Vega, D. & Coste C. 1991. Field Effectiveness and Biodegradation of Cyclic Imides in Lettuce Field Soils. Pes tic. Sci. 32: 427-438. Paul, E.A. & Clark, F.E. 1989. Soil Microbiology and Biochemistry. Academic Press, San Diego. 275 p. Tchelet, R., Levanon, D., Mingelgrin, U. & Henis, Y. 1993. Parathion degradation by a Pseudomonas sp. and a Xanthomonas sp. and by their crude enzyme extracts as affected some cations. Soil Biol. Biochem. 25: 1665-1671. Tu, C.M. 1978. Effect of pesticides on acetylene reduction and microor ganisms in sandy loam. Soil Biol. Biochem. 10: 451-456. Tu, C.M. 1993. Effect of fungicides, captafol and chlorothalonil, on microbial and enzymatic activities in mineral soil. J. Environ. Sci. Health. B28(l): 67-80. 77 Roots, mycorrtiizas and rhizosphere microbes. Variation in the amount of organic carbon in soil within a forest stand: effect of trees and implications for sampling Jari Liski Introduction Soil properties, both physical and chemical, vary considerably even within distances of a few meters (e.g. Ilvesniemi 1991, Järvinen et ai. 1993). In forests, besides the geological factors, trees are important causes for the variation (Zinke 1962). In addi tion to the importance for designing an effective soil sampling, a thorough description of the variation can also provide useful information on the processes that generate the variation. Materials and methods For studying the variatior in the amount of organic carbon (kg C/ m 2), a total of 99 soil cores (50 cm deep) were taken from a 4 x 8 m grid in a 130 year old Scots pine (Pinus sylvestris L.) stand on a glaciofluvial sand depoiit in southern Finland (Fig. 3). One 4 x 4 m half of the grid was placed under tree canopies and the other in a small within-stand opening. In order to study the effect of trees in more detail, 27 a Iditional cores were taken around three trees. Thicknesses of the horizons of the podzolized soil (F/H, E, B) were measured on the cores. Then, the cores were divided into the organic F/H and 0-10 cm (E and top of B horizon), 10-20 cm (middle layer of B horizon) and 20-40 cm (bottom of B horizon and top of C layer) mineral soil layers for analyzing the amount Department ot Forest Ecology, P.O. Box 24, FIN-00014 University of Helsinki, Finland. 78 of carbon. For 0-40 cm mineral soil layer, the amount was obtained by totalling the amounts in the sublayers. The number of samples, n, needed for given confidence, d, in 1 "2 the mean estimate was assessed using the formula n=(z s d ) , where s is the standard deviation of the data and z the 95 % fractile of Student's t-distribution (Ranta et ai. 1989). Semivariograms were used for studying spatial dependence and ordinary block kriging for interpolating the values for 20 x 20 cm blocks at the study site (Isaaks and Srivastava 1989). Results and discussion The amount of carbon varied 4-8 fold within the soil layers and the coefficients of variation ranged from 21 % to 41 % (Table 1). On the basis of the observed standard deviations, 8-9 samples result in a mean estimate that differs less than 0.5 kg/m2 from the true mean by the probability of 95 % both in the F/H and 0-40 cm layers (Fig. 1). Sample numbers in excess of 10 do not substan tially increase the accuracy of estimating the mean. Table 1. Amount of organic carbon in the different soil layers (kg/m 2 , except % for the CV), n=l26. Figure 1. 95 % confi dence limits for estimat ing the mean amount of organic carbon (kg/m 2) in the organic F/H layer and 0-40 cm mineral soil layer as a function of number of samples. layer mean med SD min max CV F/H 1.88 1.77 0.524 1.00 3.60 28.0 0-10 cm 1.41 1.43 0.312 0.668 2.42 22.1 10-20 cm 0.805 0.755 0.252 0.410 1.88 31.3 20-40 cm 0.424 0.384 0.174 0.175 1.49 41.1 0-40 cm 2.64 2.66 0.562 1.43 4.85 21.3 79 Roots, mycorrhizas and rhizosphere microbes... Figure 2. Semivario grams of the amount of organic carbon (kg/m2) in the a) organic F/H layer, b) 0-10 cm, c) 10-20 cm and d) 20-40 cm mineral soil layers. The lines rep resent models fitted to the semivariances at the distances of less than 4.5 m for the kriging in terpolation. Figure 3. Interpolated map of the amount of carbon (kg/m2) in the or ganic F/H layer. Sam pling points are indicated by the small dots, trees by the large dots and stumps by the crosses. The dashed line indi cates the division of the site into the canopy and opening halves. 80 Figure 4. Interpolated maps of a) the amount of organic carbon (kg/m2) in the 10-20 cm mineral soil layer and b) the thick ness (cm) of the E hori zon. Trees are indicated by the dots and stumps by the crosses. 81 Roots, mycorrhizas and rhizosphere microbes. The amount of carbor was spatially dependent at distances of less than 1-4 m and the spatial dependence accounted for 45-86 % of the total variance, depending on the soil layer (Figs. 2a-d). Therefore, to fulfill the criteria of statistical independence, sam ples should be taken further apart than the range of spatial depend ence. Conversely, to utilize the dependence when interpolating (kriging), samples should be taken closer than the range. The F/H layer was 15 % thicker and contained 33 % more car bon in the canopy half than in the opening. The highest carbon contents tended to occur around the trees (Fig. 3). These differ ences, due to changes in litter deposition and decomposition rates, had developed over a period of some tens of years which was, on the basis of the stumps, the age of the opening. In the 0-10 cm lay er the amount of carbon was patchy and not associated with the trees. On the other hand, the 10-20 cm and 20-40 cm layers con tained more carbon near the trees than elsewhere and the largest quantities were found in the immediate vicinities of the stems (Fig. 4a). The spatial patterning of the E horizon thickness was similar (Fig. 4b). The exceptionally thick E horizon near the stems was most probably caused by stemflow and the large amount of carbon in the B horizon below was, in turn, due to the organic compounds transported into the soil by the stemflow. Owing to the podzolic properties, the organic compounds may have re mained dissolved in water in the conditions of the E horizon and precipitated first in the E horizon. It seems, that even if the vol ume of stemflow is not more than 1-2 % of precipitation in Scots pine stands (Päivänen 1966), it still induces remarkable variation in soil properties. This is most probably due to concentrated routes of stemflow into the soil and high carbon concentration, up to 200 mg/1 (Gersper and Holowaychuk 1971) compared to an av erage of 26 mg/1 in throuj hfall (Westman et al. 1994), in the stem flow. According to these results, trees induce heterogeneity in soil properties and clearly observable alterations may develop fairly quickly considering the time scale of soil formation, namely in less than hundred years. References Gersper, P. L. & Holowaychuk, N. 1971. Some effects of stem flow from forest canopy trees on chemical properties of soils. Ecol ogy 52: 691-702. Ilvesniemi, H. 1991. Spatial and temporal variation of soil chemical characteristics in pine sites in southern Finland. Silva Fenn. 25: 99-108. 82 Isaaks, E. H. & Srivastava, R. M. 1989. Applied geostatistics. Oxford University Press, New York. 561 p. Järvinen, E., Hokkanen, T. J. & Kuuluvainen, T. 1993. Spatial heteroge neity and relationships of mineral soil properties in a boreal Pinus sylvestris stand. Scand. J. For. Res. 8: 435-445. Päivänen, J. 1966. Sateen jakaantuminen erilaisissa metsiköissä, Sum mary: The distribution of rainfall in different types of forest stands. Silva Fenn. 119: 1-37. Ranta, E., Rita, H. & Kouki, J. 1989. Biometria. 2nd edition. Yliopis topaino, Helsinki. 569 p. Westman, C. J., Fritze, H., Helmisaari, H-S., Ilvesniemi, H., Jauhiainen, M., Kitunen, V., Lehto, T., Liski, J., Mecke, M., Pietikäinen, J. & Smolander, A. 1994. Carbon in boreal coniferous forest soil. In: Kanninen, M. & Heikinheimo, P. (eds.) The Finnish research programme on climate change, second progress report. Paina tuskeskus Oy, Helsinki, pp. 177-186. Zinke, P. J. 1962. The pattern of influence of individual forest trees on soil properties. Ecology 43: 130-133. 83 Roots, mycorrhizas and rhizosphere microbes. Decomposition of fine roots of Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.) in different soils Krista Lõhmus 1 , Mari Ivask2 and Ivika Ostonen1 Introduction The most interesting resu Its of root investigations during the last decade are connected with fine-root productivity and turnover. These indicate that the production and replacement of fine roots in boreal forests can forn a major part of net primary production (Persson 1983). Therefore, root decomposition is a key process in nutrient, mass and eneigy dynamics of a coniferous forest. The paper represents a summary of the results of long-term decompo sition studies of finest and fine roots in Norway spruce and fine roots in Scots pine, which were carried out with the aim: (1) to describe the dynamics of the organic matter, ash, nitrogen and energy content during decomposition; (2) to analyze variability between different sites and incubation depths; (3) to analyze variability between different conifer species (Nor way spruce and Scots pine). Proceeding from this, the root litter-bag technique was em ployed. 1 1nstitute of Geography, Dept. of f lature Geography and Landscape Ecology, Tartu University, Ulikooli 18, EE-2400Tartu, Estonia. 2 Institute of Environmental Protec ion, Estonian Agricultural University, Riia 12, EE-2400 Tartu, Estonia. 84 Table 1. Characteristics of the permanent plots in Estonia. Materials and methods For Norway spruce the study of the decomposition of the finest (<1 mm in diameter) roots was carried out in a 40-year-old high site quality (I a ) Norway spruce (Picea abies (L.) Karst.) stand described in Ivask et al. (1991), and Löhmus and Ivask (1994) and in Table 1 (Site Voore 1). Fine root (<2 mm in diameter) decom position studies were conducted on 8 permanent plots in Estonia. A. Root decomposition and stand characteristics Site Canopy Age Mean Site Basal Remaining amount of roots after five composition (years) height quality area years ( % from the initial weight) (m) class (m 2 ) Voore 1 10S 40 19 l a 48.9 Spruce < 1 mm, 60 % Voore 2 9S1B 50 20 I 33.0 Spruce < 2 mm, 54 % Haanja 9S1B 45 20 I 43.2 Spruce < 2 mm, 54 % Väätsa 9S1B 63 25 I 29.7 — Vigala 6S 3P 43 18 I 35.6 Spruce < 2 mm, 61 % Putkaste 9S 1 B 64 19 I I 34.0 Spruce < 2 mm, 49 % Kuusnömme 5S 5P 73 11 IV-V 11.8 Spruce < 2 mm, 73 % Tipu 8S1P1B 56 22 I 44.3 Spruce < 2 mm, 53 % Pikasiiia 7P 3S 63 11 III 26.8 Spruce < 2 mm, 70 % Pine < 2 mm, 73 % Növa 10P 143 15 V 10.7 Pine < 2 mm, 69 % S - Picea abies, P - Pinus sylvestris, B - Betula pubescens B. Soil characteristics Site Soil type Parent material Humus form pH ( H 20 ) in 0 - 20 cm Voore 1 Umbric Red-brown till mull 5.5 - 6.0 Luvisol Voore 2 Umbric Red-brown till mull 4.4-5.6 Luvisol Haanja Dystric Red-brown till moder 4.3 - 5.6 Podzoluvisol Väätsa Umbric Yellow grey till mull 6.5 - 7.2 Cambisol Vigala Dystric Vawed clay moder-mull 4.4 - 4.9 Gleysoi Putkaste Gleyic Aqueous sand mor 5.3 - 5.8 Podzol Kuusnömme Rendzic Pebble till mull 7.0-8.3 Leptosol on pebble Tipu Haplo-Gleyic Fluvioglacial mor 4.5 - 5.0 Podzol sand Pikasiiia Sombri-Ferric Fluvioglacial mor-moder 4.4 - 5.3 Podzol sand Növa Sombri-Ferric- Fluvioglacial mor 5.7 - 6.0 Gleyic Podzol sand 85 Roots, mycorrtiizas and rhizosphere microbes... Stand and soil characteristics are given in Table 1 (Sites: Voore 2, Haanja, Väätsa, Vigala, Putkaste, Kuusnömme, Tipu, Pikasilla). Scots pine decomposition of fine roots (<2 mm in diameter) was studied in Pikasilla and Növa (Table 1). The initial material for decomposition was collected for the Norway spruce at high-quality spruce stands: finest roots (<1 mm in diameter) at site Voore 1 and fine roots (<2 mm in diameter) at site Haanja (Table 1); for the Scots pine the fine roots were col lected at the low quality pine stand Növa (Table 1). The methods are described in Löhmus and Ivask (1994). The mesh size of the root-litter bags was about 0.1 mm, the size of litter bags was 5x5 cm 2 for finest and BxB cm 2 for fine roots. Each bag contained 1000 mg of finest or about 500 mg of fine roots. One hundred bags of finest roots were incubated randomly under the forest floor and in subsequent 10 cm soil layers down to the depth of 40 cm in site Voore 1 in July 1986. The litterbags of fine roots were incubated in soil at a depth of 10 cm in July 1989. The bags were collected once or twice a year except for Voore 1 and Voore 2 sites, where the seasonal dynamics was investigated. In all initial and decom posing samples oven-dry weight, ash and energy content (by the macrocalorimeter KL-5) and nitrogen concentration (by the Kjel dahl method) was determined. Results 1. Finest (<1 mm in diameter) spruce root decomposition The finest spruce root decomposition dynamics was studied in site Voore 1 (Table 1) and discussed in Löhmus and Ivask (1994). The initial N, ash and lignin concentrations were 1.29 %, 5.7 % and 34.8 % respectively, caloricity was 18.48 kJ/g. By the multi ple comparison of means no significant differences were found between various depths of decomposing samples for the remain ing oven-dry and ash-free mass, caloricity and N concentration. The ash-free dry weight of the samples decreased by 14.3 % of the initial dry weight during the first month; the low caloricity of the dry weight loss indicates that the main loss was formed by soluble carbohydrates with a caloricity of 17.0 (Morowitz 1968). After five years the finest roots had lost 40 % of their initial weight, half of it during the first year. Following in time the abso lute amount of N in remaining material, the phases of leaching, accumulation and final release (Berg and Staaf 1981) are 86 observed, the mean nitrogen concentrations varied during the incubation from 1.47 to 1.78 %. 2. Fine (<2 mm in diameter) spruce root decomposition studies The initial N, ash and lignin concentrations were 0.73 %, 1.8 % and 37.0 % respectively, the caloricity was 20.0 kJ/g. The rate of fine-root decomposition in the Voore forest (Site Voore 2 in Table 1.) is somewhat different from that of the finest roots (Lsh mus and Ivask 1994). During the first month ash-free dry weight decreased by 7.3 % of the initial dry weight, which shows that the amounts of soluble compounds in fine roots are smaller than in the finest root fraction. Due to the lower initial N concentration, 0.73 % in fine roots (for finest roots 1.29 %), the change of the absolute amount of nitrogen over time is different. An accumula tion phase can be distinguished, after which a release phase (Berg and Staaf 1981) begins. During the first three years the decay rates of the finest and fine spruce roots in the same soil were similar. In different soils after the first year fine spruce roots had lost 21.0 to 32.7 % of their initial dry weight, after two years the loss was 22.5 to 43.2 %. The remaining dry weight per centages from the initial dry weight after five years are given in Table 1. In all sites the N concentration in five years was higher than the initial concentration and varied from 0.97 to 1.70 % in different sites. 3. Fine (<2 mm in diameter) pine root decomposition studies The initial N, ash and lignin concentrations were 0.47 %, 1.1 % and 22.7 % respectively, the caloricity was 19.2 kJ/g. After the first year fine pine roots had lost of their initial dry weight at Pikasilla and Nova sites 19 % and 31 %, respectively; the nitro gen concentration varied in five years from 0.62 to 1.12 %. The remaining dry weight percentages after five years are given in Table 1. We may conclude that the decay rates of the Norway spruce and Scots pine fine root litter are smaller at the sites where the site quality is lower. 87 Roots, mycorrhizas and rhizosphere microbes... References Berg, B. & Staaf, H. 1981. Leaching, accumulation and release of nitrogen in decomposing forest litter. In: Terrestrial Nitrogen Cycles. Clark, F. E. & Rosswall T. (eds.). Ecol. Bull., Stock holm, 33 pp. 163-178. Ivask, M., Löhmus, K. & Rästa, E. 1991. Below-ground tree productiv ity of Norway spruce forest: a preliminary report. In: Plant Roots & their Environment. Developments in Agricultural & Managed-Forest Ecology 24. McMichael, B.L. & Persson, H. (eds.) Elsevier, The Netherlands, pp. 213-217. Lohmus, K. & Ivask, M. 1994. Decomposition and nitrogen dynamics of fine roots of Norway spruce (Picea abies (L.) Karst. at differ ent sites. Plant and Soil (in press). Morowitz, H. J. 1968. Energy flow in biology. Acad. Press, New York, pp. 1-179. Persson, H. 1983. The distribution and productivity of fine roots in boreal forests. Plant and Soil 71: 87-101. 88 89 Roots, mycorrhizas and rhizosphere microbes. Seasonal variation of fine root biomass in Pinus syl vestris (L.) stand Kirsi Makkonen Introduction Roots, especially fine roots, have a most important role for the function of forest trees. They take up and transport water and nutrients to the aboveground tree parts. Although fine roots are so important for the tree, they have seldom been included in ecophysiological studies. One reason for this is the tremendous amount of work and processing time, which root research involves. The objective of this study was to determine the seasonal var iation of fine roots in a 35-year-old Scots pine (Pinus sylvestris L.) stand in eastern Finland. This research was a part of the re search project "Nutrient dynamics and biomass production of Scots pine" carried out by the Finnish Forest Research Institute and the University of Joensuu. The Society of Forestry in Finland supported my work, for which I am very grateful. Materials and methods Experimental stand The research was carried out in a Scots pine (Pinus sylvestris L.) stand at Ilomantsi near Mekrijärvi Research Station (62° 47' N; 30° 58' E; 144 m a.5.1.) in eastern Finland. The research stand is a naturally regenerated 35-year-old pole stage stand (Table 1). The site type is a Vaccinium-type, according to the classification of Cajander (Cajander 1949). The field layer The Finnish Forest Research Institute, Department of Forest Ecology P.O. Box 18, FIN-01301 Vantaa, Finland. 90 is dominated by Vaccinium vitis-idaea (L.) and Calluna vulgaris (L.) Hull. The bottom layer is dominated by Pleurozium schreberi (Brid.) Mitt, and some Cladonia species. The soil is an iron podsol, relatively poor in available nutri ents. The thicknesses of the soil horizons were humus 2.5 cm, elu vial horizon 5.0 cm, and illuvial horizon 11.0 cm (Helmisaari and Mälkönen 1989). Table 1. Some characteristics of the experimental stand in 1985. The climate of the research area is continental. Mean annual temperature was 1.0 °C and annual rainfall 699 mm during the study period (1985-1988). Root sampling Eleven samplings were carried out during the growing seasons 1985-1988. 20 soils cores per sampling (volumetric samples, core diameter 36 mm) were taken for fine-root biomass determi nations. The soil samples were divided into three layers: humus, 0-10 cm mineral soil and 10-30 cm mineral soil. Samples were transported to the laboratory and stored in a deep-freeze. Laboratory analysis In the laboratory roots were separated from soil by washing and sorted into Scots pine living roots, other living roots and dead roots. The sorted living roots were separated into three classes by diameter <2 mm, 2-5 mm and >5 mm and dead roots (necro mass) <2 mm and >2 mm. Only results from fine roots (<2 mm diameter) will be reported here. The classified roots were dried at 70 °C for 5 days and weighed to determine the oven-dry biomass per area basis. Age, a 35 Number of trees/ha 2980 Mean diameter, cm 7.4 Mean height, m 6.4 Basal area, m 2 /ha 14,0 Stem volume, m 3 /ha 11.3 Volume increment, m 3 /ha/a 11.3 Plot area, m 2 91 Roots, mycorrhizas and rhizosphere microbes. Results The variation of Scots pine fine-root biomass The biomass of Scots pine fine roots varied seasonally in differ ent layers (Fig. 1). Particularly the fine-root biomass in humus varied seasonally and between years. The major part of living fine roots was in the upper mineral soil layer, 53 % more than in humus and 31 % more than in the lower mineral soil layer. The seasonal variation was statistically significant only in hu mus layer in 1988: the amount of fine roots was greater in June (p<0.01) and in October (p<0.001) than in July. Differences be tween the same months of different years were statistically signif icant only in July; differences between 1985 and 1988 were rather significant (p<0.05), and between 1987 and 1988 significant (pcO.Ol). In the mineral soil layers the seasonal or between-year varia tion was not statistically significant. The variation of other living fine-root biomass The divided part "other living roots" consisted of dwarf shrubs roots and grass roots. 90 % of these roots were in humus and upper mineral soil layers (Fig 1). Seasonal variation was statistically significant only in 1988; the amount of roots was significant greater (p<0.01) in July than in October. There were no signifi cant differences between years. The variation of necromass "Necromass" included all dead roots, both dead Scots pine fine roots, dead dwarf shrubs roots and dead grass roots. The major part of dead roots was in humus and the upper mineral soil layer (Fig.l). In the humus layer the seasonal variation was statistically significant in 1987 and 1988; the fine-root necromass differed significantly between June and July 1987 (p<0.001), between July and September 1987 (p<0.01) and between June and Octo ber 1988 (pcO.Ol). The seasonal variation was significant in the mineral soil only in the upper layer between June and July 1987 (p<0.01). The amount of dead roots increased from the beginning to the end of research period. The variation between the same months of differ ent years was significant in humus in July between 1985 and 1988 (p<0.001) and between 1987 and 1988 (p<0.001). 92 Figure 1. The seasonal variation of a) fine-root biomass of Pinus sylvestris, b) biomass of other fine roots, c) necromass in different soil layers in a Scots pine stand during the research period July 1985 - October 1988. 93 Roots, mycorrhizas and rhizosphere microbes. Discussion Major part of dwarf shrub roots and grass roots were in the humus layer while Scots pine roots were in mineral soil. Similar results have been reported by Persson (Persson 1978, 1980 a, 1983). Differences between species groups depend on the strate gies of root growth. The amount of dead fine roots was larger than the amount of living fine-root biomass. In this study living fine-root biomass was only 55 % of necromass. The fine roots have been shown to replace their weight several times during the growing season (Persson 1978, 1980 a, 1980b, 1992). Fine root growth is a complex process affected by environ mental factors. In this study the year 1988 varied from the others. In humus the amount of living Scots pine fine roots decreased in July 1988, but the biomass of dwarf shrubs roots and grass roots did not decrease. The sudden decrease of living Scots pine fine roots may be related to a chance in the environmental factors. The seasonal variation of dwarf shrub roots and grass roots might mostly be related to their seasonal growth dynamics. The biomass of other fine roots decreased and the amount of dead roots in creased in October at the end of the growing season. In July 1988 the dying of Scots pine fine roots gave space to a rapid increase in dwarf shrub root and grass root biomass. References Cajander, A.K. 1949. Forest types and their significance. Acta For. Fenn. 56: 1-69. Helmisaari, H.-S. & Mälkönen, E. 1989. Acidity and nutrient content of troughfall and soil leachate in three Pinus sylvestris stands. Scand. J. For. Res. 4: 13-28. Persson, H. 1978. Root dynamics in a young Scots pine stand in Central Sweden. Oikos 30: 508-519. Persson, H. 1980 a. Death and replacement of fine roots in a mature Scots pine stand. In Persson, T. (Ed.) Structure and Function of Nothern Coniferous Forest - An Ecosystem Study. Ecol. Bull. 32: 251-260. Persson, H. 1980b. Spatial distribution of fine roots growth, mortality and decomposition in a young Scots pine stand in Central Swe den. Oikos 34: 77-87. Persson, H. 1983. The distribution and productivity of fine roots in boreal forests. Plant and Soil 71: 87-101. Persson, H. 1992. Factors affecting fine root dynamics of trees. Suo 43: 163-172. 94 95 Roots, mycorrhizas and rhizosphere microbes... Effects of mycorrhizas on the defensive chemistry in Scots pine seedlings Anne Nerg, Toini Holopainen and Jarmo K. Holopainen Introduction Mycorrhizas improve growth and survival of plants by enhancing nutrient uptake. Improved nutrient uptake allows mycorrhizal plants to allocate more carbon to shoot growth and possibly to antiherbivore defenses in the form of carbon-based defensive compounds. Carbon allocation to antiherbivore defense reduces investments for plant growth, but it may provide protection against herbivores, pathogens and fungal diseases. It is also known, that some ectomycorrhizal fungi themselves can synthe size carbon-based secondary metabolites using carbon parti tioned to roots and fungus or induce production of secondary metabolites in roots and thus protecting plants from root patho gens and belowground grazers (Jones and Last 1991). If these metabolites are also transported to shoots, they can influence the aboveground resistance as well. Plant phenolics are carbon-based metabolites and practically all higher plant phenolics are formed via the shikimic acid path way from shikimate through the intermediacy of phenylalanine (Harborne 1980). Terpenoids are also carbon-based metabolites and are formed from mevalonic acid via the acetate-mevalonate pathway after the glycolysis of carbohydrates to pyruvic acid and further to acetylCoA (Vickery and Vickery 1981). When condi tions are favorable for the plant growth, carbon is allocated to growth processes instead of these secondary metabolites (Bryant et al. 1983). Different environmental stresses may inhibit more growth potential than photosynthetic potential in plants leading to Ecological laboratory, Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. 96 increased carbohydrate concentrations. This carbohydrate can be used for secondary metabolite production (Bryant et al. 1983). Some air pollutants, nutrient stresses and water stress have their major impact on photosynthesis and thus may decrease translocat ed carbon for secondary metabolites (Chapin 1991) and also for fungal and root growth (Andersen and Rygiewicz 1991). In this study we aimed to find out the effects of mycorrhizal infection on the concentrations of some defensive compounds. The concentrations of total phenolics in Pinus sylvestris L. shoots and roots and resin acids in shoots were determined. Materials and methods Surface sterilized Pinus sylvestris L. seeds were germinated on water agar. After 20 days, the seedlings were transferred to the Petri dishes using a modified version of the sterile Petri dish technique earlier developed by Wong and Fortin (1989). The roots of six-weeks-old seedlings were inoculated with Cenococ cum graniforme or Suillus variegatus and allowed to grow nine weeks until the shoots and roots from individual seedlings were harvested for chemical analyses. The total phenolics were extracted with 80 % acetone from pine shoots and roots and ana lysed with Folin-Ciocalteu reagent as reported by Julkunen-Tiitto (1985). The absorbances of samples were measured with spectro photo-meter at 735 nm. Resin acids from pine shoots were extracted following the procedures of Gref and Ericsson (1985). Samples were analysed by gas chromatography-mass spectrome try. The infection level in roots was determined calculating the mycorrhizal and non-mycorrhizal short roots. Results The mean infection level in C. graniforme roots was 35 % and in S. variegatus roots 26 %. According to ultrastructural studies C. graniforme formed normal looking mycorrhiza, but the infection with S. variegatus was poorer. The root/shoot ratio was signifi cantly (P<0.05) higher in the seedlings inoculated with C. grani forme than in other treatments. The total phenolic concentration was higher in S. variegatus inoculated roots than in C. grani forme inoculated roots and on the other hand higher in the shoots of seedlings inoculated with C. graniforme than in seedlings inoculated with S. variegatus (Fig. 1). Abietic and neoabietic Roots, mycorrhizas and rhizosphere microbes.. 97 Figure 1. The total phe nolic concentration (mg/ g d.wt) in pine roots and shoots in different treat ments. C.gr = inoculated with Cenococcum grani forme, S.var = inoculated with Suillus variegatus. The concentrations in roots or shoots followed by different letters differ significantly at P<0.05 according to Duncan's test. The error bars indi cate standard deviations. Figure 2. The concentra tions of different resin acids (mg/g d.wt) in pine shoots in different treat ments. Treatments as in Fig. 1. The concentration of dehydroabietic acid with different letter differs significantly at P<0.05 according to Duncan's test. The error bars indi cate standard deviations. acids were the most common resin acids (Fig. 2). The concentra tion of dehydroabietic acid was significantly higher in shoots of seedlings inoculated with C. graniforme than in other treatments (Fig. 2). The total resin acid concentration in the shoots of non mycorrhizal seedlings was slightly lower than in mycorrhizal seedlings, but there were not significant differences. The propor tional quantity of palustric acid was significantly (P=0.021) higher in the shoots of seedlings inoculated with S. variegatus than in the shoots of seedlings inoculated with C. graniforme. Instead in the shoots of non-mycorrhizal seedlings the propor- 98 tional quantity of abietic acid was significantly (P=0.000) higher than in C. graniforme treatment and also significantly (P=0.003) higher than in S. variegatus treatment. Discussion Results concerning the effects of mycorrhizal infection on the concentration of phenolics in host plant roots are highly varying. Krishna and Bagyaraj (1984) found increase in total phenolics in VA-mycorrhizal Arachis hypogaea roots and they suggested that the situation is similar if a pathogenic fungus is invading a host plant. In Allium porrum L. and Ginkgo biloba L. inoculated with VA-mycorrhiza, the concentrations of cell wall bound phenolics were not changed due to mycorrhizal infection (Codignola et al. 1989). Instead Miinzenberger et al. (1990) found a reduction of phenolics in ectomycorrhizas of Norway spruce roots and they suggested that this reduction enables the mycorrhizal fungus to penetrate root tissue. If phenolics are accumulating in roots, they can gradually reduce the plasticity and elasticity of the fungal matrix (Grandmaison et al. 1993). In Scots pine, Bonello et al. (1993) found increased concentrations of some phenolics in the non-mycorrhizal roots after challenging the roots with the root pathogen. Mycorrhizal infection had a dampening effect to the induction of these compounds. In this study the only difference in total phenolics was be tween two different mycorrhizal types, both in roots and shoots. The difference between them cannot be explained by higher infec tion level because the mycorrhizal infection was lower in the roots inoculated with S. variegatus. The formation of mycorrhizas also demands part of the assimilated carbon and it may explain lower phenolic concentration in the shoots of S. variegatus seedlings. It seems that total phenolics is not sensitive enough revealing the ef fects of mycorrhizas on plant phenolics. Individual phenolic com pounds should be analysed in order to better understand possible mechanisms. There are only few studies about the effects of mycorrhizas on the biosynthesis of terpenoids. Krupa and Fries (1971) found the accumulation of volatile compounds, primarily terpenes and ses quiterpenes, in the mycorrhizal roots of Pinus sylvestris L. The authors proposed that the response of the host to mycorrhizal in fection is non-specific and similar to a wound response: increased production of volatile (terpenes etc.) and nonvolatile (phenolics, resin acids etc.) substances. If these compounds are in sufficient 99 Roots, mycorrhizas and rhizosphere microbes. concentrations, they may restrict the growth of mycorrhizal fungi and also inhibit the growth of root pathogens. Cheniclet et al. (1988) have found that fungal infection mainly induces accumu lation of volatile terpenes while wounding and insect attacks in duce the accumulation of non-volatile terpenes. In this study nonvolatile resin acid concentrations, except dehydroabietic acid, in pine shoots were not changed according to mycorrhizal infec tion. References Andersen, C.P. & Rygiewicz, P.T. 1991. Stress interactions and mycor rhizal plant response: understanding carbon allocation priorities. Environ. Pollut. 73: 217-244. Bonello, P., Heller, W. & Sandermann, H. Jr. 1993. Ozone effects on root-disease susceptibility and defence responses in mycorrhizal and non-mycorrhizal seedlings of Scots pine (Pinus sylvestris L.). New Phytol. 124: 653-663. Bryant, J.P., Chapin, F.S., 111 & Klein, D.R. 1983. Carbon/nutrient bal ance of boreal planls in relation to vertebrate herbivory. Oikos 40: 357-368. Chapin, F.S., 111. 1991. Effects of multiple environmental stresses on nutrient availability and use. In: Mooney, H.A., Winner, W.E., Pell, E.J. & Chu, E. (eds.). Response of plants to multiple stresses. Academic Press, Inc., San Diego, pp. 67-88. Cheniclet, C., Bernard-Dagan, C. & Pauly, G. 1988. Terpene biosynthe sis under pathological conditions. In: Mattson, W.J., Levieux, J. & Bernard-Dagan, C. (eds.). Mechanisms of woody plant defenses against insects - Search for pattern. Springer-Verlag, New York. pp. 117-130. Codignola, A., Verotta, L., Spanu, P., MafFei, M., Scannerini, S. & Bon fante-Fasolo, P. 1989. Cell wall bound-phenols in roots of vesic ular-arbuscular mycorrhizal plants. New Phytol. 112: 221-228. Grandmaison, J., Olah, G.M., Van Calsteren, M.-R. & Furlan, V. 1993. Characterization and localization of plant phenolics likely involved in the pathogen resistance expressed by endomycor rhizal roots. Mycorrhiza 3: 155-164. Gref, R. & Ericsson, A. 1985. Wound-induced changes of resin acid concentrations in living bark of Scots pine seedlings. Can. J.For. Res. 17: 346-349. Harborne, J.B. 1980. Plant phenolics. In: Bell, E.A. & Charlwood, B.V. (eds.). Secondary plant products. Springer-Verlag, Berlin, pp. 329-402. Jones, C.G. & Last, F.T. 1991. Ectomycorrhizas and trees: implications for aboveground herbivory. In: Barbosa, P., Krischik, V.A. & Jones, C.G. (eds.). Microbial mediation of plant-herbivore inter actions. John Wiley & Sons, Inc., New York. pp. 65-103. 100 Julkunen-Tiitto, R. 1985. Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics. J. Agric. Food Chem. 33:213-217. Krishna, K.R. & Bagyaraj, D.J. 1984. Phenols in mycorrhizal roots of Arachis hypogaea. Experientia 40: 85-86. Krupa, S. & Fries, N. 1971. Studies on ectomycorrhizae of pine. I. Pro duction of volatile organic compounds. Can. J. Bot. 49: 1425- 1431. Munzenberger, 8., Heilemann, J., Strack, D., Kottke, I. & Oberwinkler, F. 1990. Phenolics of mycorrhizas and non-mycorrhizal roots of Norway spruce. Planta 182: 142-148. Vickery, M.L. & Vickery, B. 1981. Secondary plant metabolism. The MacMillan Press Ltd, London. 335 p. Wong, K.K.Y. & Fortin, J. A. 1989. A Petri dish technique for the asep tic synthesis of ectomycorrhizae. Can. J. Bot. 67: 1713-1716. 101 Roots, mycorrhizas and rhizosphere microbes. Nitrite reductase activity in the mycorrhizal roots of Scots pine seedlings Karoliina Niemi 1 , Helvi Heinonen-Tanski 2 and Toini Holopainen 3 Abstract Nitrite reductase (NiR) activity was shown in roots of Scots pine (Pinus sylvestris) seedlings inoculated with three strains of Ceno coccum geophilum and two strains of Paxillus involutus. All mycorrhizal associations could use nitrite (NO~2) after both asep tic and non-aseptic cultivation. Nitrogen fertilization increased significantly the NiR activity in P. involutus mycorrhizas of asep tic seedlings. Introduction Nitrification is low in many acid coniferous forest soils and ammonium (NH4 + ) is the dominant form of mineral N (Adams and Attiwill 1982, Martikainen 1984). The significance of nitrate (N0 3") as a N source has, however, increaced due to NOx pollu tion and NO3" fertilization in forests and nurseries. Most NO3" assimilation in Scots pine (Pinus sylvestris L.) may occur in roots (Sarjala et al. 1987, Pietiläinen and Läh desmäki 1988, Seith et al. 1994). Therefore the mycorrhizal sym biosis formed in roots has a significant effect on N03 " nutrition of Scots pine. Nitrate utilization has been observed both in the myc orrhizal (Sarjala 1991) and non-mycorrhizal roots of Scots pine 1 Department of Plant Biology / Forest Pathology, P.O. Box 28, FIN-00014 University of Helsinki, Finland. 2 Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. 3 Ecological Laboratory, Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. 102 (Seith et ai. 1994). N03 " assimilation presumes the activity of ni trite reductase (NiR) (EC 1.7.7.1 in plants, EC 1.6.6.4 in fungi). Seith et al. (1994) showed NiR to work in the non-mycorrhizal roots of Scots pine. It is, however, quite unclear, how this enzyme works in the mycorrhizal roots of conifer trees. This study was designed to test the NiR activity in the mycor rhizal roots of Scots pine seedlings and the influence of N fertili zation on it. Materials and methods Surface sterilized Scots pine seeds, representing a central Finland provenance, were sown into a 1:1 (v:v) sterile vermiculite:peat mixture. The seedlings were grown in a controlled growth room where the day might temperatures were 26:21 °C and the light:dark cycle was 22:2 h. Seedlings were watered with sterile water when required. Non-aseptic seeds were sown into peat. The seedlings were grown in a greenhouse in the same temperature and light condi tions as the aseptic ones. Seedlings were watered with lake water. After three weeks the seedlings were inoculated with an agar bloc of mycelium taken from pure cultures of two Paxillus invo lutus (Batch.) Fr. (referred here numbers 12 and 14) and three Cenococcum geophilum (Sow.) Ferd. & Winge (36, 38 and 53) strains. All the strains were taken from the culture collection of University of Kuopio. The seedlings were fertilized twice over the experimental pe riod (6 and 8 weeks after sowing) with NaN03 . Both treatments were calculated to correspond to 50 kg/ha NO3" -N. The control seedlings received only water. NiR activity of pine mycorrhizas was determined 14 weeks after sowing. The enzyme was isolated by a modification of the method of Yoneyama and Sasakawa (1979). The roots were cut into small pieces and ground with a grinding medium composed of potassium phosphate buffer (pH 7.5), cysteine and EDTA. The homogenate was squeezed and the filtrate was centrifuged. The supernatant was used as a crude enzyme extract. The NiR activity measurements were based on the method of Ida and Morita (1973). The enzyme assay mixture contained Tris-HCI buffer, NaN0 2, methyl violagen MQ water and the enzyme. All measure ments were carried out below +4 C. The reaction was started by adding sodium dithionite in sodium bicarbonate to the reaction mixture. After 20 min of incubation in the dark at 30 C, the loss 103 Roots, mycorrhizas and rhizosphere microbes. of NC>2~ was determined by the addition of Griess reagents. Ab sorbance of the diazo color was read at 540 nm after 20 min. Results The infection of mycorrhizal fungi was obvious in each case. All five mycorrhizal associations of Scots pine seedlings could use N0 2 ". NiR activity in unfertilized pines with P. involutus mycor rhizas (12 and 14), grown in aseptic conditions, was weak, only about 60 % of that of C. geophilum strain 38 (Fig.l). N fertiliza tion (2 x 50 kg NO3" -N/ha) improved NiR activity in P. involutus mycorrhizas quite clearly. Figure 1. Nitrite reduct ase activity, N0 2 " de creased nmol/h/g the fresh weight of root mass, in different associations of aseptic Scots pine (P/- nus sylvestris) mycor rhizas (Paxillus involutus strains 12 and 14, Ceno coccum geophilum strains 36, 38 and 53). C =control (unfertilized), N =fertilized (2x50 kg N03 " -N/ha). Vertical bars rep resent SD (n = 3, except C3B n - 2). Figure 2. Nitrite reduct ase activity, N0 2 " de creased nmol/h/g the fresh weight of root mass, in different associations of non-aseptic Scots pine (Pinus sylvestris) mycor rhizas (Paxillus involutus strains 12 and 14, Ceno coccum geophilum strains 36, 38 and 53). C = control (unfertilized), N =fertilized (2x50 kg N0 3 " -N/ha). Vertical bars rep resent SD (n = 3). 104 Unfertilized control mycorrhizas of the strain 14 used N0 2 ~ least of all non-aseptic controls, about 70 % of the amount that the strain 38 used (Fig. 2). The effects of fertilization were slight, all together. Discussion The fate of N02 ~ formed in N03 " reduction is not well known. N0 2 " should be assimilated into some organic form quickly after N0 3 " reduction in the mycorrhizae, because N02 ~ and NH4 + are toxic to cells. That is why NiR activity might follow the level of NO3" reduction. In the present study all five mycorrhizal associations of Scots pine seedlings could use N02". The strains 38 and 53 of C. geophilum were isolated from NO3" and NH4 + rich soil near a pulp mill. In this study the mycorrhizas formed by these strains used N02 " the best. Fertilization (2 x5O kg/ha N03 " -N) during the growing period did not affect NiR activity in aseptic C. geophilum mycorrhizas. The stimulating effect on P. involutus was, however, significant. Seith et al. (1994) showed N03 to stimulate NiR in the non-myc orrhizal roots in aseptic conditions. In the present study NiR ac tivity was higher in non-aseptic controls than aseptic ones, which could result from some kind of association between mycorrhizas and microbes in non-aseptic soil. Fertilization did not actually improve the NiR activity of non-aseptic mycorrhizas. The total amount of NO3" in the fertilizer was propably too high and even inhibited NiR. This could be seen especially in the strains 14 and 36. NiR activities of mycorrhizas presented here are much lower than activities (N02 ~ decreased over 10 000 nmol/h/g fresh weight) of pure Hebeloma cylindrosporum (Romagn.) mycelium reported by Plassard et al. (1984). The low activities can be partly a result of conditions. The method used here has been developed for the optimal conditions in spinach leaf (Ida and Morita 1973), where NiR activity may be very high. When determining NiR ac tivities the strength of reagents and conditions were not tested and it may be that they were not ideal for NiR of pine mycorrhizas. The results of the present experiments, however, show clear NiR activity in Scots pine ectomycorrhizas. This supports earlier observations of the ability of some ectomycorrhizal fungi (Ho and Trappe 1980, Sarjala 1990) and plants (Sarjala 1990, Scheromm et. al. 1990) to assimilate NO3" and use it as a source of N. A new 105 Roots, mycorrhizas and rhizosphere microbes. approach of studing N0 3 " metabolism of mycorrhizas may be found by developing a method optimal for mycorrhizal NiR. After that the effects of N03 " fertilization and pollution on the whole N0 3 " assimilation cycle could be better understood. References Adams, M.A. & Attiwill, P.M. 1982. Nitrogen mineralization and nitrate reduction in forests. Soil Biol. Biochem. 14: 197-202. Ho, I. & Trappe, J.M. 1980. Nitrate reductase activity of non-mycor rhizal Douglas-fir rootlets and of some associated mycorrhizal fungi. Plant and Soil 54: 395-398. Ida, S. & Morita, Y. 1973. Purification and general properties of spin ach leaf nitrite reductase. Plant Cell Physiol. 14: 661-671. Martikainen, P.J. 1984. Nitrification in two coniferous forest soils after different fertilization treatments. Soil Biol. Biochem. 16: 577- 582. Pietiläinen, P. & Lähdesmäki, P. 1988. Effect of various concentrations of potassium nitrate and ammonium sulphate on nitrate reduct ase activity in the roots and needles of Scots pine seedlings in N Finland. Ann. Bot. Fenn. 25: 201-206. Plassard, C., Mousain, D. & Salsac, L. 1984. Mesure in vivo and in vitro de l'activite nitrite reductase dans les thalles de Hebeloma cylindrosporum, champignon basidiomycete. Physiologie Vege tale 22: 147-154. Sarjala, T. 1990. Effect of nitrate and ammonium concentration on nitrate reductase activity in five species of mycorrhizal fungi. Physiol. Plant. 79: 65-70. 1991. Effect of mycorrhiza and nitrate nutrition on nitrate reductase activity in Scots pine seedlings. Physiol. Plant. 81: 89- 94. - Raitio, H. & Turkki, E.-M. 1987. Nitrate metabolism in Scots pine seedlings during their first growing season. Tree Physiol. 3: 285-293. Scheromm, P., Plassard, C. & Salsac, L. 1990. Nitrate nutrition of mari time pine (Pinus pinaster Solan in Ait.) ectomycorrhizal with Hebeloma cylindrosporum Romagn. New Phytol. 114: 93-98. Seith, 8., Setzer, 8., Flaig, H. & Mohr, H. 1994. Appearance of nitrate reductase, nitrite reductase and glutamine synthetase in different organs of the Scots pine (Pinus sylvestris) seedling as affected by light, nitrate and ammonium. Physiol. Plant. 91: 419-426. Yoneyama, T. & Sasakawa, H. 1979. Transformation of atmospheric N0 2 absorbed in spinach leaves. Plant Cell Physiol. 20: 263- 266. 106 107 Roots, mycorrhizas and rhizosphere microbes.. Ectomycorrhiza develop ment: 2-dimensional analy sis of the cytoskeleton Sara Niini, Mika Tarkka and Marjatta Raudaskoski Cytoskeleton is an essential part of a functioning cell, it plays a major role i.e. in cell division, plant cell wall orientation and in fungal tip growth. The main components of cytoskeleton in eukaryotic cells are microtubules, microfilaments and intermedi ate filaments. The subunits of microtubules are a- and B-tubulin, while microfilaments are formed of actin. Intermediate filaments consist of several proteins. Immunological research has shown that microtubules and microfilaments are common structural components in plant and fungal cells, and there is data about the occurrence of intermediate filaments in plant cells. In addition to the main components the cytoskeleton consists of accessory pro teins which can be subdivided in assembly controlling, linkage and motor proteins. The interest in the function of microtubules and microfila ments in mycorrhizal associations originates from the ability to identify the cytoskeletal components by immunological methods both in the fungal and plant cells. Indirect immunofluorescence microscopy has been applied to study the cytoskeletal elements in fungal pure cultures (Runeberg et ai. 1986, Raudaskoski et ai. 1988, Salo et ai. 1989, Niini and Raudaskoski 1993) and also in mycorrhizal associations (Timonen et ai. 1993). One dimensional electrophoresis and immunoblotting with specific monoclonal antibodies reveals cytoskeletal proteins in all analyzed ectomyc orrhizal fungi (Niini and Raudaskoski 1993) and in ectomycor rhizal associations (Timonen et ai. 1993). It is possible to separate Pinus sylvestris and Suillus bovinus tubulins in 1-D electrophoresis with a sliigth difference in molecular weight. Department of Botany, University of Helsinki, P.O. Box 7, FIN-00014 University of Helsinki, Finland. 108 A good correlation has been shown to exist between the po lymerization state of the cytoplasmic microtubules and the growth of the fungi (Niini and Raudaskoski 1993). This supports the idea that intact cytoplasmic microtubules are involved or needed for the extension growth of the fungal apical cells. A change of microtubule pattern from a strand-like to more reticu late in the fungal cells has been observed to be related to the for mation of the Hartig net (Timonen et ai. 1993). The role of cytoskeleton in the development of ectomycorrhiza is an interest ing question as the formation of coralloid mycorrhizas typical to Pinus sylvestris-Suillus bovinus symbiosis involves meristematic activity of the root cells, which first leads to the development of dichotomous short roots and later to the coralloid mycorrhizas with numerous root tips. It may be assumed that the plant cell cy cle in the short roots is affected by the fungus, especially since no coralloid roots are found in the non-mycorrhizal pine roots. In order to obtain a more detailed understanding of the changes especially in the cytoskeletal proteins during the ecto mycorrhiza development 2-dimensional electrophoresis gels and their immunoblots were prepared and analyzed from main roots and short roots of Pinus sylvestris seedlings with or without myc orrhizas. In addition pure cultures and extramatrical mycelium of Suillus bovinus were also used. The mycorrhizas were classified in four groups on the basis of their developmental morphology: undivided short root tips with Al) exposed meristem or A 2) complete fungal sheath, B) dichotomously branching mycorrhiza and C) coralloid mycorrhiza with three or more tips. Extra care was taken in sample preparation and by using several proteinase inhibitors during the extraction procedure (Äström et al. 1991). Careful solubilization of the proteins ensured the successful im munodetection of cytoskeletal proteins. The analysis of the total polypeptide patterns indicate that the different developmental stages of ectomycorrhiza share 19 polypeptides that are not present in the seedling or fungus, and thus they could be putative ectomycorrhizins. Relative intensity of the OD readings of the silver stained gels reveal that these ec tomycorrhizins can be constitutive during the development of the mycorrhiza or they can be present only in the early phase or late stage. Their amount may also increase through the different stag es being most abundant in the coralloid phase. The immunoblots of ectomycorrhizal extracts with a-tubulin antibody revealed seven isotypes of a-tubulin, four of which are of fungal origin and three of plant origin. Plant a-tubulin could be recognized by its faster migration than that of fungal a-tubu- 109 Roots, mycorrhizas and rhizosphere microbes.. lin, which has already been shown in 1-D gels (Timonen et ai. 1993). Two strongly expressed a-tubulin isotypes and two weak er signals were detected in the immunoblots of the mycelial ex tracts of Suillus. There was a marked increase in the expression of the weaker fungal a-tubulin signals in the samples from the mycorrhizal roots. In the immunoblots with actin antibody two plant and two fungal actins could be visualized. It seems that in the early mycorrhizal developmental stage the plant actins are strongly dominating species but as the mycorrhiza development proceeds to dichotomous and coralloid stage the fungal actins be come visible. The resolution of B-tubulins in the immunoblots is not as good as that of actin and in the immunoblots of the mycor rhizal samples the plant and fungal B-tubulins overlap, thus mak ing the quantification difficult. Deducing of the B-tubulin immunoblots there could be two plant and two fungal B-tubulins distinguished in the mycorrhizas. References Niini, S.S. & Raudaskoski, M. 1993. Response of ectomycorrhizal fungi to benomyl and nocodazole: growth inhibition and micro tubule depolymerization. Mycorrhiza 3: 83-91. Raudaskoski, M., Salo, V. & Niini, S.S. 1988. Structure and function of the cytoskeleton in filamentous fungi. Karstenia 28: 49-60. Runeberg, R, Raudaskoski, M. & Virtanen, I. 1986. Cytoskeletal ele ments in the hyphae of the homobasidiomycete Schizophyllum commune visualized with indirect immunofluorescence and NBD-phallacidin. Eur. J. Cell Biol. 41: 25-32. Salo, V., Niini, S.S., Virtanen, I. & Raudaskoski, M. 1989. Comparative immunocytochemistry of the cytoskeleton in filamentous fungi with dikaryotic and multinucleate hyphae. J. Cell Sci. 94: 11-24. Timonen, S., Finlay, R.D., Söderström, B. & Raudaskoski, M. 1993. Identification of cytoskeletal components in pine ectomycor rhizas. New Phytol. 124: 83-92. Äström, H., Virtanen, I. & Raudaskoski, M. 1991. Cold stability in the pollen tube cytoskeleton. Protoplasma 160: 99-107. 110 111 Roots, mycorrhizas and rhizosphere microbes. Ectomycorrhiza: carbohy drates, minerals and hor mones Jan-Erik Nylund Frank (1885) assumed that (ecto)mycorrhiza received its carbo hydrate nourishment from the host, but his ideas took long to be proved; most of his contemporaries believed that the mycobiont took an active part in decomposition. It was only Melins many physiological experiments which showed that Frank, in this as in so many other of his observations, had been (almost) right. Now, when data on the precise share for the fungus of the plant's total carbohydrate are forthcoming at an accelerating pace, there is also increasing evidence for fungal decomposition of organic matter (cf. Abuzinadah and Read 1987), albeit this is of minor, but not marginal importance to the overall energy budget of the mycobiont. However, Melin's findings and other current physiological work in the '3os made Hatch (1937) propose that carbohydrate supply depends on the host's mineral nutrition status in a paper as hard to access as central in its theory; which in turn led Björkman (1942) to formulate his theory for the role of carbohydrate in my corrhiza formation: Mycorrhiza develops only where the host has a "surplus" of carbohydrate available. There is much to say about this hypothesis (cf Nylund 1988), which for a long time was not subject to experimental testing to the extent it would have merited. Björkman himself was so con vinced of its validity that he never questioned the generalizability of his very limited proof. Yet, even his opponents tacitly acknowl edged the central role of carbohydrates in regulating the symbio sis, they only tried to attack the assumed link to N and P nutrition. Dept. of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S-750 07 Uppsala, Sweden. 112 The chief alternative theory, proposing auxin to be the primary regulator rather than N and P, was drawn up by Slankis (summary in Slankis 1973), while Meyer (1962) presented confusing results where mineral nutrition seemed to correlate with ectomycorrhiza just contrary to Bjorkman's statements. The story is not very edifying: a poorly supported theory was contested with even less evidence in favour of the counterpropos al; it has been told elsewhere (Nylund 1988). This paper will try to disentagle the controversy and set it in an ecological context, based on recent research, mainly in the Uppsala lab. The original research is presented in a series of articles by Nylund and Wal lander (Nylund and Wallander 1989, Wallander and Nylund 1991, 1992, Wallander, et al. 1992, 1994, Nylund et ai. 1994). The three interactions In a long series of hydroponic culture experiments based in Ingestad's well known principles of steady-state nutrition, the following picture emerged: 1. Nitrogen (superoptimal) suppresses the development of myc orrhiza, and has some influence on the carbohydrate pool. 2. Mycorrhiza. Increases in mycorrhizal biomass reduce the root carbohydrate pool. 3. Increased carbohydrate availability boosts mycorrhiza devel opment. Surplus N usually reduces the carbohydrate pool in uninfected seedlings, as does severe starvation. The reason why the N effect on the carbohydrate pool is not so easy to see in mycorrhizal plants is that the depression of the mycorrhiza reduces the demand for carbohydrate counteracting the direct nutrition effect. The mechanisms of nitrogen depression of mycorrhiza will be discussed in detail in a following paper by Hakan Wal lander, only the implications will be treated here. How does this compare with Bjorkman's statements? Regard ing N and P, he showed a pronounced N optimum, and a weaker but nevertheless clear optimum for P; the two factors clearly in teracted. He also showed that reduced light and impeded carbon translocation caused mycorrhiza to vanish. Yet, we find no evi dence of his claim that surplus cabohydrate, i.e. a large pool, would be present in mycorrhizal roots; both we and colleagues have repeatedly found the opposite. Strong mycorrhiza develop- 113 Roots, mycorrhizas and rhizosphere microbes. ment, caused by "virulent" fungi, always reduces the host carbo hydrate pool compared with controls and weaker mycorbionts. Available carbohydrate is normally taken care of by the fungus, never allowing a pool to build up. If we had methods of monitor ing the carbohydrate flow, it would most probably be found to in crease in proportion to mycorrhiza development. But in my opinion, the surplus-N-and-reduced-P-situation provides enough circumstantial evidence for Bjorkman's underlying standpoint that carbohydrate sets a limit to mycorrhiza development: N as such depresses mycorrhizal growth, and may tend to reduce car bohydrate availability by requiring carbon skeletons for assimila tion, a basic assumption by Björkman, taken over from Hatch. But P starvation reduces the amount of AMP available for making ATP and fuelling all metabolic processes, thereby reducing growth and causing photosynthetic carbon to accumulate even to the point of feedback inhibition of photosynthesis. This more than offsets the N effect, the result being a vigorous development of mycorrhiza. This was empirically found by Stenström et ai. (1990) a few years before our work: the success of nursery inocu lations required a reduction of P supply compared with standard nursery practices, while attempt at reducing N had no comparable effect. The elusive auxin So, we come out in support of Björkman on a more fundamental plane, while rejecting some of his more specific statements. But what about the hormone theory? Even its originator considers carbohydrate supply to be the ultimate regulator, but proposes that this is controlled by auxin action. Based on root culture experiments, Slankis (1973) claimed that (1) lAA of fungal ori gin causes mycorrhizal morphogenesis and (2) induces a flow of carbohydrate from host to fungus, proportional to the lAA sup ply. This, in turn, (3) was considered to be influenced by the nitrogen availability. The first statement may well be true. The second may be par tially true but oversimplified. The last statement is completely un tenable. We have run a series of assays, using the only reliable method available: GC-MS with inner standards. Added nitrogen actually increases the secretion of lAA and the pool in the host, since the hormone is manufactured from tryptophan, which of course becomes more abundant when other amino acids abound. Carbohydrate certainly flows to a plant part to which lAA is ap- 114 plied, but we could only detect reduced, not the predicted in creased lAA pools in mycorrhizal short roots and root systems. On the other hand, there is increasing evidence that lAA pools are really of no significance at all, turnover being potentially a better indicator, positional effects and hormone-receptor relationships being the important terms in which lAA effects are to be analysed. The monitoring of pools or even production may be trivial and lead nowhere. We expected much from a series of experiments with lAA hy perproducing mutants. The hypothesis was that the high lAA se cretion would lead to high pools in the roots, and an enhanced carbohydrate drain from the host to the fungus. This would be seen as reduced growth of the host and strong growth of the my cobiont. Unfortunately, we found nothing, which most probably was due to the physiology of the mutation, enhancing tryptophan accumulation, not lAA production directly. So, whatever the interactions between lAA, carbohydrates and mycorrhiza development may be, they are difficult to demonstrate, and far more complex than Slankis thought. Yet, there is circum stantial evidence that lAA does have a role in the symbiosis. Application of the theory But why so much noise about a half-century old theory, which in spite of faltering original evidence was found correct? Well, above all since nutritional interactions are central to the under standing of pollution and possible fertilization effects, a red hot subject in Swedish debate today, where the Forest Service and landowners' groups want to remedy the situation by liming, but are doggedly resisted by many researchers, particularly at the Agricultural University. Our studies have reference to one of the fundamental processes which are affected: the carbohydrate cycling in the tree-mycorrhiza system. Whatever the immediate effect of, say pollution, may be: wax layer and stomata damage by acid; ozone poisoning of photosynthesis or nitrogen satura tion: effects on the carbohydrate flow to the roots affect the entire rhizosphere-mycorrhiza-root system. Disturbed carbohydrate allocation to the root has several consequences. First, fruit body development is affected. In our work, even a slight increment of nitrogen concentrations in the medium (far less than standard nursery applications) suppressed fructification in the "hungry" Laccaria bicolor. This is also seen in the field: fertilization sup presses mushroom and toadstool (but not conch!) formation, 115 Roots, mycorrhizas and rhizosphere microbes. while our recent results (Karen and Nylund, unpublished) reveal only marginal effects on the species composition of mycorrhiza. The study by Fortin and coworkers (Godbout and Fortin 1992) very nicely illustrated how fruitbodies constitute a direct sink for photosynthetic carbohydrate. Secondly, soil mycelium develop ment is suppressed. This was analysed in detail by us, but exten sive research by the Lumi team (Söderström 1991) has also dem onstrated a suppression varying with species in nitrogen-rich soils. Ultimately, the fungal biomass on the root tips, or mycor rhiza proper, is affected, but even very heavy doses (200 ppm) could not entirely remove mycorrhiza in our culture trays. Simi larly, in the field, mycorrhizal roots displayed very thin mantles, and morphotypes became ever more difficult to discern, but no single case of non-mycorrhizal short roots could be detected in the Skogaby trials, where the ammonium sulphate treatments amounted to more than one hundred kg of each element N and S per hectare a year. As we see it, the main culprit is the nitrogen. Acidification may have many negative effects on cuticles and soil cation satu ration, but neither lab or field studies demonstrate any notable ef fects on the mycorrhiza itself. This implies that any policies of forest "life-saving" have to address the nitrogen issue, not only the acidification. This most probably applies to fine root development also: the observed reductions seem almost entirely be a response to the nitrogen load, or in more severe cases also to disturbed pho tosynthesis, but not to acid as such (direct poisoning in Central Europe is another case not discussed here). And the theory makes no provision for pH effects (tested but not discussed here): what ever there is, is indirect. So far, our discussion has concerned generalizations. A main point is of course that species and strains are quite different. Some, such as Laccaria and Pisolithus are very "virulent", ex creting much lAA in pure culture, drain the hosts carbohydrate re serves, and take a strong hold on their roots. Some are nitrogen tolerant, other refuse to grow into N fertilized soil even from a well colonized root in low-nutrient medium. pH optima certainly vary, but due to the conditioning effect of roots and soil mycelia on their immediate environment, in vitro studies may be of limited value. The fungi also differ in their soil mycelium, some forming hydrophobic strands and mycelial mats, other having a diffuse hy drophilic mycelial structures hard to detect, while some seem to lack a stronger connection with the soil at all. Returning to nitro gen-stressed mycorrhiza, the smooth brown mycorrhiza type into which so many species turn seems to do with little soil mycelium, 116 perhaps a reaction to the abundancy of the regulatory mineral nu trient in their immediate surroundings. References Abuzinadah, R.A. & Read, D.J. 1989. Carbon transfer associated with assimilation of organic nitrogen sources by silver birch (Betula pendula (Roth.). Trees 3: 17-23. Björkman, E. 1942. Über die Bedingungen der Mykorrhizabildung bei Kiefer und Fichte. Symbola Botanica Upsaliensia VI. Frank, A.B. 1885. Ueber die auf Wurzelsymbiose beruhende Ernährung gewisser Bäume durch unterirdische Pilze. Berichten der Deut schen Botanischen Gesellschaft 3: 128-145. Godbout, c. & Fortin, A. 1992. Effects of nitrogen fertilization and pho toperiod on basidiome formation on Laccaria bicolor associated with container-grown jack pine seedlings. Can. J. Bot. 70: 181- 185. Hatch, A.B. 1937. The physical basis of mycotrophy in the genus Pinus. Black Rock Forest Bulletin 6: 1-168. Meyer, F.H. 1962. Die Buchen- und Fichtenmykorrhiza in ver schiedenen Bodentypen, ihr Beeinflussigung durch Mineraldun gung sowie fiir die Mykorrhizabildung wichtige Faktoren. Mit teilungen der Bundesforschungsanstalt fur Forst- und Holzwis senschaft 54: 1-73. Nylund, J-E. 1988. The regulation of mycorrhiza formation - carbohy drate and hormone theories reviewed. Scand. J. For. Res. 3: 465- 479. Nylund, J. -E. & Wallander, H. 1989. Effects of ectomycorrhiza on host growth and carbon balance in a semi-hydroponic cultivation sys tem. New Phytol. 112: 389-398. Nylund, J. -E., Wallander, H., Sundberg, B. & Gay, G. 1994. lAA over producer mutants of Hebeloma cylindrosporum Romagnesi mycorrhizal with Pinus pinaster (Ait) Sol. and Pinus sylvestris L. in hydroponic culture. Mycorrhiza 4: 247-250. Slankis, V. 1973. Hormonal relationships in mycorrhizal development. In: Marks, G.C. & Kozlowski, T.T. (eds.) Ectomycorrhizae. Academic Press, New York & London, p 231-298. Stenström, E., Ek, M. & Unestam, T. 1990. Variation in field response of Pinus sylvestris to nursery inoculation with four different ectomycorrhizal fungi. Can. J. For. Res. 20: 1796-1803. Söderström, B. 1991. The fungal partner in the mycorrhizal symbiosis. In: Marcus Wallenberg Foundation Symposia Proceedings 7. Falun 1991. Wallander, H. & Nylund, J.-E. 1991. Effects of excess nitrogen on car bohydrate content and mycorrhizal development in Pinus sylves tris L. seedlings. New Phytol. 119: 405-411. 117 Roots, mycorrhizas and rhizosphere microbes... Wallander, H. & Nylund, J.-E. 1992. Effects of excess nitrogen and phosphorus starvation on the extramatrical mycelium ectomyc orrhizas of Pinus sylvestris L. New Phytol. 120: 495-503. Wallander, H., Nylund, J.-E. & Sundberg, B. 1992. Ectomycorrhiza and nitrogen effects on root lAA: results contrary to theory. Mycor rhiza 1: 91-92 Wallander H., Nylund J.-E. & Sundberg B. 1994. Influence of endog enous lAA, carbohydrates and minerals on ectomycorrhizal development in Pinus sylvestris in relation to nutrient supply. New Phytol. 127: 521-528. 118 119 Roots, mycorrhizas and rhizosphere microbes. The role of roots in carbon cycling in forests Hans Persson Available data in literature on forest trees indicate a substantial fine-root production with different seasonal patterns in needle or leaf production. Root growth is sensitive to different climatic fac tors imposed or strengthened by human activities. Carbon incor poration into the soil by dead roots is an important pathway in the total carbon flow. Factors that lead to growth suspension may or may not be the same as those that result in root shedding or senescence. Sequential core sampling, ingrowth cores (initially root free cores with sifted mineral soil removed on successive sampling oc casions) and minirhizotrons were used in coniferous forest stands in order to investigate how the fine-root turnover rate was influ enced by liquid fertilization (IF) with a complete set of essential nutrients with the aim of eliminating water and mineral nutrients as growth-limiting factors. The data reveal a more substantial total fine-root production in the IF treated plots than in the control (C) plots. As regards the increments in fine-root length of the <1 mm diameter fraction, the production proceeded much more rapidly in the IF-plots subjected to "optimum fertilization with irrigation" than in the C plots. It was demonstrated that major environmental problems result from the lack of understanding of the part played by tree roots in the total carbon flow. New pathways of carbon and nutrient cycling require further assessment. Background Large diameter roots in forest trees die with the tree itself, while the fine roots have been shown to be in a constant flux, often with Department of Ecology and Environmental Research, Swedish University of Agricultural Sciences, Box 7072, S-750 07 Uppsala, Sweden. 120 a high rate of death and renewal (Persson 1979, 1992). Roots < 1 mm in diameter consist of fine ramifications with mycorrhizal root tips that are morphologically very distinctive from the rest of the root system and include both mycorrhizal host and fungal mantle tissues. Comparable with definitions for populations, a birth rate, death rate and average life expectancy can be defined for fine roots (Majdi and Persson, manuscript). The most widely used approach for estimating fine-root production and mortality has been from changes in dry weight in live and dead roots, often less than 1 or 2 mm in diameter (Vogt and Persson 1991). Closely related to the cost-benefit balance of the forest trees are different selective tactics in growth patterns and structures. Although no one would refute the important role played by roots in water and nutrient uptake, species that minimize the investment of energy into these functions seem to be suitable for silvicultural practice. Fine-root growth dynamics Growth dynamics of fine roots may differ considerably between different sites, tree species and from one year to another (Santan tonio and Hermann 1985, Persson 1980). Most earlier works on the temporal pattern of fine-root growth indicate a considerable variation in the amount of fine-roots during the growing season, as a consequence of a high turnover rate (cf. Persson 1980). These turnover figures constitute the background for the long term fluctuations in the soil organic matter during the life of a forest since the dead root material constitutes one important source of organic input into the soil environment. The main fac tors that may influence root growth of forest trees are age and type of tree species, carbon economy, nutrient and water supply, other abiotic factors such as soil temperature, soil strength and aeration, and finally chemical toxicity and allelopathy (Persson 1992). Besides local climatic and edaphic factors, silvicultural practice may vary the picture considerably. Site quality may sig nificantly affect the relationship between the amount of fine roots and foliage produced annually (Santantonio et al. 1977). In closed canopy stands, there is a consistent, strong negative rela tionship in dry matter partitioning between fine roots and stems (Santantonio and Grace 1987). The carbon cost necessary for the uptake of water and nutrients by the fine roots appears to be in balance with the carbon partitioning to the foliage (Santantonio and Grace 1987). The annual turnover of fine-roots in a young and a mature Scots pine stand was shown to be at least be twice 121 Roots, mycorrhizas and rhizosphere microbes. and equal to the average fine-root biomass (Persson 1979). Avail able data suggest that the fluctuations and fine-root production are higher in young Scots pine stands than in a mature ones. The respiration costs increases furthermore considerable with age of the trees. The mechanisms resulting in the rapid disappearance of roots upon death are of great interest since a high amount of car bon is involved. Carbon economy An indication of the importance of root production in the total carbon flow may be obtained from carbon budgets (Agren and Axelsson 1979, Ägren et ai. 1980, Table 1). The root production may then be obtained as a rest fraction if growth, respiration and the net photosynthesis are measured. The carbon budget reveals that 13-57 % of the carbon that is assimilated annually by Scots pine trees of varying age is used for the growth of root systems; the related respiration cost is 5-25 %. Investigations by Raich and Nadelhoffer (1989) show that live-root respiration can be a major contributor to the total soil respiration, up to one-third to two third of the annual carbon release from the forest soil. The carbohydrate storage capacity in tree roots is generally fairly high (as much as 5-25 % of the dry weight consists of starch - Ericsson and Persson 1980). Within fine roots variation in starch content is considerable, also due to varying diameter (Wargo 1976, Ericsson and Persson 1980). It may be concluded that fine root growth leads to a reduction in starch concentration (Ericsson and Persson 1980). Marshall and Waring (1985) developed a model to predict fine-root production and turnover from soil tem- Table 1. Annual carbon budget for an average tree in eight forest stands in Jädraäs, Central Sweden (Agren and Axelsson 1979). Estimates are given as g C year-1. Stand age 17 32 40 56 83 90 106 122 Net photosynthesis 1551 2738 6315 3348 7914 9880 10695 11653 Roots <5 mm 885 307 790 1075 4300 4619 4276 6479 (57%) (11%) (13%) (32%) (54%) (47%) (40%) (56%) Growth 592 1861 4186 1768 2848 4067 4992 4115 (38%) (68%) (66%) (53%) (36%) (41%) (47%) (35%) Respiration 74 570 1338 504 765 1194 1427 1059 (5%) (21%) (21%) (15%) (10%) (12%) (13%) (9%) 122 perature and starch depletion of the fine roots. The following hy potheses were tested (i) that the growth of fine roots is accompanied by starch accumulation rather than depletion; (ii) that a fully developed fine root meets its maintenance require ments wholly from its starch and sugar reserves, and (iii) that the root dies when its starch and sugar reserves are exhausted. From the work by the latter authors one may conclude: that initial starch concentration and soil temperature are key variables estimating fine-root turnover and fine-root biomass. The general lack of insight into the relationship between above- and belowground production in forests results from the relatively few reliable measurements of belowground production (cf. Santantonio et al. 1977, Santantonio and Hermann 1985, Pers son 1980). Fine-root production and mortality can occur simulta neously in small soil volumes and can therefore not be separated on time or space (Santantonio and Grace 1987). Only limited at tempts have been made so far to test their statistical precision of existing data (Persson 1979). Estimation of fine-root production is usually a part of studies to quantify total stand production in plan tations and natural forests. Although most studies have been com parative e.g. good versus poor sites (Keyes and Grier 1981), quantity and form of available nitrogen (Finer 1992, Helmisaari 1990, Nadelhoffer et al. 1985) few studies have so far been exper imental. Results from minirhizotrone investigations (Fig. 1) on the cumulative fine-root production and mortality in the control and "optimum" fertilized plots with liquid fertilizers" (IF at Skogaby, cf. Persson et al. in press) make it clear that the produc tion and mortality proceeded much more rapidly in the IF-plots than in the C-plots. Results from related studies suggest that a greater amount or proportion of the total net primary production goes to the fine roots, when site conditions are less favourable for growth (Persson et al. in press). Data on fine-root growth in response to the presence of plant nutrients in the vicinity of the tree roots are available from exper imentally manipulated areas from many field experiments in Swe den (cf. e.g. Ahlström et al. 1988, Persson 1979, Persson and Ahlström 1990, Persson et al. in press). From these areas has been shown that an increased needle mass in stands with a high nitro gen supply often corresponds to a reduced amount of fine roots and mycorrhizal frequency. Liquid fertilization using drop tubes indicates a positive effect on fine root growth in the area nearest the drop tube. Other experimental treatments such as liming and compensatory fertilization may result in both negative and posi tive effects on the growth and development of fine roots, depend- 123 Roots, mycorrhizas and rhizosphere microbes. Figure 1. Cumulative fine-root production and mortality (mm) in different treatment plots from Au- gust 1991 to August 1992 at Skogaby to a depth of 30 cm. The estimates were obtained on the 1.35 1.8 cm area of ob- servation and Filled symbols refer to production and unfilled symbols refer to mortali- ty. ing on the soil type and the dose at which the liming or fertilization agents are applied (Persson and Ahlström 1994). Investigations of long term effects of forest liming on fine root growth dynamics show a tendency to increased specific root length and slightly thinner roots (Clemensson-Lindell and Pers son 1992). Similar effects have furthermore been shown by liquid fertilization (Persson 1980). Liming, however, does not seem to have a persistent long term effect on the fine-root development (Persson and Ahlström 1994). N-fertilization in most cases will cause persistent negative effects on both fine-root and mycorrhiz al development (Ahlström et ai. 1988, Persson and Ahlström 1990). A sufficient water uptake by some part of the root system can provide the necessary water for the whole tree. Therefore, some roots of the same root system may grow through dry zones when the water uptake and supply is guaranteed by other roots. In dry soils, roots have a tendency to grow towards more humid zones and are generally found at greater depth than in moist soils (Lyr and Hoffmann 1967, Persson et al. in press). In dry sites the whole soil profile appears to be more occupied by fine roots than in wet sites, although the total fine-root production may not differ signif icantly (cf. Santantonio 1979). Roots are forced to penetrate large soil volumes, often against mechanical resistance of densely packed soil layers (cf. Clemensson-Lindell and Persson 1992). Root members in compacted soil are often less branched and have thicker long root tips, compared to normal conditions. In a mixed forest stand (two, three species or more, the tree-root system may 124 overcome the soil resistance better and grows deeper than in mo nocultural forests. Root damage A destabilization of the root systems in the forest soil frequently occurs as a result of human activities (cf. Puhe et ai. 1986). Root damage is often observed as a decline in the amount of living fine-roots, an increase in the amount of dead versus live fine roots (a lower live/dead ratio) and an increasing amount of dead medium and coarse roots. The most important factors which may cause a reduction in fine-root growth and mycorrhizal develop ment are: (i) ion-imbalances, viz. high nitrogen/nutrient ratios; (ii) A 1 toxicity, viz. elevated Al/cation ratios and (iii) an increased sensivity of the root systems to environmental stress (drought, wind-break, nutrient shortage, etc.). The processes of ion uptake are dependent of the degree of penetration of fine roots and mycorrhizas into the soil. If the up take process is hampered, the growth of the whole tree may be af fected. Root damage, in this context, may underline a predisposing stress, thus reducing water and mineral nutrient up take. Root damage often seems to be related to poor soil condi tions with generally low nutrient availability (Ulrich 1990). Future root investigations Many major environmental problems result from the lack of understanding of the part played by tree roots, in the total carbon flow (Persson 1991). New pathways of carbon and mineral nutri ent cycling require further assessment. Some important areas for future root investigations in forest ecosystems are: i) Carbon and mineral nutrient allocation pattern in trees. ii) How long the nutrients remain in any of the belowground compartments (i.e. the resistence time)? iii) Are there physiological differences between young and old trees in the growth dynamics of fine roots? iv) Links between soil, rhizosphere and fine-root chemistry. v) Exudation from tree roots. vi) The uptake by mycorrhizal roots. vii) The pattern of distribution and movement of water in the for est soil and in the rhizosphere. Roots, mycorrhizas and rhizosphere microbes. 125 References Ägren, G. & Axelsson, B. 1979. Annual carbon budgets for Scots pine. In: Linder, S. (Ed.) Understanding and predicting tree growth. Swedish Conif. For. Proj., Techn. Rep. 25: 145-155. Ägren, G., Axelsson, 8., Flower-Ellis, J.G.K., Linder, S., Persson, H., Staaf, H. & Troeng, E. 1980. Annual carbon budget for a young Scots pine. In: Persson, T. (Ed.) Structure and Function of Northern Coniferous Forests - An Ecosystem Study. Ecol. Bull. (Stockholm) 32: 307-313. Ahlström, K., H. Persson & Börjesson, I. 1988. Fertilization in a mature Scots pine (Pinus sylvestris L.) stand - effects on fine roots. Plant and Soil 106: 179-190. Clemensson-Lindell, A. & Persson, H. 1992. Long-term effects of for est liming on fine-root standing crop and mineral nutrients in the soil. Scand. J. For. Res. Ericson, A. & Persson, H. 1980. Investigations of structural properties and dynamics of Scots pine stands. Ecol. Bull. (Stockholm) 32: 125-138. Finer, L. 1992. Biomass and nutrient dynamics on a drained ombryo trophic bog. Academic Diss., University of Joensuu, 93 p. Helmisaari, H.-S. 1990. Nutrient retranslocation within Pinus sylves tris. Academic Diss., University of Joensuu, 107 p. Keyes, M.R. & Grier, C.C. 1981. Above- and belowground net produc tion in 40-year-old Douglas-fir stands on low and high produc tivity sites. Can. J. For. Res. 11: 599-605. Lyr, H. & Hoffmann, G. 1967. Growth rates and growth periodicity of tree roots. Int. Rev. Forest. Res. N.Y. 2: 181-236. Majdi, H. & Persson, H. Manuscript. Fine-root dynamics in response to nutrient applications in a Norway spruce (Picea abies L. (Karst.)) stand in SW Sweden. Marshall, J.D. & Waring, R.H. 1985. Predicting fine root production and turnover by monitoring root starch and soil temperature. Can. J. For. Res. 15: 791-800. Nadelhoffer, K.J., Aber, J.D. & Melillo, J.M. 1985. Fine roots, net pri mary production and soil nitrogen availability: a new hypothe sis. Ecology 66: 1377-1390. Paavilainen, E. 1966. On the effect of drainage on the root systems of Scots pine on peat soils. Comm. Inst. For. Fenn. 61. (In Finnish, English Summary) Persson H. 1979. Fine-root production, mortality and decomposition in forest ecosystems. Vegetatio 41: 101-109. Persson H. 1980. Fine-root dynamics in a Scots pine stand with and without near-optimum nutrient and water regimes. Acta Phyto geogr. Suec. 68: 101- 110. Persson, H. (Ed.) 1991. Above and below-ground interactions in forest trees in acidified soils. Air Pollution Research Report 32, 257 p. 126 Persson, H. 1992. Factors affecting fine root dynamics of trees. Suo 43: 163-172. Persson, H. & Ahlström, K. 1990. The effects of forest liming and fer tilization on fine-root growth. Water, Air Soil Pollut. 54: 365- 375. Persson, H. & Ahlström, K. 1990. The effects of alkalizing compounds on fine-root growth in a Norway spruce stand in SW Sweden. J. Env. Sciences & Health 29: 803-820. Persson, H. Majdi, H & Clemensson-Lindell, A. Effects of acid deposi tion on tree roots. Ecol. Bull. (Stockholm) (in press). Persson, H., von Fircks Y., Majdi, H. & Nilsson, L.O. Root distribution in a Norway spruce stand subjected to varying nutrient supplies. Plant and Soil (in press). Puhe, J., Persson, H. & Börjesson, I. 1986. Wurzelwachtum und Wur zelshäden in Skandinavischen Nadehväldern. Allgemeine Forst Zeitschrift 20: 488-492. Raich, J.W. & Nadelhoffer K.J. 1989. Belowground carbon allocation in forest ecosystems: global trends. Ecology 70: 1346-1354. Santantonio, D. 1979. Seasonal dynamics of fine roots in mature stands of Douglas-fir of different water regimes - A preliminary report. In: Riedacker, A. & Gaignard-Michard, J. (eds.) Root Physiol ogy and Symbiosis. INRA-CNRF, Nancy-Chamenoux, pp. 190- 203. Santantonio, D., Hermann, R.K. & Overton, W.S. 1977. Root biomass studies in forest ecosystems. Pedobiologia 17: 1-31. Santantonio, D. & Grace, J.C. 1987. Estimating fine-root production and turnover from biomass and decomposition data: a compart ment flow model. Can. J. For. Res. 17: 900-908. Santantonio, D. & Hermann, R.K. 1985. Standing crop, production, and turnover of fine roots on dry, moderate, and wet sites of mature Douglas-fir in western Origon. Ann. Sci. For. 42: 113-142. Santantonio, D. & Santantonio, E. 1987. Effects of thinning on produc tion and mortality of fine roots in a Pinus radiata plantation on a fertile site in New Zealand. Can. J. For. Res. 17: 919-928. Ulrich, B. 1990. An ecosystem approach to soil acidification. In: Ulrich, B. &. Sumner, M.E. (eds.) Soil Acidity. - Springer-Ver lag, Berlin, pp. 28-79. Wargo, P.M. 1976. Variation of starch content among and within roots of red and white oak trees. Forest Science 22: 468-471. Vogt, K.A. & H. Persson, 1991. Measuring growth and development of roots, in: Hincley, T. & Lassoie, J.P. (eds.). Techniques and Approaches in Forest Tree Ecophysiology. CRC-Press, Inc., Florida, pp. 477-501. 127 Roots, mycorrhizas and rhizosphere microbes. Effect of shadelight quality on dry weight allocation and mycorrhizal development in Scots pine seedlings Tania M. de la Rosa 12 , Tarja Lehto1 and Pedro J. Aphalo1 Introduction In their natural habitat, plants are exposed to continual changes in the light environment. Shading of canopies or neighbouring plants not only reduces the total amount of radiation (total flux density), but also alters the composition of light due to selective spectral absorption and reflection of leaves, particularly in the red and far red region. Low red/far-red photon ratios (R/FR) characteristic of shading or neighbouring vegetation are sensed by plants through phytochromes. The perception of low R/FR usually promotes stem elongation and alters the biomass alloca tion - e.g. reduced leaf/stem dry weight ratio and increased shoot/ root ratio. Root system growth and formation of mycorrhiza are depend ent on carbohydrate supply from the shoot. Any effect of light quality on the allocation of resources might affect root develop ment. Low light intensities decrease ectomycorrhizal formation (Björkman 1942, Ekwebelam and Reid 1983), and this effect has been attributed to carbohydrate availability. To the best of our knowledge, the effect of light quality on the growth of the root system and mycorrhiza development has not been studied before, but the results discussed above indicate that such an effect could be possible. The aim of this study was to examine early changes in dry weight allocation, morphology and formation of mycorrhizas in 1 Finnish Forest Research Institute, FIN-77600 Suonenjoki, Finland. 2 Department of Botany, FIN-00014 University of Helsinki, Helsinki. 128 Scots pine (Pinus sylvestris L.) seedlings in relation to light qual ity. Material and methods Pinus sylvestris L. seedlings were grown from seeds in a sub strate containing 1 part sand and 2 parts unfertilised peat (v/v), in a greenhouse (26/15 °C max. day / min. night temperatures and 18 hours light regime from natural light plus metal halide lamps). Within a randomised design, four seedlings per treatment within each of six blocks were placed under the following light quality treatments: control and supplemental far-red (FR+). Starting 1 wk after germination, FR light was provided by an additional far red sidelight source, filtered through two glass cuvettes contain ing water (to prevent difference in temperature) and red plus blue polyester films. The photosynthetic photon flux density (/PAR) at the top of the plants was approx. 250 |J.mol m 2 s l . A complete nutrient solution (modified Ingestad solution, Riddoch et al. 1991) was provided at a rate of 1.2 mg N wk -1 per seedling throughout the whole experiment. Forty two days after germina tion, all plants were inoculated with Suillus bovinus (strain K4 ex Robin Sen), by placing fungal culture (Hagem agar) in direct contact with fine roots to ensure availability of inoculum. The height of plants was measured to the nearest millimetre with a ruler twice a week throughout the experiment. Seedlings were harvested 93 d after the light treatment was started. The root systems were carefully washed and two subsamples at 30 - 50 and 105- 125 mm depths were taken from each seedling. The subsam ples were observed under a stereomicroscope for total count of short root tips and characterisation of mycorrhizas. The develop ment of mycorrhizas was assessed by microscopy after staining (Koske and Gema 1989), and root length was estimated by the grid intersection method according to Tennant (1975). Short root tip classification included the following categories: non mycor rhizal tips; developing mycorrhiza (mantle not yet developed, but Hartig net visible after staining); mycorrhiza with mantle and/or external mycelium. Using the dry weight of the subsamples and of the whole root system, estimates of root length and numbers of short root tips and mycorrhizas in the whole plant were calculated. The root subsamples as well as the remaining root system, stem and needles were dried at 65° C for 48 hours before weighing. 129 Roots, mycorrtiizas and rhizosphere microbes... Results Light quality had a significant effect on the growth of young Scots pine seedlings: FR+ increased stem height by 17% (P = 0.010*) (Table 1). The total dry weight of the plant was 564 mg in control and 482 mg in FR+ (P = 0.073), but the dry weight ratio of stem was larger in the FR+ and that of roots was smaller (Fig. 1). Total root length (data not shown) and specific root length (Table 1) were not affected by FR+ treatment. Mycorrhizal categories as percentage of the total number of short root tips and the number of root tips expressed per unit of length were not affected by light quality (Table 1). However, the estimated total number of short root tips and developing mycor rhizas per seedling were somewhat smaller in the FR+ seedlings. There were very few mycorrhizas with mantle and/or external mycelium (Table 1 and 2). Table 1. Stem length, specific root length, number of short root tips per root length unit and mycorrhizal categories as percentage of the total number of short root tips. Probabilities from ANOVA. Table 2. Estimated total number of short root tips and mycorrhizas per seedling. Probabilities from ANOVA. CONTROL FR+ P Stern length (mm) 107.02 124.84 0.010 Specific root length (m g 1 ) 71.4 73.8 0.632 Short roots (tips nr 1 ) 175 189 0.200 Non mycorrhizal tips (%) 0.27 0.14 0.434 Developing mycorrhiza (%) 99.5 99.56 0.995 Mycorrhiza with mantle ami/ or external mycelium (%) 0.02 0.01 0.593 CONTROL FR+ P Total short root tips 2184 1591 0.096 Non mycorrhizal tips 4.09 1.61 0.190 Developing mycorrhiza 2167 1573 0.096 Mycorrhiza with mantle and/ 0.40 0.37 0.578 or external mycelium 130 Figure 1. Dry weight ra tio of Scots pine seedling as affected by supple mentary FR+. Discussion The Scots pine seedlings used in this study responded to FR light by increasing stem elongation, a common "shade avoidance" behaviour, observed in many herbaceous and tree species includ ing Monterey pine (Warrington et al. 1989). In contrast to previ ous studies on herbaceous plants (Ballare et al. 1991, Smith 1994), dry weight allocation to needles did not differ between light treatments, however in FR+ seedlings allocation to stems increased at the expense of roots. This suggests that, in this case, increased stem elongation does represent a cost in terms of car bon allocation, but to roots rather than to needles. This different behaviour could be related to differences in life span and compe tition strategies of annual and perennial plants. Reduction in the number of short root tips and developing my corrhiza paralleled the decrease in root biomass. Root morpholo gy or the stage of development of mycorrhizas did not change due to FR+. As Ekwebelam and Reid (1983) found an increase in the percent of mycorrhizas with increasing light intensity, our results suggest that the effects of light quality and of total irradiance on mycorrhizas are different. The ability of Scots pine seedlings to sense changes in light quality could enhance success in competition with other plants. However, one should bear in mind that allocation to roots and aboveground parts is strongly affected by soil nutrients and water availability. 131 Roots, mycorrhizas and rhizosphere microbes... References Ballare C.L., Scopel, A.L. & Sanchez, R.A. 1991. On the opportunity cost of the photosynthate invested in stem elongation reactions mediated by phytochrome. Oecologia 86: 561-567. Björkman, E. 1942. Über die Bedingungen der Mykorrhizabildung bei Kiefer und Fichte. Symbolae Botanici Upsaliensis 6: 1-191. Ekwebelam S.A. & Reid, C.RR 1983. Effect of light, nitrogen fertiliza tion, and mycorrhizal fungi on growth and photosynthesis of lodgepole pine seedlings. Can. J. For. Res. 13: 1099-1106. Koske, R.E. & Gema, J.N. 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycol. Res. 92: 486-505. Riddoch, 1., Lehto, T. & Grace, J. 1991. Photosynthesis in tropical tree seedlings in relation to light and nutrient supply. New Phytol. 119: 137-147. Smith, H. 1994. Sensing the light environment: the functions of the phytocrome family. In: Kendrick, R.E. & Kronenberg, G.H.M. (eds.). Photomorphcigenesis in Plants - 2nd Edition. Kluwer Academic publishers, Dordrecht, pp. 377-416. Tennant, D. 1975. A test of a modified line intersect method for esti mating root length. J. Ecol. 63: 995-1001. Warrington, 1.J., Rook, D.A., Morgan, D.C. & Turnbull, H.L. 1989. The influence of simulated shadelight and daylight on growth, devel opment and photosynthesis of Pinus radiata, Agathis australis and Dacrydium cupressinum. Plant Cell Environ. 12: 343-356. 132 133 Roots, mycorrtiizas and rhizosphere microbes. Biomass and turnover of roots in a mesotrophic fen Timo Saarinen Introduction Estimates of the below-ground biomass and production of mire plants are scarce. Recently reported values both from a minero trophic fen (Sjörs 1991) and an ombrotrophic bog (Wallen 1986) indicate that over 90 % of the total biomass may be located below-ground. The aim of this work was to measure the vertical distribution of biomass and production of vascular plants in a mesotrophic fen. Special attention was paid to fine roots and deep growing roots and their significance to peat accumulation. Material and methods The study site is located on the Suurisuo mire complex, Janak kala, southern Finland. A mesotrophic fen community dominated by Carex rostrata and Potentilla palustris was chosen for the study. The indirect 14 C labelling method described by Wallen (1986) was applied to estimate the below-ground biomass of the vascular plants in the uppermost 30 cm in June 1992. The deepest roots (between 30-230 cm) were sorted manually from cores collected in June 1993. The 14 C turnover method developed by Milchunas and Lauenroth (1992) was modified to measure the below-ground productivity. Long-term 14 C sample plots established in July 1992 were sampled regularly. 14 C activities were analysed both in structural and non-structural carbon fractions of roots and the pro portion of labelled fine roots were calculated with autoradiogra phy. Below-ground production was estimated by dividing Department of Botany, Ecological laboratories, P.O. Box 4, FIN-00014 University of Helsinki, Finland. 134 Fig. 1. Vertical distribu tion of biomass of Carex rostrata and PotentiHa palustris in a mesotrophic fen of Suurisuo mire com plex, Janakkala, south ern Finland. Fig. 2. Percentage of 14 C labelled fine roots of Carex rostrata. The sam ple plots were pulse la belled in July 1992. 135 Roots, mycorrhizas and rhizosphere microbes... biomass by 14 C turnover time. Above-ground biomass and pro duction were measured using demographic methods. Individual shoots were tagged on 10 sample plots in May 1993. The length of the shoots was measured and new shoots tagged monthly dur ing the growing seasons. Biomass was calculated using the rela tionship between length and weight of shoots. Results and discussion The total living biomass of Carex and Potentilla was 2280 g m" 2 and 420 g m" 2 , respectively (Fig. 1). The below-ground biomass of both species was considerably high, no less than 92% and 88% of the total biomass Carex and Potentilla, respectively. Fine roots comprised 78% of the biomass of Carex. The living roots of Carex reached at least the depth of 230 cm. This is clearly deeper than the values reported in earlier studies (e.g. Metsävainio 1931). As decomposition of peat is slow in ca totelm, even the relatively small biomass of these deepest roots (68 g m" 2 between 30 and 230 cm) may affect the accumulation of peat. In the beginning of May 1993, the majority of the 14 C label of fine roots (labelled in July 1992) was in the structural carbon frac tion. The label in living roots may disappear only when old roots die and new roots develop. During the summer period (May-Sep tember), the percentage of the labelled fine roots decreased from 81 % to 24 %, which refers to a relatively rapid turnover of fine roots (Fig. 2). However, no decrease could be observed in the win ter time (September-May). Due to a relatively rapid turnover of fine roots, the annual production of Carex rostrata is high (1340 g m" 2 a _1 ) in this fen Table 1. Biomass and production of Carex rostrata in a mesotrophic fen on Suu risuo mire complex, Janakkala, southern Finland. Biomass Turnover Production (9 m" 2 ) (a 1 ) (g m" 2 a" 1 ) Shoots 185 - 176 Rhizomes 185 0.47 87 Coarse roots 135 0.47 63 Fine roots 1717 0.59 1014 136 (Table 1). With an average carbon content of 48%, there is a car -1 1 bon input of approximately 640 gC m a . Fine roots contribute to 76 % of the total production. References Metsävainio, K. 1931. Untersuchungen iiber das Wurzelsystem der Moorpflanzen. Ann. Bot. Soc. Zool.-Bot. Fenn. Vanamo 1(1). Milchunas, D.G. & Lauenroth, W.K. 1992. Carbon dynamics and esti mates of primary production by harvest, 14 C dilution and 14 C turnover. Ecology 73: 593-607. Sjörs, H. 1991. Phyto- and necromass above and below ground in a fen. Holarct. Ecol. 14: 208-218. Wallen, B. 1986. Above and below ground dry mass of the three main vascular plants on hummocks an a subarctic peat bog. Oikos 46: 51-56. 137 Roots, mycorrhizas and rhizosphere microbes. Microbial biomass in the rhizosphere of trees Aino Smolander, Ilari Lumme and Heljä-Sisko Helmisaari Introduction The rhizosphere can be defined as that part of the soil adjacent to a plant root, which is different from the surrounding soil because of the chemical, physical and biological activity of plant root. The effect of root is caused by several processes such as water and nutrient uptake, gas exchange and excretion of organic com pounds. There is evidence that plants can stimulate microbial activity through the supply of organic subtrate in root exudates, but they may also limit microbial activity through depletion of mineral nutrients (Bääth et ai. 1978, van Veen et ai. 1989, Parme lee et ai. 1993). The aim was to study the effect of tree roots on soil microbial biomass and activities related to carbon and nitrogen cycles. Pre liminary microbial biomass results are discussed in this paper. Materials and methods Pot experiment The effect of the roots of Norway spruce (Picea abies Karst.) seedlings on soil microbial biomass was studied in connection with nitrogen allocation studies using 15 N isotope (Lumme 1994). Three-year-old potted seedlings of a Norway spruce clone were grown for 3 months in acid sandy soil. Nitrogen ((NH4)2SO4) was applied to the soil in the seedling pot and in the corresponding pots without seedlings twice a week, the total amount of nitrogen applied being 200 mg N per seedling, corre sponding to approximately 40 kg/ha/year. Soil without plants was Finnish Forest Research Institute, Department of Forest Ecology, P.O. Box 18, FIN-01301 Vantaa, Finland. 138 treated similarly. At the end of the experiment, roots were sepa rated from the soil by hand picking, and microbial biomass in the remaining soil was determined using the fumigation extraction (FE) technique (Vance et al. 1987). Field studies The gradual development of rhizosphere microbial population with the growing roots and mycorrhizas is under study in some Norway spruce and Scots pine (Pinus sylvestris L.) stands using the root ingrowth core method (Persson 1990). In this method, a nylon net mesh is inserted into the hole in soil, made by a cylin drical corer, and filled with sorted and rootless mineral soil. After one to four growing seasons, the cores are removed, and the bio mass of roots determined. In the present study, roots were sepa rated from the soil by hand picking, and microbial biomass in the remaining soils was determined using both the fumigation extrac tion (Vance et al. 1987) and substrate induced respiration (SIR) (Andersson and Domsch 1978) techniques, as described earlier by Smolander et al. (1994). Results and discussion Soil microbial biomass was almost six-fold in pots planted with spruce seedlings compared to those without seedlings (Fig. 1). The increase in soil microbial biomass C in planted pots indi- Figure 1. FE-derived mi crobial biomass C in sandy soil where Norway spruce seedlings had grown for 3 months, and in corresponding soil without plants. Mean ± SD for 3 replicate pots. 139 Roots, mycorrhizas and rhizosphere microbes. cated the importance of plant derived substrate in the mineral soil with a low organic matter content. Accordingly, Parmelee et al. (1993) showed the positive effect of pine roots on microbial bio mass in mineral soil but got opposite results in organic soil. Results of the first sampling of root ingrowth cores showed a slight decrease with depth in both FE- and SIR-derived microbial biomass C. No clear relationship between root biomass and mi crobial biomass was yet observed. In the pot experiment and field studies, soil microbial biomass values are probably underestimations because part of mycorrhiza and the microbes attached to roots and mycorrhiza are not includ ed. A few replicate root ingrowth cores with roots were also sub jected to microbial biomass determination; removal of roots had decreased the SIR-derived biomass C values 0-26 %. References Anderson, J.P.E. & Domsch, K.H. 1978. A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biol. Biochem. 10: 2:15-221. Bääth, E., Lohm, U., Lundgren, 8., Rosswall, T., Sohlenius, B. & Wiren, A. 1978. The effect of nitrogen and carbon supply on the development of soil organism populations and pine seedlings: a microcosm experiment. Oikos 31: 153-163. Lumme, I. 1994. Nitrogen uptake of Norway spruce (Picea abies Karst.) seedlings from simulated wet deposition. For. Ecol. Manag. 63: 87-96. Parmelee, R.W., Ehrenfeld, J.G. & Tate 111, R.L. 1993. Effects of pine roots on microorganisms, fauna, and nitrogen availability in two soil horizons on a coniferous forst spodosol. Biol. Fertil. Soils 15: 113-119. Persson, H. 1990. Methods of studying root dynamics in relation to nutrient cycling. In: Harrison, S.F., Ineson, P. & Heal, O.W. (Eds.) Nutrient cycling in terrestrial ecosystems. Field methods, application and interpretation, pp. 198-217. Elsevier Applied Science. Smolander, A., Kurka, A., Kitunen, V. & Mälkönen, E. 1994. Microbial biomass C and N, and respiratory activity in soil of repeatedly limed, and N- and P-fertilized Norway spruce stands. Soil Biol. Biochem. 26: 957-962. Vance, E.D., Brookes, P.C. & Jenkinson, D.S. 1987. An extraction method for measuring soil microbial biomass C. Soil Biol. Bio chem. 19: 703-707. Van Veen, J.A., Merckx, R. & van de Gein S.C. 1989. Plant and soil related controls of the flow of carbon from roots through the soil microbial biomass. Plant and Soil 115: 179-188. 140 141 Roots, mycorrtiizas and rhizosphere microbes. A new hypothesis to explain the negative influence of nitrogen on ectomycorrhizal development Håkan Wallander Abstract Nutrient uptake by forest trees is largely dependent on their asso ciated ectomycorrhizal fungi. The presence of extramatrical myc elium produced by ectomycorrhizal fungi allows trees to exploit a larger soil volume. Elevated levels of nitrogen strongly inhibit the development of extramatrical mycelium. To explain reduced ectomycorrhizal development under con ditions of high levels of N supply, it is suggested that the the fun gus consumes the available carbohydrate in order to take up and assimilate nitrogen. Only if there is a surplus of carbohydrates, fungal mycelium and fruit bodies can be produced. The present hypothesis proposes that it is the fungus, rather than the host which adjusts its carbon allocation patterns to the N supply. Introduction Ectomycorrhizal roots are characterized by fungal hyphae grow ing between root cortical cells to form a Hartig net and by several layers of hyphae ensheathing the root to form a mantle (a good overview of mycorrhizal structure and function is provided by Harley and Smith 1983). An extramatrical mycelium is radiating out from the mantle into the soil. In this way, the soil volume exploited by the tree is increased substantially (Read et al. 1985, Microbial Ecology, Department of Ecology, Lund University, Ecology Building, S-223 62 Lund, Sweden. 142 Harley 1989). It has been suggested that the host allocates less carbohydrate to the mycobiont at high levels of nutrient supply owing to a greater demand for carbon by the growing shoots under such conditions (originally proposed by Björkman 1942). In the present paper, an alternative hypothesis is presented that may explain nitrogen effects on mycorrhizal development. Influence of nitrogen supply on production of extramatrical mycelium In general, high nitrogen availability, due to anthropogenic N deposition (Termorshuizen and Schaffers 1991, Arnolds 1988) or forest fertilization (Wasterlund 1982, Ohenoja 1988) reduces the production of ectomycorrhizal fruit bodies. However, fruit body production does not necessarily reflect the amount or activity of ectomycorrhizal roots. We have for a long time investigated mineral nutrient effects on mycorrhizal development in a steady-state, semi-hydroponic cultivation system. Fungal biomass of mycorrhizal seedlings have consistently been reduced at elevated levels of N supply (Nylund and Wallander 1989, Wallander and Nylund 1991,1992). This re duction was more pronounced for extramatrical mycelium than for fungal biomass in mycorrhizal roots (Wallander and Nylund 1992). Whereas mycorrhizal roots continued to develop, but at a lower rate, the growth of extramatrical mycelium was completely inhibited and in some cases the mycelium even died when sup plied with excess nitrogen. Similar results were found by Arne brant (1994) who showed that the growth of extramatrical mycelium of an unidentified white ectomycorrhizal fungus was totally inhibited once it reached peat amended with 1, 2 or 4 mg N g" 1 ((NH4)2SO4 or NaN03) whereas Paxillus involutus colo nized peat with N amendments of up to 4mg Ng' 1 . Gorissen et al. (1991) found that Douglas-fir seedlings fertilized with 200 kg N ha" 1 had the same mycorrhizal frequency as seedlings fertilized with 50 kg N ha" 1 , but that the respiration of labelled 14 C by root and fungal tissue was reduced by 60% in the high N treatment. As these investigators pointed out, this reduction could have been due to a decrease in fungal activity at high N levels that was not re vealed by counting mycorrhizal root tips. 143 Roots, mycorrhizas and rhizosphere microbes. Figure 1. Hypothetical patterns of carbon flow between host and fungus at low or high levels of N supply, a) At a low level of N supply most host carbon is used for fungal growth. At high N supply levels a number of ef fects on carbon flow are possible: b) Total carbon flow to the fungus is re duced, leading to re duced fungal growth, c) The fungus is forced to allocate more carbon to N-assimilation and less to fungal growth. N avail ability does not influence total carbon flow to the fungus, d) The fungus has a low N-assimilation rate and can maintain a high rate of fungal growth at elevated levels of N supply. Possible ways to explain the negative influence of nitrogen on extramatrical mycelium Increasing the levels of N supply may have a number of effects on carbon flow between host and fungus. Björkman (1942) sug gested that the host would allocate less carbohydrate to the myco biont at high levels of nitrogen and phosphorus availability owing to a greater demand for carbon by growing shoots under such conditions (Fig. 1 a,b). By contrast, we found that carbohydrate pools in roots increased in response to elevated N levels; still, mycorrhizal development was reduced (Wallander and Nylund 1991). This finding indicates that fungal growth was inhibited for reasons other than carbohydrate deficiency. Björkman (1942) assumed that nitrogen was assimilated in the host and that all car bohydrates transferred to the fungus were used for its develop ment. However, since then it has been convincingly demonstrated that ammonium is assimilated in the fungal tissue (France and Reid 1983, Finlay et al. 1988). Host carbohydrates allocated to 144 the mycorrhizal fungus are used in growth processes and as car bon skeletons and energy sources in the process of ammonium assimilation. The assimilated N is either used in growth processes by the fungus, stored as amino acids or protein in the fungal man tle or returned to the host in the form of amino acids (Fig. 1). In our work we found that growth of the extramatrical mycelium of L.bicolor resumed once the N supply had been lowered (Wal lander and Nylund 1992). These findings suggest that an excess supply of nitrogen might not be detrimental to the fungus. Thus when faced with an oversupply of nitrogen the fungus might allo cate its carbohydrate reserves to amino acid biosynthesis, whereas at low levels of nitrogen supply the carbohydrates could be used for growth. The hypothesis can be expressed as follows: Ectomycorrhizal fungi are adapted to N-limited environments. Host carbohydrates available to the fungus are preferably used to assimilate ammonium-N into amino acids. The surplus of the car bohydrates can be used to produce fungal mycelium and fruit bodies. In contrast with the general suggestion that elevated N levels inhibit mycorrhizal fungi, it has been shown that Lactarius rufus, Laccaria laccata and P. involutus, among other species, can sub stantially increase sporocarp production after N additions (Ohe noja 1978, 1988). N-tolerant fungi may reduce the allocation of host carbon to N assimilation in other (unknown) ways in order to favour fungal growth (Fig. Id). To test the hypothesis we are now investigating a number of strains of P. involutus and Suillus variegatus. The main question is: Do ectomycorrhizal fungi which are more sensitive to nitrogen have a high rate of N uptake and a high rate of translocation of assimilated N to the host. And vice versa: Do nitrogen tolerant species have a possibility to reduce the N uptake and N transloca tion when the N input is high? In conclusion: It seems clear that mycorrhizal fungi allocate more carbon to the process of N assimilation (carbon skeletons and energy) when N supply levels are high, leading to reduced fungal growth. However, the response of fungal species are likely to differ in this respect (see Fig. 1), depending on their capacity to assimilate N, their sensitivity to toxic levels of N and, probably, on other unknown factors as well. References Arnebrant, K. 1994. Nitrogen amendments reduce the growth of extram atrical ectomycorrhizal mycelium. Mycorrhiza (in press). 145 Roots, mycorrhizas and rtiizosphere microbes. Arnolds, E.J.M. 1988. The changing macromycete flora in the Nether lands. Transactions of the British Mycological Society. 90: 391- 406. Björkman, E. 1942. Über die bedingungen der Mykorrhizabildung bei Kiefer und Fichte. Symbolae Botanicae Upsalienses 6: 2. France, R.C. & Reid, C.RR 1983. Interactions of nitrogen and carbon in the physiology of ectomycorrhizae. Can. J. Bot. 61: 964-984. Gorissen, A., Joosten, N.N. & Jansen, A.E. 1991. Effects of ozone and ammonium sulphate on carbon partitioning to mycorrhizal roots of juvenile Douglas lir. New Phytol. 119: 243-250. Harley, J.L. & Smith, S.E. 1983. Mycorrhizal symbiosis. Academic Press, London. Harley, J.L. 1989. The significance of mycorrhiza. Mycol. Res. 92: 129-139. Nylund, J.-E. & Wallander, H. 1989. Effects of ectomycorrhiza on host growth and carbon balance in a semi-hydroponic cultivation sys tem. New Phytol. 112: 389-398. Ohenoja, E. 1978. Mushrooms and mushroom yields in fertilized for ests. Ann Bot Fenn. 15: 38-46. Ohenoja, E. 1988. Effects of forest management procedures on fungal fruit body production in Finland. Acta Bot. Fenn. 136: 81-84. Read, D.J., Francis, R. & Finlay, R.D. 1985. Mycorrhizal mycelia and nutrient cycling in plant communities. In: Ecological interac tions in soil. A.H. Fitter, D. Atkinson, D.J. Read & M.B. Usher (eds.). Blackwell Scientific Publications, London. Termorshuizen, A.J. & Schaffers, A. 1991. The decline of carpophores of ectomycorrhizal fungi in stands of Pinus sylvestris L. in the Netherlands: possible causes. Nova Hedwigia. Wallander, H. & Nylund, J.-E. 1991. Effects of excess nitrogen on car bohydrate concentration and mycorrhizal development of Pinus sylvestris L. seedlings. New Phytol. 119: 405-411. Wallander, H. & Nylund, J.-E. 1992. Effects of excess nitrogen and phosphorus starvation on the extramatrical mycelium of Pinus sylvestris L. ectomycorrhiza. New Phytol. 120: 495-503. Wästerlund, I. 1982. Hur päverkar skogens skötsel förekomsten av storsvampars fruktkroppar? Svensk Botanisk Tidskrift 83: 1 OS IO. 146 147 Roots, mycorrhizas and rhizosphere microbes. Mycorrhizal root coloniza tion and ergosterol content in an experimentally pol luted subarctic birch-pine forest Martin Zobel1 - 2 , Olav Sarv12 and Martti Komulainen2 Introduction Deposition of Cu and Ni and acid rain have had a considerable effect on plant communities in the subarctics (Kozlov et ai. 1993). Both arbuscular mycorrhiza (AM, Gildon and Tinker 1983) and ectomycorrhiza (Denny and Wilkins 1987) influence the uptake of heavy metals by plants. If pollution is changing the degree of mycorrhizal root colonization, the response of plants can be in correlation with it's mycorrhizal status. The aim of this study was to gather preliminary information about the effects of acid rain and Cu-Ni pollution induced changes to ectomycorrhiza and AM of the two common plant species of a subarctic forest. Materials and methods The study was conducted near the Kevo Subarctic Research Sta tion in Finnish Lapland. In June, 1991, 4x4 m plots with a simi lar field layer vegetation located in a mountain birch forest were randomly assigned to different treatments. A factorial design incorporated two factors - acid and heavy metal treatments. Each 1 Department of Botany & Ecology University of Tartu, Lai St 40, EE-2400 Tartu, Estonia. 2 Laboratory of Ecological Zoology Department of Biology, University of Turku, FIN-20500 Turku, Finland. 148 treatment combination was replicated five times. Treatments were applied by irrigating plots twice a week (about 5 mm/irri gation event) during the periods 20 July - 27 August 1991 and 11 June - 28 August 1992. Control plots received water from lake Kevojärvi (pH about 7). The acid water was acidified with sul phuric acid to pH 3. CUNI water was prepared by adding Cu (as CuS0 4 ) and Ni (as NiS04) to the raw water to give a deposition/ irrigation event of 8.3 and 5.0 mg/m 2 for Cu and Ni, respectively. The mycorrhiza of two relatively abundant plant species - Betula pubescens ssp. tortuosa and Linnaea borealis - were stud ied. During July 20-28,1992, roots were sampled from each study plot (20 in total). Mycorrhizal root tips of mountain birches were collected. For studying AM, root samples of L. borealis were tak en. In all cases, root samples of one species from one experimental plot were pooled because it was impossible to separate individu als. The ergosterol assay was used to quantify mycorrhiza. The analysis was restricted to birch root tips displaying a predominat ing smoke gray pinnate type of ectomycorrhiza characteristic of Lactarius spp. (Ingleby et al. 1990). Ergosterol was measured ac cording to Nylund and Wallander (1992) using HPLC. In addi tion, roots of L. borealis were stained by trypan blue, the percent of root colonization was determined by microscope slide method. The data were analyzed by a mixed ANOVA model, in which metal and acid rain were fixed effects and study area was random and nested within two others. Results and discussion Based on ANOVA, none of the factors had a significant (p<0.05) impact on the ergosterol content of the Lactarius mycorrhiza of mountain birch (Tables 1 and 2). However, the influence of acid rain treatment was marginally significant at the level 0.077 - resulting in clearly lower content of ergosterol. No statistically significant effect of the heavy metal treatment alone or in combi nation with the acid rain treatment was apparent. In plots receiv ing heavy metals, the content of ergosterol in root tips was slightly lower than in irrigated control although the lowest recorded mean concentration was detected in the acid rain and CuNi treated plots. AM colonization of the roots of L. borealis was very sparse (2-3 % of the root length). The percent colonization was strongly influenced by acid rain treatment; significantly lower root coloni- 149 Roots, mycorrhizas and rhizosphere microbes... Table 1. Content of ergosterol (µg/mg) in birch root tips (A) and in roots of Lin naea borealis (B) and percent root colonization of Lborealis (C) in case of four experimental treatments (IR - irrigated control, A3 - simulated acid rain, M - metal (Cu and Ni) treatment, A3M - combination of acid rain and metal treat ments), mean and standard error. Table 2. Impact of experimental acid rain and Cu-Ni treatments on mycorrhizal status of mountain birch and Linnaea borealis - results of ANOVA. A3 - simulat ed acid rain, M - metal (Cu and Ni) treatment, B - experimental area (block). A3 and M are fixed effects and B(A3*M) is random and nested within A3 and M. zation was observed in acid treated plots. Heavy metal treatment had no significant effect on percent colonization, though the mean colonization rate was lower in metal treated plots (Table 2). A B C IC 2.45 ± 0.37 0.99 ±0.17 2.37 ± 0.52 A3 1.92 ±0.43 0.97 ±0.16 1.53 ±0.63 M 2.29 ± 0.30 0.53 ± 0.08 1.87 ±0.78 A3 M 1.58 ± 0.26 1.35 ±0.29 1.66 ±0.68 Source of DF F value P variation Content of ergosterol in mycorrhiza root tips of Betula pubescens ssp. tortuosal A3 1 3.57 0.077 M 1 0.81 0.380 B(A3*M) 15 0.00 0.480 A3* M 1 1.03 0.956 Content of ergosterol in rootlets of Linnaea borealis A3 1 0.00 0.980 M 1 0.55 0.594 B(A3*M) 15 1.03 0.660 A3*M 1 0.00 0.958 Percent colonization of rootlets of Linnaea borealis by AM A3 1 11.14 0.001 M 1 1.34 0.247 B(A3*M) 15 16.87 0.000 A3*M 1 4.02 0.046 150 When the content of ergosterol in rootlets of L. borealis was considered, very different results were observed. The content of ergosterol in L. borealis was more than two fold lower than in birch root samples. According to the ANOVA, none of the factors had a significant influence on the ergosterol content. Heavy metal treatment resulted in a slightly lower content. We did not find any significant correlation (r=0.094, p=0.700) between the percentage of root colonization and the content of ergosterol. Our results suggest a negative impact of acid rain on mycor rhizal root colonization of two common plant species in a subarc tic forest. Still, the ergosterol analysis did not give satisfactory results in case of L.. borealis, which is probably due to nonselec tive nature of this method. Rootlets of L. borealis were surround ed and sometimes also penetrated by different hyphae. Under the light microscope only mycorrhizal fungi were counted, but chem ical method, in turn, takes all these fungi into account. Acknowledgements We thank the staff of Kevo Subarctic Research Institute for their help in the field. Drs. R.Sen, D.Richter and J. -E.Nylund kindly commented the manuscript. Financial support was obtained from the Finnish Academy of Sciences. References Denny, H.J. & Wilkins, D.A. 1987. Zinc tolerance in Betula spp. IV. The mechanism of ectomycorrhizal amelioration of zinc toxic ity. New Phytol. 106: 545-553. Gildon, A. & Tinker, P.B. 1983. Interactions of vesicular-arbuscular mycorrhizal infection and heavy metals in plants. I. The effects of heavy metals on the development of vesicular-arbuscular mycorrhizas. New Phytol. 95: 247-261. Ingleby, K., Mason, P.A., Last, F.T. & Fleming, L.V. 1990. Identifica tion of Ectomycorrhizas. Edinburgh Research Station, Edin burgh. Kozlov, M.V., Haukioja, E. & Yarmishko, V.T., eds. 1993. Aerial Pollu tion in Kola Peninsula: Proceedings of the International Work shop. April 14-16. 1992. St.-Petersburg. Nylund, J.-E. & Wallander, H. 1992. Ergosterol analysis as a means of quantifying mycorrhizal biomass. In: Methods in Microbiology, vol. 24. Norris, J.R., Read, D.J. & Varma, A.K. (eds.) . Acad. Press, London. 151 Roots, mycorrhizas and rhizosphere microbes.. 152 Recyclable product with low emissions during production Hakapaino Oy, Helsinki 1995 ISBN 951—40-1408-1