Ne%v Phytol.

(1992), 120, 105-115

Utilization of organic and inorganic nitrogen sources by ectomycorrhizal fungi in pure culture and in symbiosis with Pinus contorta Dougl. ex Loud.
A. FROSTEGARD AND A.-M. S O N N E R F E L D T Department of Microhial Ecology, University of Lund, Helgonavdgen 5, S-223 62 Lund, Sweden
BY R. D. F I N L A Y ,

{Received 8 March 1991; accepted 31 July 1991)

SUMMARY

The growth of ten species of ectomycorrhizal fungi was measured in liquid media containing different organic and inorganic nitrogen sources. All fungi grew well on ammonium. Growth on nitrate was generally lower, although there was considerable variation between different isolates of the same species. Suillus variegatus, Piloderma croceum, Paxillus involutus, Hebeloma crustuliniforme and unidentified pink and white isolates often grew as well on organic nitrogen sources as on ammonium. Growth of the other species was more variable. Isolates of Thelephora terrestris and Lactarius rufus varied in their ability to use bovine serum albumen (BSA) but two Laccaria species were poor at using the protein as a nitrogen source. The ability of mycorrhizal and nonmycorrhizal Pinus contorta Dougl. ex Loud, plants to utilize BSA was also examined. Non-mycorrhizal plants and mycorrhizal plants infected with either T. terrestris or the unidentified pink ectomycorrhizal symbiont were supplied either with ammonium or with BSA. Growth of plants supplied with BSA was significantly increased by mycorrhizal infection with the pink symbiont and not significantly different from that of plants supplied with ammonium, but non-mycorrhizal plants were unable to use the protein as a nitrogen source and had significantly lower yields and nitrogen contents than infected plants. In contrast, mycorrhizal infection with T. terrestris had no effect on growth or nitrogen contents of plants supplied with protein. The results are discussed in relation to possible physiological differences between ectomycorrhizal fungi occurring at different successional stages of forest development. Key words: Ectomycorrhiza, nitrogen metabolism, Pinus contorta, protein, succession.

INTRODUCTION

enable conversion of nitrogen into forms which are ... ... . , ^, w\/i * D . A*

Nitrogen availability is frequently a major factor limiting forest growth and ectomycorrhizal fungi are now thought to contribute to the nitrogen nutrition of their host plants in both a quantitative and qualitative manner. The modified spatial geometry and increased surface area of ectomycorrhizal root systems (Bowen, 1973) increases the efficiency of absorption and translocation of inorganic nitrogen to the root (France & Reid, 1983). This may be particularly important in situations where concentrations of available ammonium are low or where diffusion is severely restricted in drier soils (Clarke & Barley, 1968). The effect of ectomycorrhizal fungi, however, is not simply restricted to physical changes in the area and distribution of absorptive surfaces for nutrient uptake. Ectomycorrhizal infection may also alter the efficiency of nitrogen assimilation and

more readily utilized by the root (Martin, Kamstedt & Sdderball, 1987). Experiments by Finlay et al. (1988, 1989) suggest that much of the nitrogen absorbed by mycorrhizal roots may be in organic form following assimilation of inorganic nitrogen by the mycorrhizal mycelium at sonie distance from the root and translocation in the form of amino acids to the host fungus interface. The capacity of ectomycorrhizal fungi to use more recalcitrant, organically bound forms of nitrogen is probably low (Lundeberg, 1970). Experiments by Leake & Read (1990) suggest that the ability to use chitin as a sole nitrogen source is very limited in ectomycorrhizal fungi compared with ericoid mycorrhizal fungi. However, it is now established that the ability of ectomycorrhizal fungi to utilize soluble proteins is much greater than hitherto appreciated, Potential nitrogen sources thus include both simple

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R. D. Finlay, A. Frostegard and A.-M.

Sonnerfeldt
MATERIALS AND METHODS

organic forms of nitrogen such as soluble amino acids and peptides (Ahuzinadab & Read, 1988) and polymeric forms such as soluble proteins (Abuzinadah & Read, 1986 a, b, 1989 a, b: Abuzinadah, Finlay & Read, 1986). Ahuzinadah & Read (1986 a) identified marked differences in proteolytic ability between the species of fungi they tested and speculated that under certain situations a succession of fungi of the type observed by Mason et al. (1983) might he predicted and explained in terms of soil-derived selection pressures. The proteolytic capacity of fungi characteristically occupying different successional niches might thus be related to the changes in resource quality taking place during forest development. Declining resource quality and accumulation of organic matter during forest development should thus select against fungi with limited proteolytic capacit\- and favour those which are able to compete successfully for limited, organic nitrogen sources. Successions of mycorrhizal fungi during forest development have been reported widely (Mason et aL, 1982; Dighton & Mason, 1985; Dighton, Poskitt & Howard, 1986). This concept of 'mycorrhizal succession' has led to the adoption of the terms 'early-stage' and 'late-stage" to describe fungi commonly found at these particular stages of forest development (Deacon, Donaldson & Last, 1983; Fleming, 1983, 1984; Last et al. 1983, 1984, 1985; Mason et al, 1983, 1987). Whilst there is general agreement that changes in the diversity and composition of the ectomycorrhizal flora can occur with time there has heen much controversy as to whether tbese terms are appropriate or adequate. Interpretation of the sequential appearance of fruiting structures is complicated by the fact that different fungi produce fruiting structures at different ages (Harper & Webster, 1964), Furthermore, as many workers have pointed out, while tbe presence of reproductive structures is always associated with the presence of mycelia, tbe absence of fruit bodies does not necessarily imply the absence of mycelia. One further caution against undue generalization is that there may he important differences hetween species in first rotation sites and those previously occupied hy trees (Mason et al. 1987).

Fungal species The fungal species used were Laccaria bicolor, Laccaria proxima, Thelephora terrestris, and Hebeloma crustuliniforme (typically found in early stages of forest development), Paxillus involutus (occurring in both early and late stages of forest development), Piloderma croceum, Lactarius rufus, Suillus variegatus (often found in later stages of forest development) and unidentified pink and white isolates from two different pine stands 40 and 130 years old. Two isolates of each species were used except for L. bicolor, and the unidentified pink and white isolates. Authorities and isolation details, where available, are given in Table 1. Experiment 1

In the first experiment the growtb of a range of different fungi on different nitrogen sources was examined in pure culture using methods similar to Abuzinadah & Read (1986 a). A modified MelinNorkrans liquid medium was used as the basal medium. Ammonium and malt extract were omitted from the medium and the different nitrogen sources were added individually to the medium to provide a final concentration of 60 mg 1"^ N. The nitrogen sources chosen were ammonium sulphate (0-284 g 1"^), calcium nitrate (0-5 g I"'), and bovine serum albumen (BSA, molecular weight 67000, N content 16o) 0-375 g l " ' . Glucose was added as the sole carbon source at 3 g 1~^ for the ammonium and nitrate treatments and 2-62 g T' for BSA to give a final carbon to nitrogen ratio of 20:1. Tbe media containing inorganic nitrogen were adjusted to pH 4-5 by addition of H^SO^ or NaOH and then autoclaved at 0-7 atm for 30n:iin. The BSA was dissolved in a portion of the basal medium, adjusted to pH 4-5, and added to autoclaved basal medium through a 0-2 ftm millipore filter. Eight fungal species were used, L. bicolor, L. proxima, H. crustuliniforme, T. terrestris, S. variegatus, P. croceum, P. involutus and an unidentified pink strain, isolated from a 40year-old pine stand. This isolate formed a mycorrhizal association with pine which has been classified as Pinirhiza rosea hy Uhl (1988). Discs of fungal Further experimentation is required to relate inoculum were cut with a 6 mm diameter cork borer functional differences in physiology to edaphic site from the edge of actively growing 2-3 week old characteristics. Information so far gathered on the colonies on agar plates, The discs of inoculum were proteolytic activity of ectomycorrhizal fungi is transferred under aseptic conditions to sterile Petri restricted to a relatively narrow range of species. The dishes and placed on centrally positioned droplets of object of the present study was to examine a wider sterile liquid agar to prevent movement of the range of ectomycorrhizal fungi characteristically inoculum, and to ensure that the fungal plug was just occupying different successional stages of forest above the surface of the liquid medium. As soon as development and to evaluate their nitrogen metabthe agar had solidified, 20 ml of liquid medium olism with respect to their ability to utilize organic containing the different nitrogen sources were added nitrogen sources. to each Petri dish. There were fifteen replicate disbes of eacb combination of species and nitrogen source.

Utilization of organic and inorganic N by ectomycorrhizal fungi Table 1. Fungal species used in the study together with details of their hosts and original habitats, where available
Species Heheloma crustuliniforme (Bull, ex St. Amans.) Quel Hebeloma crustuliniforme Laccaria bicolor (R. Mre). Orton Laccaria proxima (Boud.) Pat. Laccaria proxima Paxillus involutus Isolate 85.021 85.023 S-238a S-472 87.018 87.017 86.006 85.009 85.029 88.009 88.007 87.016 85.001 Lr 88-01 85.013 88.015 88.016 Details of isolation, host and origin Isolated from fruitbody under Fagus sylvatiea, Amance, France (CNRF, Nancy) Isolated from spores from a fruitbody under 5-year-old Picea sitchensis on a brown earth soil Isolated from fruitbody under Tsuga mertensiana stand Crater Lake National Park, Oregon, USA Isolated from fruitbody under Pinus contorta stand, Tillamook Co., Oregon, USA Re-isolated from 2-year-old Picea sitchensis plants in a pot experiment. Bush Estate, Scotland Isolated from a fruitbody in a coal waste with 15-30-year-old Betula pendula trees, Midlothian, Scotland Isolated from a fruitbody under a 20-year-old plantation of Picea abiesjPicea sitchensis Isolated from roots of Pinus sylveslris under a 40-year-old stand Torrmyra, Sweden Isolated from roots of Pinus syhestris under a 40-year-old stand Torrmyra, Sweden Isolated from fruitbody under a 110-year-old stand of Pinus syhestris, Istaby, Sweden Isolated from fruitbody under a UO-year-old stand of Pinus syhestris, Istaby, Sweden Isolated from roots of Picea sitchensis ex R. Jackson Isolated from roots of Pinus sylvestris Glasfynydd forest, UK Isolated from fruitbody under 80-year-old Pinus sylvestris stand with Picea ahies & Betula pendula, Nasien, Sweden Isolated from fruitbody under a 40-year-old stand of Pinus syhestris, Torrmyra, Sweden Isolated from roots of Pinus syhestris under a 40-year-old stand, Torrtnyra, Sweden Isolated from roots of Pinus sylvestris under a 130-year-oid stand, Kroksbo, Sweden

(Fr.) Fr
Paxillus i?ivolutus Piloderma croceum Erikss. & Hjorts. Piloderma croceum Suillus variegatus

(Fr.) O. Kuntze
Suillus variegatus Thelephora terrestris (Fr.) Thelephora terrestris Lactarius rufus (Scop, ex Fr.) Fr. Lactarius rufus Unidentified pink isolate Unidentified white isolate

The dishes were incubated at 18-20 C in sealed plastic bags and five replicate dishes from each treatment combination were harvested after 20, 40 and 60 days of incubation. The agar plug was cut away and the fungal colony carefully transferred to pre-weighed pieces of aluminum foil. These were oven dried at 80 C and the fungal dry weights recorded. The pH value of the medium was also measured after the final harvest.

Experiment 2 A second experiment was conducted using a slightly wider range of organic nitrogen sources. The basal medium was as for Experiment 1 above. Eight different nitrogen sources were chosen; ammonium, nitrate and BSA, as above, plus alanine and asparagine (neutral amino acids), glutamic acid (an acidic amino acid), arginine (a basic amino acid) and the protein gliadin, an ethanol-soluble proiamine (molecular weight 30000, N content 14%). The last 6 compounds were added in appropriate amounts to

autoclaved medium (pH 45) by sterile filtration through a 0-2 fiva millipore filter to give 60 mg N 1~^. Appropriate amounts of glucose had previously been added to give a constant C: N ratio of 20:1. The fungal species examined were, P. ini^olutus (87.017), T. terrestris (87.016) and the unidentified white isolate. Time courses of fungal growth were examined for each species and the different species were harvested at the time-points corresponding to maximum biomass production. These were 20, 30 and 40 days respectively. The methods used were as in Experiment 1 above. Five replicate plates were used for each combination of nitrogen treatment and species. Experiment 3 In a third experiment the ability of mycorrhiza] and non-mycorrhizal Pinus contorta plants to use BSA as a nitrogen source was tested. The methods used in this experiment followed those described by Abuzinadah, Finlay and Read (1986). Seeds of P. contorta were surface sterilized with hydrogen per-

108

R. D. Finlay, A. Frostegdrd and A.-M.

Sonnerfeldt mean nitrogen content of freshly germinated P. contorta seeds was determined in the same manner to calculate the initial amount of N available to the growing plants.

oxide and placed on water agar until germination occurred. Erlenmeyer flasks (250 ml) were filled with 150 ml perlite and 50 ml of a modified MelinNorkrans solution containing 2-5 g T^ glucose {20 mg C per flask) from which nitrogen (NH4)2HPO^) had been omitted. A small amount of 'starter' nitrogen was added to the flasks at time zero, prior to application of the main nitrogen regimes, in order to facilitate mycorrhiza synthesis. Flasks which were subsequently to receive ammonium were supplied with 1 mg N 'starter' nitrogen as ammonium sulphate. All flasks were then autoclaved and those flasks to receive BSA were supplied with 1 mg N as arginine, added by milHpore filtration (0-2 firo). At time zero half of the flasks were inoculated with discs of fungaJ mycelium taken from the actively growing margins of agar plate cultures of the unidentified pink isolate. After allowing 20 days for growth of the fungus, three aseptically germinated pine seedlings were added to each fiask and a further period of 40 days was allowed for the dei'elopment oi mycorrhiza] infection. The main nitrogen treatments were then applied by adding 4 mg N, either as ammonium sulphate or as BSA. The nitrogen sources were dissolved in 5 tnl of the basal M M N solution and added by sterile filtration through a 0 2 //tn millipore filter. There were thus four main treatments, mycorrhizal with ammonium or BSA (Amm/M,BSA/M) and non-mycorrhizal with ammonium or BSA (Amm/NM,BSA/NM). Two additional control treatments were set up concurrently with those above to examine the separate effects of starter nitrogen and BSA on non-mycorrhizai plants. In these treatments non-mycorrhizal plants received either no nitrogen at all (NM/-N) or BSA but no initial starter nitrogen (BSA/NM-S). There were thus six different treatments in total (Amm/M; BSA/M; Amm/NM; BSA/NM; NM-N; BSA/NM-S) and eight replicate flasks of each. Two harvests were originally intended, but, owing to contamination of some flasks, only 5-7 replicates of each treatment were available and one harvest was finally taken. This experimental design was repeated in an additional experiment using T. terrestris (87.016) instead of the pink isolate. In this experiment the two additional non-mycorrhizal controls (NM-N) and BSA/NM-S) were omitted and the nitrogen and mycorrhizal treatments were combined in a simple factorial design with 5 replicates of each treatment. Plants from both experiments were harvested after an additional 10 weeks growth following addition of the main treatment nitrogen. Plants were partitioned into shoots and roots which were oven dried before determination of dry weights. The total nitrogen content of the seedlings was determined by flow injection analysis following digestion in a 3:1 (v/v) mixture of HgSO, and nitrogen-free HgOj. The

RESULTS

Experiment 1 The dry weight yields of the fungi in the first pure culture study after 20, 40 and 60 days are shown in Tables 2, 3 & 4 respectively. Although there was considerable variation between strains of the same fungus some patterns of nitrogen utilization were evident. Growth on ammonium was generally much higher than on nitrate, although this was less evident at later harvests, possibly reflecting a time lag in the induction of nitrate reductase activity. Certain fungi, including H. crustuliniforme, L. proxima and P. involutus, grew almost as well on nitrate as ammonium but L. bicolor and P. croceum showed only intermediate growth on nitrate. Individual isolates of T. terrestris and S. variegatus grew well while other isolates of these two species, L. rufus and the unidentified pink isolate were relatively poor at utilizing nitrate. Patterns of protein utilization were also evident. Final yields on BSA were as high, or higher than those on ammonium for P. croceum, S. variegatus and the pink isolate. This was true despite iarge overall variation in growth rates both between individual species and between different isolates of the same species. H. crustuliniforme grew well on BSA with yields comparable to those on ammonium in both isolates. P. involutus and T. terrestris both grew well on BSA, although individual isolates of both species clearly differed in their capacity to utilize the protein. L. bicolor and the two isolates of L. proxima, species both typical of early successional stages of forest development, both grew poorly on BSA. Final pH values of the media are shown in Table 5. Not surprisingly there was an overall decrease in pH in the ammonium treatments and an overal] increase in the nitrate treatments. The pH changes were generally smaller with BSA as the N source. There was no apparent relationship between fungaj yieJd and the size and direction of the pH shifts.

Experiment 2 The maximum dry weight yields and growth rates of the three ectomycorrhizal fungi used in the second pure culture study are shown in Figures 1 a & 1 6. All three species grew well on ammonium. Faxillus involutus produced final yields approximately 50% lower on nitrate, whilst Thelephora terrestris and the unidentified white isolate grew poorly on this nitrogen source. Growth on the single amino acid nitrogen sources was generally good, with several

Utilization of organic and inorganic N by ectomycorrhizal fungi Table 2. Yields {mg dry weight) of 9 different fungal species after 20 days growth in liquid media containing different nitrogen sources. Figures in parentheses indicate standard errors. Yields followed by different superscripts within rows are significantly different atP< 0.05 according to Fisher's protected least significant difference Nitrogen source Fungal species Hebeloma crustuliniforme Hebeloma crustuliniforme Laccaria bicolor Laccaria proxima Laccaria proxima Paxillus ini'olutus Paxillus involutus Piloderma croceum Piloderma croceum Suillus variegatus Suillus variegatus Thelephora terrestris Thelephora terrestris Unidentified pink isolate Lactarius rufus Lactarius rufus Strain 85.021 85.023 S-238a S-472 87.018 87.017 86.006 85.009 85.029 88.009 88.007 87.016 85.001 88.015 Lr 88-01 85.013 Ammonium Nitrate 7'5(l-0r 5-9 (0-7)" 20-8 (0 7)" 14-2 {0-S)22-4 (0-6)" 24-9 (17)" 6-8 (0'5)" 9-2 (0-3)" 8-5 (0-3)" 21-8(0-4)" 26-0 (0-6)'' 19-9(0-7)" 15-5(1-8) 6-5 (0-2)" 21-0 (0-8) 18-5 (0-3)'' 7-2 (0-2)" 5-5 (0-6)" 6-1 (0-5)^ 14-6 (0'3)" 4-9 (0-2)" 12-2 (0'5)* 7-0 (0'2)'' 4-5 (0-3)" 3-1 (0-4)'' 3-8 (0-4)" 3-9 (0-2)^ 3-3 (0-3)^ 6-6 (0-6)" 3-6(0-1)" 5-6 (0-2)" 2-9(0-1)" BSA 6'2 (0-3)'' !3'2(0-4)'' 41 (0-2)^ 4-5 (0-3)^ 4'2 (0-3)" 21-5(1-1)" 4-4(0-1)" 8'3 (0-5)" 7-7(0-1)" 19-1 {\-9)'' 8-9 (1-4)" 5-7 (0-2)" 7-8 (0-5)" 5-9 (0-3)" 5-7 (0-2)" 3-4(0-1)"

Table 3. Yields {mg dry weight) of 9 different fungal species after 40 days growth in liquid media containing different nitrogen sources. Figures in parentheses indicate standard errors. Yields followed by different superscripts within rows are significantly different at F < 0.05 according to Fisher's protected least significant difference Nitrogen source Fungal species Hebeloma crustuliniforme Hebeloma crustuliniforme Laccaria bicolor Laccaria proxima Laccaria proxima Paxillus involutus Paxillus involutus Piloderma croceum Piloderma croceum Suillus variegatus Suillus variegatus Thelephora terrestris Thelephora terrestris Unidentified pink isolate Lactarius rufus Lactarius rufus Strain 85.021 85.023 S-238a S-472 87.018 87.017 86.006 85.009 85.029 88.009 88.007 87.016 85.001 88.015 Lr 88-01 85.013 Ammonium Nitrate 16-8(0-7)" 12-5 (0-2)^ 26-0 (1-8) 19-2(0-8)'' 23-4 (0-4) 20-1 (0-3)" 19-8 (0-3) 17-1 (0-4)" 23-6 (0-4)" 15-0 (0-2)" 17-8 (0-7)" 18-9(0-3)" 19-4 (0-8)" 9-6 (0-1) 22-2 (0-2)" 26-5 (0-2)" 17-1 (0-6)" 13-8 (0-5)" 8-8 (1-2)" 15-5(07)" 13-5 (0-2)" 15-2 (0-8)" 10-3 (0-2)'' 7-6 (0-3)^ 6-4 (0-3)" 7-9 (1-0)^ 4-1 (0-4)' 3-3(0-2)' 10-5 (0-4)" 3-1 (0-6)<4-8 (0-4)^ 2-7 (O-I)" BSA 14-7 (07)" 15-3 (0-2)"
3-7 (0-2)^ 6-6 (0-6)^ 4-8 (0-2)^ 21-0(0-3)'' 4-7 (0-2)^

15-5 (07)" 19-4(0-6)" 20-9 (0-4)" 20-7(0-1)" 9-6 (0-7)" 18-0(0-3)" 6-8 (0-3)'* 14-7 (2-3)" 3-9 0-2)"

exceptions. T. terrestris grew poorly on glutamic acid while P . involutus showed reduced growth on alanine and no growth at all on asparagine. Growth on arginine was good and P. involutus showed the highest growth rate of all the tested fungi on this

nitrogen source. There were clear differences in the capacity of the different fungi to use the proteins as nitrogen sources. P. involutus and the unidentified white isolate both grew well on BSA, producing yields which were even higher than those on

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T a b l e 4. Yields {mg dry weight) of 9 different fungal species after 60 days growth in liquid media containing different nitrogen sources. Figures in parentheses indicate standard errors. Yields followed by different superscripts within rows are significantly different a/ P < 0.05 according to Fisher's protected least significant difference Nitrogen source Fungal species Hebeloma crustuliniforme Hebeloma crustuliniforme Laccaria bicolor Laccaria proxima Laccaria proxima Paxillus involutus Paxillus involutus Piloderma croceum Piloderma croceum Suillus variegatus Suillus variegatus Thelephora terrestris Thelephora terrestris Unidentified pink isolate Lactarius rufus Lactarius rufus Strain 85.021 85.023 S-238a S-472 87.018 87.017 86.006 85.009 85.029 88.009 88.007 87.016 85.001 88.015 Lr 88-01 85.013 Ammonium Nitrate 15-5(17)" 14-9 {0-2)'' 27-5 (0-5)18-2 (0-5)" 20-0 (0-2) 15'8(O-5)'' 20.0 (0-8)'' 16-4 (0-3)" 21-8 (0-20" 14-3 (0-8)* 16-2 (0-3)" 19-6 (0-3)" 17-1 (1-6)^ 11-9 (0-9)" 20-1 (0-3)" 207 (0-2)'' ] 5-4 (0-8)" 13-1 (0-3)'12-8 (0-6)'' 16-4 (0'6)^ 36-1 (0-4)" 16'9(0-8)'' 12-3 (0-1)" 10-6 (0'3)^ 107(0 5)" 17-3(07)" 4-] (0-3)* 4'0 (0-4)^ 137 (0'2)' 3-5 (0-2)^ 5-5 (0-4r 3 0(0-3)^ BSA 17-1 (17)" 16-1 (0-1) 4-3 (0-2)'9'1 (0-5r 4-4 (0-3r 18-5 (0-4)" 11-4(2-5)* 18-4 (0-2)" 21-8(0-3)" 18-1 (0'2)'] 6-6 (0-4) 13-1 (1-5)* 20-1 (0-4)" 7-4 (0-5)" 16-9(17)" 6-2 (0-2)"

T a b l e 5. Final pH values of liquid culture media containing different nitrogen sources following growth of 9 different fungal species for 60 days. Standard errors (n = 5) were all < 0-1 pH unit Nitrogen source Fungal species Hebeloma crustuliniforme Hebeloma crustuliniforme Laccaria bicolor Laccaria proxima Laccaria proxima Paxillus involutus Paxillus involutus Piloderma croceum Piloderma croceum Suillus variegatus Suillus variegatus Thelephora terrestris Thelephora terrestris Unidentified pink isolate Lactarius rufus Lactarius rufus Strain 85.021 85.023 S-238a S-472 87.018 87.017 86.006 85.009 85.029 88.009 88.007 87.016 85.001 88.015 Lr 88-01 85.013 Ammonium Nitrate 3-0 3-0 2-7 2-8 3-0 2-9 27 27 2-6 3-3 3-5 2-8 2-9 2-8 2-9 2-9 6-5 6-2 6-3 6-4 6-4 6-3 6-2 5-6 5-2 3-7 4-0 3-9 4-2 4-3 4-3 4-1 BSA 5-6 5-9 4-9 5-0 5-2 5'1 4-9 4-5 4-2 3-8 6-6 4-0 5-1 5-8 4-9 5-0

a m m o n i u m . T h e same fungi were also able to grow on gliadin although the final yields were slightly lower. T h e growth rates of P . involutus were faster than those of the white isolate on both proteins. In contrast, the third fungus tested, T. terrestris, grew very poorly on both BSA and gliadin.

Experiment

The dry weight yields of P. contorta plants grown in association with the unidentified pink isolated are shown in Figure 2a, b, c. Total plant yields (Fig 2c) were highest for mycorrhizal plants grown with

Utilization of organic and inorganic N by ectomycorrhizal fungi

(a)

30 20

o 10 o
-

10

BSA/NM-S NM/-N Amm/M Amm/NMBSA/MBSA/NM

>

(W
1,2 1,0 0,8 0,6

in

BSA/NM-S NM/-NAmm/MAmm/NMBSA/IVIBSA/NM

c _, 0,4 .c DJ 0,2
0,0

amm

nit

arg

Ic)

glu

asn

BSA Gliadin

N source

Figure 1. {a) Histogram showing maximum dn- weight yields of three different ectomycorrhizal fungi grown on a variety of different nitrogen sources; ammonium sulphate (amm). calcium nitrate (nit), alanine (ala), arginine (arg), glutamic acid (glu), asparagine (asn), bovine serum albumen (BSA) and gliadin. The fungi, Paxillus involutus, ( ) Thelephora terrestris ( 0 ) and an unidentified white isolate ( ) were harvested after 20, 30 and 40 days, respectively. Bars indicate standard errors, (h) Histogram showing the rates of dry weight increase per day of the three ectomycorrhizal fungi in Figure 1 a.

ammonium as the sole nitrogen source but there was no significant difference between mycorrhizal plants grown with BSA as the sole nitrogen source and nonmycorrhizal plants grown with amtnonium. Nonmycorrhizal plants grew significantly less well when BSA was the sole nitrogen source and the yields did not differ significantly from those of nonmycorrhizal plants grown with no nitrogen (NM-N), suggesting that plants were unable to utilize BSA as a nitrogen source. The yields of non-mycorrhizal plants grown on BSA with no 'starter" nitrogen (BSA/NM-S) did not differ significantly from those of similar plants grown with 'starter' nitrogen, suggesting that the effect of this additional 'starter' nitrogen on final yields was insignificant. Differences in root weight between mycorrhizal and nonmycorrhizal plants grown on BSA were larger than those between shoot weights. The larger contribution of differences in root yield to overall differences in total plant yield suggests direction of resources into root growth in response to nutrient stress. The root; shoot ratio of mycorrhizal plants supplied with ammonium was lowest, suggesting that these plants were least nutrient stressed. The

BSA/NM-S NM/-N Amm/MAmm/NM BSA/M BSA/NM Figure 2. Histograms showing dry weight yields of Pinus contorta plants grown in association with an unidentified pink ectomycorrhizal isolate on media containing ammonium (Amm) or BSA as the sole nitrogen source. Plants were either non-mycorrhizal (NM) or mycorrhizal (M); NM-N indicates non-infected plants with no nitrogen, BSA/NM-S indicates non-mycorrhiza! plants supphed with BSA but no starter nitrogen, (a) shoot weight, (b) root weight, (c) total weight. Bars indicate standard errors. DifFerent letters above the bars indicate a significant {P < 0-05) difference between the yields (Duncan's Multiple Range test).

shoot and root nitrogen concentrations of the plants are shown in Figure 3 a. The levels of nitrogen in the tissues of mycorrhizal plants which had received ammonium were highest at approximately 11'7 and 9-9 mg g"^ for shoots and roots respectively. The shoot N levels in non-mycorrhizal plants receiving ammonium were not significantly different but the root levels were significantly lower ( P < 0 ' 0 1 ) . Mycorrhi2al plants receiving BSA had intermediate levels of nitrogen which were lower than mycorrhizal plants receiving amnnonium but not significantly different from the levels in roots of the nonmycorrhizal plants receiving ammonium. Nonmycorrhizal plants supplied with BSA had the lowest nitrogen concentrations which were not significantly different from those of non-mycorrhizal plants which had received no nitrogen at all. The addition of the small amount of starter nitrogen also had no significant effect on the final levels of nitrogen. The total nitrogen contents of the plants are shown in Figure 36 where they are related to the initial

R.D. Finlay, A. Frostegdrd and A.-M.

(a) Pink isolate 12 ro o 4 2 10

Sonnerfeldt
(a)

Amm/M

Amm/NM

BSA/M

BSA/NM

0 BSA/NM-S NM/-N Amm/M Amm/NM BSA/M BSA/NM 20 r Pink isolate

Seed N content Amm/M Amm/NM BSA/M BSA/NM

(c) 40 BSA/NM-S NM/-N Amm/M Amm/NM'BSA/M BSA/NM ^ 30

Figure 3. (o) Histogram showing the nitrogen concentrations in shoots ( 0 ) and roots ( P ) of Pinus contorta plants grown m association with an unidentified pink ectomycorrhizal isolate on media containing ammonium (Amm) or BSA as the sole nitrogen source. Plants were either non-mycorrhizai (NM) or mycorrhizal (M); NM-N indicates on-infected plants with no nitrogen, BSA/NMS indicates non-mycorrhizal plants supplied with BSA but no starter nitrogen. Bars indicate standard errors, (b) Histogram showing the total nitrogen contents of the same plants as in Figure 3 a. The horizontal line indicates the initial nitrogen content of the seed, the standard error of which is too small to be displayed.

I 20
10

nitrogen contents of the seeds. The non-mycorrhizal plants supplied with BSA showed no net assimilation of nitrogen from the substrate. The other nonmycorrhizal controls receiving no nitrogen at all, or BSA but no starter nitrogen, also contained total amounts of N which were not significantly different from the initial seed content. Mycorrhizal plants supplied with ammonium contained the largest amounts of nitrogen followed by the non mycorrhizal plants receiving ammonium. The mycorrhizal plants supplied with BSA assimilated significant quantities of nitrogen and had final contents which were not significantly different from the non-mycorrhizal plants supplied with ammonium. Levels of mycorrhizal miection were similar in plants receii'ing the different nitrogen treatments, making it unlikely that differences in the degree of infection could have influenced the results. Patterns of growth and nitrogen utilization in the plants infected with T. terrestris were different. The dry weight yields are shown in Figure 4a, b, c. Mycorrhizal plants supplied with ammonium again had the highest total dry- weights but none of the

Amm/M Amm/NM BSA/M BSA/NM Figure 4. Histograms showing dry weight yields of Pinus contorta plants grown in association with the ectomycorrhizal fungus Thelephora terrestris on media containing ammonium (Amm) or BSA (BSA) as the sole nitrogen source. Plants were either non-mycorrhizal (NM) or mycorrhizal (M) (a) shoot weight, (b) root weight, (c) total weight. Bars indicate standard errors. Different letters above the bars indicate a significant (P < 0-05) difference between the yields (Duncan's Multiple Range test).

other three treatments differed significantly from each other. The root/shoot ratio of non-mycorrhizal plants supplied with ammonium was lower than in other treatments but mycorrhizal infection had no effect on the yields of plants supplied with BSA despite the fact that levels of mycorrhizal infection in plants supplied with ammonium and BSA were similar. The levels of nitrogen in roots and shoots of plants supplied with ammonium (Fig. 5 a) were similar for both mycorrhizal and non-mycorrhizal plants. Roots of non-mycorrhiza! plants were smaller and had slightly higher concentrations of N. Plants supplied with BSA had consistently lower concentrations of root and shoot nitrogen, irrespective of whether or not they were mycorrhizaJ. The total amounts of nitrogen are shown in relation to seed nitrogen content in Figure 5 b. Ammonium treated plants showed assimilation of significant amounts of the added nitrogen whereas plants supplied with BSA showed no significant assimilation of nitrogen

Utilization of organic and inorganic N by ectomycorrhizal fungi

(a) 20

10

Am m/M
\b)

Amm/N M

BS A/M

BSA/NM

Seed N content

100

Amm/M

Amm/NM

BSA/M

BSA/NM

Figure 5. {a) Histogram showinjf the nitrogen concentrations in shoots ( ^ ) and roots ( ^ ) of Pinus cnntorta plants grown in association with Thelephora terrestris on media containing ammonium (Amm) or BSA (BSA) as the sole nitrogen source. Plants were either non-mycorrhizai (NM) or mycorrhizal (M). Error bars indicate standard errors, {b) Histogram showing the total nitrogen contents of the same plants as in Figure 5 a. The horizontal line indicates the initial nitrogen content of the seed, the standard error of which is too small to be displayed.

source. Differences in proteolytic capability may be important in determining the distribution of ectomycorrbizal fungi in time and space (Grier et al., 1981) and, as Abuzinadah & Read (1986fl) pointed out, may help to explain the types of 'succession' observed during forest development (Mason et al., 1983; Dighton & Mason, 1985). In soils with increasing organic content selection for proteolytic activity would be expected and this might thus be more highly developed among fungi typically associated with later stages of forest development than those which are common during the initial stages. This does not preclude the possibility of similar proteoiytic activity in 'early stage' fungi isolated from soils with a high organic content relative to the available inorganic nitrogen. The proteolytic capacitj' of a particular mycorrhiza] isolate may have practical significance with regard to selection of suitable mycorrhizal inoculants and their persistence following transplantation of inoculated trees to forest soils of low fertility. Chu-Chou & Grace (1990) found that ectomycorrhizal isolates common on Pinus radiata seedlings in relatively nutrient rich nursery soils were frequently replaced by other species following transplantation of seedlings to forest soils of lower fertility and this may, in part, reflect a poorly developed ability to use organic resources, leading to competitive exclusion by indigenous species. The variable nature of the results from the present study highlights the need for further experimentation and for better information about the dynamics of organic nitrogen sources in the forest soils from which different fungi are isolated. In particular the results indicate the need to avoid unwarranted generalizations about 'early' and Mate stage' fungi. In the present study H. crustuliniforme and T. terrestris, which have been classified as 'early-stage' fungi, both displayed the ability to grow on BSA in pure culture. In other experiments however, Dighton, Thomas & Latter (1987) found that decomposition of hide powder and cellulose was higher in the presence of Suillus luteus than Hebeloma crustuliniforme. In the experiments reported here two isolates of Laccaria proxima and one of L. bicolor (both considered to be ' early-stage' fungi) were poor at using protein as a nitrogen source, although other workers (Ahmad et al., 1990) have shown that L. bicolor can use a range of different amino acids as a sole nitrogen source. Results from the pure culture studies described here also clearly illustrate the importance of the time of harvesting on the outcome of growth experiments since, in some cases, there were lag phases of different length prior to rapid utilization of the substrate. Considerable differences in the ability to use BSA were found between isolates. Isolate 87.016 of Thelephora terrestris grew well on BSA in pure culture whereas isolate 85.001 grew less well.

supplied in this form, irrespective of their mycorrhizal status.

DISCI'SSION

The results of the present study confirm those of Abuzinadah & Read (1986a) and Abuzinadah, Finlay & Read (1986) and extend them using a w'ider range of fungal species. Results from Experiment 1 confirm the ability of H. crustuliniforme to utilize protein (Abuzinadah & Read, 19866, 1989a, b). The data for P. involutus confirm previous experiments (e.g. Lundeberg, 1970) in that the fungus was able to utilize nitrate as a sole nitrogen source. The inability of P. inziolutus to utilize asparagine in the present study supports previous observations (Laiho, 1970; Lundeberg, 1970; Finlay et al., 1988) that asparagine and aspartic acid are poor nitrogen sources for this species. The confounding effect of pH shifts complicate interpretation of the relative grow^th on nitrate and ammonium. The final pH values of media containing protein may also have been above the optimum for the protease systems involved, although no relationship was evident between final yields and the size or direction of the pH change. Large differences were found in the ability of the fungi examined to utilize protein as a nitrogen

114

R. D. Finlay, A. Frostegard and A.-M. Sonnerfeldt heterotrophic carbon assimilation from organic sources. The general implications of the above processes for nutrient cycling may be broad (Read, Leake & Langdaie, I9S9; Dighton & Boddy, 1989), but they have particular bearing on the successful selection of appropriate mycorrhizal fungi for optimization of forest tree growth in different soils and at different stages of forest development. Further systematic investigation of differences in physiological activity in relation to measured soil parameters is an important priority in future research.

However the former isolate, when grow-n in association with P. contorta, appeared unable to transfer any nitrogen to the host plant. Similar results were obtained by Abuzinadah & Read (1989a, b) working with Hebeloma crustuliniforme, Amanita muscaria and Paxillus involutus. In their experiments all fungi grew well on the protein BSA in pure culture but the obligately mycorrhizal fungi H. crustuliniforme and A. muscaria were more efficient at transferring the assinnilated nitrogen to their host plants than the facultatively mycorrhizal Paxillus involutus which is known to have some saprotrophic capability and retained much of the assimilated N within its own tissues. In other experiments by Dighton et al. (1987) the effect ofthe mycorrhizal fungi Hebeloma crustuliniforme and Suillus luteits on the decomposition of three organic substrates was minimal in the absence of host plant roots but the presence of roots considerably enhanced decomposition. These results underscore the importance of working with intact mycorrhizal associations as w-ell as pure cultures when attempting to determine the importance of heterotrophic protein assimilation to mycorrhizal plants. There has been much speculation about physiological differences which might exist between ectomycorrhizai fungi present at different successional stages of forest development but fewer experimental studies and little attempt made to relate physiological properties of ectomycorrhizal fungi to the edaphic characteristics of the sites from which they are isolated. This in part is due to restricted information about the dynamics and pool sizes of different soil organic nitrogen sources. Culture experiments and observations of fruit body size suggest that early stage fungi have a lower dennand for, or reduced access to, host derived assimilates. Gibson & Deacon (1990) examined establishment of mycorrhizal roots in aseptic culture in relation to effects of different nutrients. In the presence of adequate mineral nutrients 'early-stage' mycorrhizal fungi formed mycorrhizal roots at low or moderate glucose levels whilst four of the five ' late-stage' isolates tested required moderate or high glucose levels for successful mycorrhizal development. In other experiments low P levels suppressed mycorrhiza formation by late stage fungi more than by early stage fungi although low levels of N suppressed mycorrhiza formation in all ofthe tested isolates. The apparently high glucose dependence of late stage fungi may also explain the efficiency of infection by mycelia growing from food bases provided by established trees. The important role of the mycorrhizal mycelium as a source of inoculum and the potential for photosynthetic assimilate movement through mycelial strands connecting root systems have been discussed by Read, Francis & Finiay (1985) and Finiay & Read (1986). There is now also evidence that some of the carbon cost of mycorrhizal infection may be met by

ACKNOWLEDGEMENTS

Financial support from the Swedish National Environmental Protection Agency is gratefully acknowledged.

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