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. BY R. D. FINLAY, A. FROSTEGARD AND A.-M. SONNERFELDT Department of Microhial Ecology, University of Lund, Helgonavdgen 5, S-223 62 Lund, Sweden {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 non- mycorrhizal 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. enable conversion of nitrogen into forms which are INTRODUCTION ... ... . , ^, w\/i * D . A* more readily utilized by the root (Martin, Kamstedt Nitrogen availability is frequently a major factor & Sdderball, 1987). Experiments by Finlay et al. limiting forest growth and ectomycorrhizal fungi are (1988, 1989) suggest that much of the nitrogen now thought to contribute to the nitrogen nutrition absorbed by mycorrhizal roots may be in organic of their host plants in both a quantitative and form following assimilation of inorganic nitrogen by qualitative manner. The modified spatial geometry the mycorrhizal mycelium at sonie distance from the and increased surface area of ectomycorrhizal root root and translocation in the form of amino acids to systems (Bowen, 1973) increases the efficiency of the host fungus interface. absorption and translocation of inorganic nitrogen to The capacity of ectomycorrhizal fungi to use more the root (France & Reid, 1983). This may be recalcitrant, organically bound forms of nitrogen is particularly important in situations where concentra- probably low (Lundeberg, 1970). Experiments by tions of available ammonium are low or where Leake & Read (1990) suggest that the ability to use diffusion is severely restricted in drier soils (Clarke & chitin as a sole nitrogen source is very limited in Barley, 1968). The effect of ectomycorrhizal fungi, ectomycorrhizal fungi compared with ericoid mycor- however, is not simply restricted to physical changes rhizal fungi. However, it is now established that the in the area and distribution of absorptive surfaces for ability of ectomycorrhizal fungi to utilize soluble nutrient uptake. Ectomycorrhizal infection may also proteins is much greater than hitherto appreciated, alter the efficiency of nitrogen assimilation and Potential nitrogen sources thus include both simple
Transcript
Ne%v Phytol. (1992), 120, 105-115
Utilization of organic and inorganicnitrogen sources by ectomycorrhizal fungiin pure culture and in symbiosis with Pinuscontorta Dougl. ex Loud.
BY R. D. F I N L A Y , 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
{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 andinorganic nitrogen sources. All fungi grew well on ammonium. Growth on nitrate was generally lower, althoughthere was considerable variation between different isolates of the same species. Suillus variegatus, Pilodermacroceum, Paxillus involutus, Hebeloma crustuliniforme and unidentified pink and white isolates often grew as wellon organic nitrogen sources as on ammonium. Growth of the other species was more variable. Isolates ofThelephora terrestris and Lactarius rufus varied in their ability to use bovine serum albumen (BSA) but twoLaccaria species were poor at using the protein as a nitrogen source. The ability of mycorrhizal and non-mycorrhizal Pinus contorta Dougl. ex Loud, plants to utilize BSA was also examined. Non-mycorrhizal plants andmycorrhizal plants infected with either T. terrestris or the unidentified pink ectomycorrhizal symbiont weresupplied either with ammonium or with BSA. Growth of plants supplied with BSA was significantly increased bymycorrhizal infection with the pink symbiont and not significantly different from that of plants supplied withammonium, but non-mycorrhizal plants were unable to use the protein as a nitrogen source and had significantlylower yields and nitrogen contents than infected plants. In contrast, mycorrhizal infection with T. terrestris hadno effect on growth or nitrogen contents of plants supplied with protein. The results are discussed in relation topossible physiological differences between ectomycorrhizal fungi occurring at different successional stages offorest development.
enable conversion of nitrogen into forms which areINTRODUCTION ... ... . , ^, w\/i * D . A*
more readily utilized by the root (Martin, KamstedtNitrogen availability is frequently a major factor & Sdderball, 1987). Experiments by Finlay et al.limiting forest growth and ectomycorrhizal fungi are (1988, 1989) suggest that much of the nitrogennow thought to contribute to the nitrogen nutrition absorbed by mycorrhizal roots may be in organicof their host plants in both a quantitative and form following assimilation of inorganic nitrogen byqualitative manner. The modified spatial geometry the mycorrhizal mycelium at sonie distance from theand increased surface area of ectomycorrhizal root root and translocation in the form of amino acids tosystems (Bowen, 1973) increases the efficiency of the host fungus interface.absorption and translocation of inorganic nitrogen to The capacity of ectomycorrhizal fungi to use morethe root (France & Reid, 1983). This may be recalcitrant, organically bound forms of nitrogen isparticularly important in situations where concentra- probably low (Lundeberg, 1970). Experiments bytions of available ammonium are low or where Leake & Read (1990) suggest that the ability to usediffusion is severely restricted in drier soils (Clarke & chitin as a sole nitrogen source is very limited inBarley, 1968). The effect of ectomycorrhizal fungi, ectomycorrhizal fungi compared with ericoid mycor-however, is not simply restricted to physical changes rhizal fungi. However, it is now established that thein the area and distribution of absorptive surfaces for ability of ectomycorrhizal fungi to utilize solublenutrient uptake. Ectomycorrhizal infection may also proteins is much greater than hitherto appreciated,alter the efficiency of nitrogen assimilation and Potential nitrogen sources thus include both simple
106 R. D. Finlay, A. Frostegard and A.-M. Sonnerfeldt
organic forms of nitrogen such as soluble aminoacids and peptides (Ahuzinadab & Read, 1988) andpolymeric forms such as soluble proteins(Abuzinadah & Read, 1986 a, b, 1989 a, b:Abuzinadah, Finlay & Read, 1986).
Ahuzinadah & Read (1986 a) identified markeddifferences in proteolytic ability between the speciesof fungi they tested and speculated that under certainsituations a succession of fungi of the type observedby Mason et al. (1983) might he predicted andexplained in terms of soil-derived selection pres-sures. The proteolytic capacity of fungi charac-teristically occupying different successional nichesmight thus be related to the changes in resourcequality taking place during forest development.Declining resource quality and accumulation oforganic matter during forest development shouldthus select against fungi with limited proteolyticcapacit\- and favour those which are able to competesuccessfully for limited, organic nitrogen sources.
Successions of mycorrhizal fungi during forestdevelopment have been reported widely (Masonet aL, 1982; Dighton & Mason, 1985; Dighton,Poskitt & Howard, 1986). This concept of 'mycor-rhizal succession' has led to the adoption of theterms 'early-stage' and 'late-stage" to describe fungicommonly found at these particular stages of forestdevelopment (Deacon, Donaldson & Last, 1983;Fleming, 1983, 1984; Last et al. 1983, 1984, 1985;Mason et al, 1983, 1987). Whilst there is generalagreement that changes in the diversity and com-position of the ectomycorrhizal flora can occur withtime there has heen much controversy as to whethertbese terms are appropriate or adequate. Interpret-ation of the sequential appearance of fruiting struc-tures is complicated by the fact that different fungiproduce fruiting structures at different ages (Harper& Webster, 1964), Furthermore, as many workershave pointed out, while tbe presence of reproductivestructures is always associated with the presence ofmycelia, tbe absence of fruit bodies does notnecessarily imply the absence of mycelia. One furthercaution against undue generalization is that theremay he important differences hetween species in firstrotation sites and those previously occupied hy trees(Mason et al. 1987).
Further experimentation is required to relatefunctional differences in physiology to edaphic sitecharacteristics. Information so far gathered on theproteolytic activity of ectomycorrhizal fungi isrestricted to a relatively narrow range of species. Theobject of the present study was to examine a widerrange of ectomycorrhizal fungi characteristicallyoccupying different successional stages of forestdevelopment and to evaluate their nitrogen metab-olism with respect to their ability to utilize organicnitrogen sources.
MATERIALS AND METHODS
Fungal species
The fungal species used were Laccaria bicolor,Laccaria proxima, Thelephora terrestris, andHebeloma crustuliniforme (typically found in earlystages of forest development), Paxillus involutus(occurring in both early and late stages of forestdevelopment), Piloderma croceum, Lactarius rufus,Suillus variegatus (often found in later stages offorest development) and unidentified pink and whiteisolates from two different pine stands 40 and 130years old. Two isolates of each species were usedexcept for L. bicolor, and the unidentified pink andwhite isolates. Authorities and isolation details,where available, are given in Table 1.
Experiment 1
In the first experiment the growtb of a range ofdifferent fungi on different nitrogen sources wasexamined in pure culture using methods similar toAbuzinadah & Read (1986 a). A modified Melin-Norkrans liquid medium was used as the basalmedium. Ammonium and malt extract were omittedfrom the medium and the different nitrogen sourceswere added individually to the medium to provide afinal concentration of 60 mg 1"̂ N. The nitrogensources chosen were ammonium sulphate(0-284 g 1"̂ ), calcium nitrate (0-5 g I"'), and bovineserum albumen (BSA, molecular weight 67000, Ncontent 16°o) 0-375 gl" ' . Glucose was added as thesole carbon source at 3 g 1~̂ for the ammonium andnitrate treatments and 2-62 g T' for BSA to give afinal carbon to nitrogen ratio of 20:1. Tbe mediacontaining inorganic nitrogen were adjusted topH 4-5 by addition of H^SO^ or NaOH and thenautoclaved at 0-7 atm for 30n:iin. The BSA wasdissolved in a portion of the basal medium, adjustedto pH 4-5, and added to autoclaved basal mediumthrough a 0-2 ftm millipore filter. Eight fungal specieswere used, L. bicolor, L. proxima, H. crustuliniforme,T. terrestris, S. variegatus, P. croceum, P. involutusand an unidentified pink strain, isolated from a 40-year-old pine stand. This isolate formed a mycor-rhizal association with pine which has been classifiedas Pinirhiza rosea hy Uhl (1988). Discs of fungalinoculum were cut with a 6 mm diameter cork borerfrom the edge of actively growing 2-3 week oldcolonies on agar plates, The discs of inoculum weretransferred under aseptic conditions to sterile Petridishes and placed on centrally positioned droplets ofsterile liquid agar to prevent movement of theinoculum, and to ensure that the fungal plug was justabove the surface of the liquid medium. As soon asthe agar had solidified, 20 ml of liquid mediumcontaining the different nitrogen sources were addedto each Petri dish. There were fifteen replicate disbesof 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, whereavailable
Species Isolate Details of isolation, host and origin
Heheloma crustuliniforme(Bull, ex St. Amans.) Quel
Hebeloma crustuliniforme
Laccaria bicolor(R. Mre). Orton
Laccaria proxima (Boud.) Pat.
Laccaria proxima
Paxillus involutus(Fr.) Fr
Paxillus i?ivolutus
Piloderma croceumErikss. & 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
85.021 Isolated from fruitbody under Fagus sylvatiea, Amance, France(CNRF, Nancy)
85.023 Isolated from spores from a fruitbody under 5-year-oldPicea sitchensis on a brown earth soil
S-238a Isolated from fruitbody under Tsuga mertensiana stand CraterLake National Park, Oregon, USA
S-472 Isolated from fruitbody under Pinus contorta stand, TillamookCo., Oregon, USA
87.018 Re-isolated from 2-year-old Picea sitchensis plants in a potexperiment. Bush Estate, Scotland
87.017 Isolated from a fruitbody in a coal waste with 15-30-year-oldBetula pendula trees, Midlothian, Scotland
86.006 Isolated from a fruitbody under a 20-year-old plantation ofPicea abiesjPicea sitchensis
85.009 Isolated from roots of Pinus sylveslris under a 40-year-oldstand Torrmyra, Sweden
85.029 Isolated from roots of Pinus syhestris under a 40-year-oldstand Torrmyra, Sweden
88.009 Isolated from fruitbody under a 110-year-old stand ofPinus syhestris, Istaby, Sweden
88.007 Isolated from fruitbody under a UO-year-old stand ofPinus syhestris, Istaby, Sweden
87.016 Isolated from roots of Picea sitchensis ex R. Jackson85.001 Isolated from roots of Pinus sylvestris Glasfynydd forest, UK
Lr 88-01 Isolated from fruitbody under 80-year-old Pinus sylvestrisstand with Picea ahies & Betula pendula, Nasien, Sweden
85.013 Isolated from fruitbody under a 40-year-old stand ofPinus syhestris, Torrmyra, Sweden
88.015 Isolated from roots of Pinus syhestris under a 40-year-oldstand, Torrtnyra, Sweden
88.016 Isolated from roots of Pinus sylvestris under a 130-year-oidstand, Kroksbo, Sweden
The dishes were incubated at 18-20 °C in sealedplastic bags and five replicate dishes from eachtreatment combination were harvested after 20, 40and 60 days of incubation. The agar plug was cutaway and the fungal colony carefully transferred topre-weighed pieces of aluminum foil. These wereoven dried at 80 °C and the fungal dry weightsrecorded. The pH value of the medium was alsomeasured after the final harvest.
Experiment 2
A second experiment was conducted using a slightlywider range of organic nitrogen sources. The basalmedium was as for Experiment 1 above. Eightdifferent nitrogen sources were chosen; ammonium,nitrate and BSA, as above, plus alanine and as-paragine (neutral amino acids), glutamic acid (anacidic amino acid), arginine (a basic amino acid) andthe protein gliadin, an ethanol-soluble proiamine(molecular weight 30000, N content 14%). The last6 compounds were added in appropriate amounts to
autoclaved medium (pH 45) by sterile filtrationthrough a 0-2 fiva millipore filter to give 60 mg N 1~̂ .Appropriate amounts of glucose had previously beenadded to give a constant C: N ratio of 20:1. Thefungal species examined were, P. ini^olutus (87.017),T. terrestris (87.016) and the unidentified whiteisolate. Time courses of fungal growth wereexamined for each species and the different specieswere harvested at the time-points corresponding tomaximum biomass production. These were 20, 30and 40 days respectively. The methods used were asin Experiment 1 above. Five replicate plates wereused for each combination of nitrogen treatment andspecies.
Experiment 3
In a third experiment the ability of mycorrhiza] andnon-mycorrhizal Pinus contorta plants to use BSA asa nitrogen source was tested. The methods used inthis experiment followed those described byAbuzinadah, Finlay and Read (1986). Seeds of P.contorta were surface sterilized with hydrogen per-
108 R. D. Finlay, A. Frostegdrd and A.-M. Sonnerfeldt
oxide and placed on water agar until germinationoccurred. Erlenmeyer flasks (250 ml) were filled with150 ml perlite and 50 ml of a modified Melin-Norkrans 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 timezero, prior to application of the main nitrogenregimes, in order to facilitate mycorrhiza synthesis.Flasks which were subsequently to receive am-monium were supplied with 1 mg N 'starter' nitro-gen as ammonium sulphate. All flasks were thenautoclaved and those flasks to receive BSA weresupplied with 1 mg N as arginine, added by milHporefiltration (0-2 firo). At time zero half of the flaskswere inoculated with discs of fungaJ mycelium takenfrom the actively growing margins of agar platecultures of the unidentified pink isolate. Afterallowing 20 days for growth of the fungus, threeaseptically germinated pine seedlings were added toeach fiask and a further period of 40 days wasallowed for the dei'elopment oi mycorrhiza] in-fection. The main nitrogen treatments were thenapplied by adding 4 mg N, either as ammoniumsulphate or as BSA. The nitrogen sources weredissolved in 5 tnl of the basal MMN solution andadded by sterile filtration through a 0 2 //tn milliporefilter. There were thus four main treatments,mycorrhizal with ammonium or BSA(Amm/M,BSA/M) and non-mycorrhizal with am-monium or BSA (Amm/NM,BSA/NM). Two ad-ditional control treatments were set up concurrentlywith those above to examine the separate effects ofstarter nitrogen and BSA on non-mycorrhizai plants.In these treatments non-mycorrhizal plants receivedeither no nitrogen at all (NM/-N) or BSA but noinitial starter nitrogen (BSA/NM-S). There werethus six different treatments in total (Amm/M;BSA/M; Amm/NM; BSA/NM; NM-N;BSA/NM-S) and eight replicate flasks of each. Twoharvests were originally intended, but, owing tocontamination of some flasks, only 5-7 replicates ofeach treatment were available and one harvest wasfinally taken.
This experimental design was repeated in anadditional experiment using T. terrestris (87.016)instead of the pink isolate. In this experiment thetwo additional non-mycorrhizal controls (NM-N)and BSA/NM-S) were omitted and the nitrogen andmycorrhizal treatments were combined in a simplefactorial design with 5 replicates of each treatment.Plants from both experiments were harvested afteran additional 10 weeks growth following addition ofthe main treatment nitrogen. Plants were partitionedinto shoots and roots which were oven dried beforedetermination of dry weights. The total nitrogencontent of the seedlings was determined by flowinjection analysis following digestion in a 3:1 (v/v)mixture of HgSO, and nitrogen-free HgOj. The
mean nitrogen content of freshly germinated P.contorta seeds was determined in the same manner tocalculate the initial amount of N available to thegrowing plants.
RESULTS
Experiment 1
The dry weight yields of the fungi in the first pureculture study after 20, 40 and 60 days are shown inTables 2, 3 & 4 respectively. Although there wasconsiderable variation between strains of the samefungus some patterns of nitrogen utilization wereevident. Growth on ammonium was generally muchhigher than on nitrate, although this was less evidentat later harvests, possibly reflecting a time lag in theinduction of nitrate reductase activity. Certain fungi,including H. crustuliniforme, L. proxima and P.involutus, grew almost as well on nitrate as am-monium but L. bicolor and P. croceum showed onlyintermediate growth on nitrate. Individual isolates ofT. terrestris and S. variegatus grew well while otherisolates of these two species, L. rufus and theunidentified pink isolate were relatively poor atutilizing nitrate. Patterns of protein utilization werealso evident. Final yields on BSA were as high, orhigher than those on ammonium for P. croceum, S.variegatus and the pink isolate. This was true despiteiarge overall variation in growth rates both betweenindividual species and between different isolates ofthe same species. H. crustuliniforme grew well onBSA with yields comparable to those on ammoniumin both isolates. P. involutus and T. terrestris bothgrew well on BSA, although individual isolates ofboth species clearly differed in their capacity toutilize the protein. L. bicolor and the two isolates ofL. proxima, species both typical of early successionalstages of forest development, both grew poorly onBSA. Final pH values of the media are shown inTable 5. Not surprisingly there was an overalldecrease in pH in the ammonium treatments and anoveral] increase in the nitrate treatments. The pHchanges were generally smaller with BSA as the Nsource. There was no apparent relationship betweenfungaj yieJd and the size and direction of the pHshifts.
Experiment 2
The maximum dry weight yields and growth rates ofthe three ectomycorrhizal fungi used in the secondpure culture study are shown in Figures 1 a & 1 6. Allthree species grew well on ammonium. Faxillusinvolutus produced final yields approximately 50%lower on nitrate, whilst Thelephora terrestris and theunidentified white isolate grew poorly on thisnitrogen source. Growth on the single amino acidnitrogen 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 daysgrowth in liquid media containing different nitrogen sources. Figures inparentheses indicate standard errors. Yields followed by differentsuperscripts within rows are significantly different atP< 0.05 according toFisher's protected least significant difference
Fungal species
Hebeloma crustuliniformeHebeloma crustuliniforme
Laccaria bicolorLaccaria proximaLaccaria proxima
Paxillus ini'olutusPaxillus involutus
Piloderma croceumPiloderma croceum
Suillus variegatusSuillus variegatus
Thelephora terrestrisThelephora terrestris
Unidentified pink isolate
Lactarius rufusLactarius rufus
Nitrogen
Strain
85.02185.023
S-238aS-47287.018
87.01786.006
85.00985.029
88.00988.007
87.01685.001
88.015
Lr 88-0185.013
source
Ammonium
7'5(l-0r5-9 (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)''
Nitrate
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 daysgrowth in liquid media containing different nitrogen sources. Figures inparentheses indicate standard errors. Yields followed by differentsuperscripts within rows are significantly different at F < 0.05 according toFisher's protected least significant difference
exceptions. T. terrestris grew poorly on glutamic acidwhile P . involutus showed reduced growth on alanineand no growth at all on asparagine. Growth onarginine was good and P. involutus showed thehighest growth rate of all the tested fungi on this
nitrogen source. There were clear differences in thecapacity of the different fungi to use the proteins asnitrogen sources. P. involutus and the unidentifiedwhite isolate both grew well on BSA, producingyields which were even higher than those on
110 R. D. Finlay, A. Frostegdrd and A.-M. Sonnerfeldt
Table 4. Yields {mg dry weight) of 9 different fungal species after 60 daysgrowth in liquid media containing different nitrogen sources. Figures inparentheses indicate standard errors. Yields followed by differentsuperscripts within rows are significantly different a/ P < 0.05 according toFisher's protected least significant difference
Fungal species
Hebeloma crustuliniformeHebeloma crustuliniforme
Laccaria bicolorLaccaria proximaLaccaria proxima
Paxillus involutusPaxillus involutus
Piloderma croceumPiloderma croceum
Suillus variegatusSuillus variegatus
Thelephora terrestrisThelephora terrestris
Unidentified pink isolate
Lactarius rufusLactarius rufus
Nitrogen
Strain
85.02185.023
S-238aS-47287.018
87.01786.006
85.00985.029
88.00988.007
87.01685.001
88.015
Lr 88-0185.013
source
Ammonium
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)''
Nitrate
] 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 (0-4r3 0(0-3)^
BSA
17-1 (17)"16-1 (0-1)°
4-3 (0-2)'-9'1 (0-5r4-4 (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)"
Table 5. Final pH values of liquid culture media containing differentnitrogen sources following growth of 9 different fungal species for 60 days.Standard errors (n = 5) were all < 0-1 pH unit
ammonium. T h e same fungi were also able to growon gliadin although the final yields were slightlylower. T h e growth rates of P . involutus were fasterthan those of the white isolate on both proteins. Incontrast, the third fungus tested, T. terrestris, grewvery poorly on both BSA and gliadin.
Experiment 3
The dry weight yields of P. contorta plants grown inassociation with the unidentified pink isolated areshown 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
- 10
(a)
- (W> •
•a
£
in
oc_,.cDJ
1,2
1,0
0,8
0,6
0,4
0,2
0,0I
amm nit asn BSA Gliadinarg glu
N sourceFigure 1. {a) Histogram showing maximum dn- weightyields of three different ectomycorrhizal fungi grown on avariety of different nitrogen sources; ammonium sulphate(amm). calcium nitrate (nit), alanine (ala), arginine (arg),glutamic acid (glu), asparagine (asn), bovine serumalbumen (BSA) and gliadin. The fungi, Paxillus involutus,( • ) Thelephora terrestris ( 0 ) and an unidentified whiteisolate ( • ) were harvested after 20, 30 and 40 days,respectively. Bars indicate standard errors, (h) Histogramshowing the rates of dry weight increase per day of thethree ectomycorrhizal fungi in Figure 1 a.
ammonium as the sole nitrogen source but there wasno significant difference between mycorrhizal plantsgrown with BSA as the sole nitrogen source and non-mycorrhizal plants grown with amtnonium. Non-mycorrhizal plants grew significantly less well whenBSA was the sole nitrogen source and the yields didnot differ significantly from those of non-mycorrhizal plants grown with no nitrogen (NM-N),suggesting that plants were unable to utilize BSA asa nitrogen source. The yields of non-mycorrhizalplants grown on BSA with no 'starter" nitrogen(BSA/NM-S) did not differ significantly from thoseof similar plants grown with 'starter' nitrogen,suggesting that the effect of this additional 'starter'nitrogen on final yields was insignificant. Differencesin root weight between mycorrhizal and non-mycorrhizal plants grown on BSA were larger thanthose between shoot weights. The larger contri-bution of differences in root yield to overalldifferences in total plant yield suggests direction ofresources into root growth in response to nutrientstress. The root; shoot ratio of mycorrhizal plantssupplied with ammonium was lowest, suggestingthat these plants were least nutrient stressed. The
„ 30
- 20
o 10o
BSA/NM-S NM/-N Amm/M Amm/NMBSA/MBSA/NM
BSA/NM-S NM/-NAmm/MAmm/NMBSA/IVIBSA/NM
Ic)
BSA/NM-S NM/-N Amm/MAmm/NM BSA/M BSA/NMFigure 2. Histograms showing dry weight yields of Pinuscontorta plants grown in association with an unidentifiedpink ectomycorrhizal isolate on media containing am-monium (Amm) or BSA as the sole nitrogen source. Plantswere either non-mycorrhizal (NM) or mycorrhizal (M);NM-N indicates non-infected plants with no nitrogen,BSA/NM-S indicates non-mycorrhiza! plants supphedwith BSA but no starter nitrogen, (a) shoot weight, (b) rootweight, (c) total weight. Bars indicate standard errors.DifFerent letters above the bars indicate a significant {P <0-05) difference between the yields (Duncan's MultipleRange test).
shoot and root nitrogen concentrations of the plantsare shown in Figure 3 a. The levels of nitrogen in thetissues of mycorrhizal plants which had receivedammonium were highest at approximately 11'7 and9-9 mg g"̂ for shoots and roots respectively. Theshoot N levels in non-mycorrhizal plants receivingammonium were not significantly different but theroot levels were significantly lower (P<0 '01) .Mycorrhi2al plants receiving BSA had intermediatelevels of nitrogen which were lower than mycorrhizalplants receiving amnnonium but not significantlydifferent from the levels in roots of the non-mycorrhizal plants receiving ammonium. Non-mycorrhizal plants supplied with BSA had the lowestnitrogen concentrations which were not significantlydifferent from those of non-mycorrhizal plants whichhad received no nitrogen at all. The addition of thesmall amount of starter nitrogen also had nosignificant effect on the final levels of nitrogen. Thetotal nitrogen contents of the plants are shown inFigure 36 where they are related to the initial
R.D. Finlay, A. Frostegdrd and A.-M. Sonnerfeldt
(a) (a)Pink isolate
ro
12
10
o
4
2
0BSA/NM-S NM/-N Amm/M Amm/NM BSA/M BSA/NM
Pink isolate
Seed Ncontent
BSA/NM-S NM/-N Amm/M Amm/NM'BSA/M BSA/NM
Figure 3. (o) Histogram showing the nitrogen concen-trations in shoots ( 0 ) and roots ( P ) of Pinus contortaplants grown m association with an unidentified pinkectomycorrhizal isolate on media containing ammonium(Amm) or BSA as the sole nitrogen source. Plants wereeither non-mycorrhizai (NM) or mycorrhizal (M); NM-Nindicates on-infected plants with no nitrogen, BSA/NM-S indicates non-mycorrhizal plants supplied with BSA butno starter nitrogen. Bars indicate standard errors, (b) His-togram showing the total nitrogen contents of the sameplants as in Figure 3 a. The horizontal line indicates theinitial nitrogen content of the seed, the standard error ofwhich is too small to be displayed.
nitrogen contents of the seeds. The non-mycorrhizalplants supplied with BSA showed no net assimilationof nitrogen from the substrate. The other non-mycorrhizal controls receiving no nitrogen at all, orBSA but no starter nitrogen, also contained totalamounts of N which were not significantly differentfrom the initial seed content. Mycorrhizal plantssupplied with ammonium contained the largestamounts of nitrogen followed by the non mycorrhizalplants receiving ammonium. The mycorrhizal plantssupplied with BSA assimilated significant quantitiesof nitrogen and had final contents which were notsignificantly different from the non-mycorrhizalplants supplied with ammonium. Levels of mycor-rhizal miection were similar in plants receii'ing thedifferent nitrogen treatments, making it unlikely thatdifferences in the degree of infection could haveinfluenced the results.
Patterns of growth and nitrogen utilization in theplants infected with T. terrestris were different. Thedry weight yields are shown in Figure 4a, b, c.Mycorrhizal plants supplied with ammonium againhad the highest total dry- weights but none of the
Amm/M Amm/NM BSA/M BSA/NM
20 r
Amm/M Amm/NM BSA/M BSA/NM
(c)
40
^ 30
I 20
10
Amm/M Amm/NM BSA/M BSA/NMFigure 4. Histograms showing dry weight yields of Pinuscontorta plants grown in association with the ectomycor-rhizal fungus Thelephora terrestris on media containingammonium (Amm) or BSA (BSA) as the sole nitrogensource. Plants were either non-mycorrhizal (NM) ormycorrhizal (M) (a) shoot weight, (b) root weight, (c) totalweight. Bars indicate standard errors. Different lettersabove the bars indicate a significant (P < 0-05) differencebetween the yields (Duncan's Multiple Range test).
other three treatments differed significantly fromeach other. The root/shoot ratio of non-mycorrhizalplants supplied with ammonium was lower than inother treatments but mycorrhizal infection had noeffect on the yields of plants supplied with BSAdespite the fact that levels of mycorrhizal infection inplants supplied with ammonium and BSA weresimilar. The levels of nitrogen in roots and shoots ofplants supplied with ammonium (Fig. 5 a) weresimilar for both mycorrhizal and non-mycorrhizalplants. Roots of non-mycorrhiza! plants were smallerand had slightly higher concentrations of N. Plantssupplied with BSA had consistently lower concen-trations of root and shoot nitrogen, irrespective ofwhether or not they were mycorrhizaJ. The totalamounts of nitrogen are shown in relation to seednitrogen content in Figure 5 b. Ammonium treatedplants showed assimilation of significant amounts ofthe added nitrogen whereas plants supplied withBSA showed no significant assimilation of nitrogen
Utilization of organic and inorganic N by ectomycorrhizal fungi
(a)
20
10
Am m/M Amm/N M BS A/M BSA/NM
\b)
Seed N content
100 •
Amm/M Amm/NM BSA/M BSA/NMFigure 5. {a) Histogram showinjf the nitrogen concen-trations in shoots ( ^ ) and roots (^ ) of Pinus cnntortaplants grown in association with Thelephora terrestris onmedia containing ammonium (Amm) or BSA (BSA) as thesole nitrogen source. Plants were either non-mycorrhizai(NM) or mycorrhizal (M). Error bars indicate standarderrors, {b) Histogram showing the total nitrogen contentsof the same plants as in Figure 5 a. The horizontal lineindicates the initial nitrogen content of the seed, thestandard error of which is too small to be displayed.
supplied in this form, irrespective of their mycor-rhizal status.
DISCI'SSION
The results of the present study confirm those ofAbuzinadah & Read (1986a) and Abuzinadah, Finlay& Read (1986) and extend them using a w'ider rangeof fungal species. Results from Experiment 1 confirmthe ability of H. crustuliniforme to utilize protein(Abuzinadah & Read, 19866, 1989a, b). The data forP. involutus confirm previous experiments (e.g.Lundeberg, 1970) in that the fungus was able toutilize nitrate as a sole nitrogen source. The inabilityof P. inziolutus to utilize asparagine in the presentstudy supports previous observations (Laiho, 1970;Lundeberg, 1970; Finlay et al., 1988) that asparagineand aspartic acid are poor nitrogen sources for thisspecies. The confounding effect of pH shifts com-plicate interpretation of the relative grow t̂h onnitrate and ammonium. The final pH values ofmedia containing protein may also have been abovethe optimum for the protease systems involved,although no relationship was evident between finalyields and the size or direction of the pH change.
Large differences were found in the ability of thefungi examined to utilize protein as a nitrogen
source. Differences in proteolytic capability may beimportant in determining the distribution of ecto-mycorrbizal fungi in time and space (Grier et al.,1981) and, as Abuzinadah & Read (1986fl) pointedout, may help to explain the types of 'succession'observed during forest development (Mason et al.,1983; Dighton & Mason, 1985). In soils withincreasing organic content selection for proteolyticactivity would be expected and this might thus bemore highly developed among fungi typically associ-ated with later stages of forest development thanthose which are common during the initial stages.This does not preclude the possibility of similarproteoiytic activity in 'early stage' fungi isolatedfrom soils with a high organic content relative to theavailable inorganic nitrogen. The proteolytic ca-pacitj' of a particular mycorrhiza] isolate may havepractical significance with regard to selection ofsuitable mycorrhizal inoculants and their persistencefollowing transplantation of inoculated trees to forestsoils of low fertility. Chu-Chou & Grace (1990)found that ectomycorrhizal isolates common onPinus radiata seedlings in relatively nutrient richnursery soils were frequently replaced by otherspecies following transplantation of seedlings toforest soils of lower fertility and this may, in part,reflect a poorly developed ability to use organicresources, leading to competitive exclusion by in-digenous species.
The variable nature of the results from the presentstudy highlights the need for further experimen-tation and for better information about the dynamicsof organic nitrogen sources in the forest soils fromwhich different fungi are isolated. In particular theresults indicate the need to avoid unwarrantedgeneralizations 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 inpure culture. In other experiments however,Dighton, Thomas & Latter (1987) found thatdecomposition of hide powder and cellulose washigher in the presence of Suillus luteus than Hebelomacrustuliniforme. In the experiments reported heretwo isolates of Laccaria proxima and one of L. bicolor(both considered to be ' early-stage' fungi) were poorat using protein as a nitrogen source, although otherworkers (Ahmad et al., 1990) have shown that L.bicolor can use a range of different amino acids as asole nitrogen source. Results from the pure culturestudies described here also clearly illustrate theimportance of the time of harvesting on the outcomeof growth experiments since, in some cases, therewere lag phases of different length prior to rapidutilization of the substrate.
Considerable differences in the ability to use BSAwere found between isolates. Isolate 87.016 ofThelephora terrestris grew well on BSA in pureculture whereas isolate 85.001 grew less well.
114 R. D. Finlay, A. Frostegard and A.-M. Sonnerfeldt
However the former isolate, when grow-n in as-sociation with P. contorta, appeared unable totransfer any nitrogen to the host plant. Similarresults were obtained by Abuzinadah & Read(1989a, b) working with Hebeloma crustuliniforme,Amanita muscaria and Paxillus involutus. In theirexperiments all fungi grew well on the protein BSAin pure culture but the obligately mycorrhizal fungiH. crustuliniforme and A. muscaria were moreefficient at transferring the assinnilated nitrogen totheir host plants than the facultatively mycorrhizalPaxillus involutus which is known to have somesaprotrophic capability and retained much of theassimilated N within its own tissues. In otherexperiments by Dighton et al. (1987) the effect ofthemycorrhizal fungi Hebeloma crustuliniforme andSuillus luteits on the decomposition of three organicsubstrates was minimal in the absence of host plantroots but the presence of roots considerably en-hanced decomposition. These results underscore theimportance of working with intact mycorrhizalassociations as w-ell as pure cultures when attemptingto determine the importance of heterotrophic proteinassimilation to mycorrhizal plants.
There has been much speculation about physio-logical differences which might exist between ecto-mycorrhizai fungi present at different successionalstages of forest development but fewer experimentalstudies and little attempt made to relate physiologicalproperties of ectomycorrhizal fungi to the edaphiccharacteristics of the sites from which they areisolated. This in part is due to restricted informationabout the dynamics and pool sizes of different soilorganic nitrogen sources. Culture experiments andobservations of fruit body size suggest that earlystage fungi have a lower dennand for, or reducedaccess to, host derived assimilates. Gibson & Deacon(1990) examined establishment of mycorrhizal rootsin aseptic culture in relation to effects of differentnutrients. In the presence of adequate mineralnutrients 'early-stage' mycorrhizal fungi formedmycorrhizal roots at low or moderate glucose levelswhilst four of the five ' late-stage' isolates testedrequired moderate or high glucose levels for suc-cessful mycorrhizal development. In other experi-ments low P levels suppressed mycorrhiza formationby late stage fungi more than by early stage fungialthough low levels of N suppressed mycorrhizaformation in all ofthe tested isolates. The apparentlyhigh glucose dependence of late stage fungi may alsoexplain the efficiency of infection by mycelia growingfrom food bases provided by established trees. Theimportant role of the mycorrhizal mycelium as asource of inoculum and the potential for photo-synthetic assimilate movement through mycelialstrands connecting root systems have been discussedby Read, Francis & Finiay (1985) and Finiay & Read(1986). There is now also evidence that some of thecarbon cost of mycorrhizal infection may be met by
heterotrophic carbon assimilation from organicsources.
The general implications of the above processesfor nutrient cycling may be broad (Read, Leake &Langdaie, I9S9; Dighton & Boddy, 1989), but theyhave particular bearing on the successful selection ofappropriate mycorrhizal fungi for optimization offorest tree growth in different soils and at differentstages of forest development. Further systematicinvestigation of differences in physiological activityin relation to measured soil parameters is animportant priority in future research.
ACKNOWLEDGEMENTS
Financial support from the Swedish National Environ-mental Protection Agency is gratefully acknowledged.
REFERENCES
ABVZINADAH. R . A . & RE.'\D, D . J. (1986ai. The role of proteins inthe nitrogen nutrition of eciomycorrhizaJ p]anT:s. J. VtUizationof peptides and proteins by ectomycorrhizal fungi. NewPhytologist 103,-iHi^93.
ABUZiNAr>,\H, R, A. & READ, D , J, (1986fr). The role of proteins intbe nitrogen nutrition of ectomycorrhizal plants, HI, Proteinutilization by Betula, Picea and Pinus in mycorrhizal associationwith Hebetoma crustuliniforme. New Phytologist 103, 506 514,
ABI'ZINID,*H, R, A, & READ, D , J, (1988). Amino acids as nitrogt-nsources for ectomycorrhizal fungi: utilization of individualamino acids. Transactions of the British Mycological Society 91.473^79,
.-\BI'ZINADAH. R, .A. & READ, D . J, (1989a). The role of proteins inthe nitrogen nutrition of ectomycorrhizat plants. IV. Theutilization of peptides by birch {Betula pendula L.) infectedwith different mycorrhizal fungi. Nezv Phytologist 112, 55-60.
ABIZINADAH, R . A . & READ, D . J, (19896), Tbe role of proteins inthe nitrogen nutrition nf ectomycorrhizal plants, V, Nitrogentransfer in bircb {Betula pendula L,) grown in association withdifFereni mycorrhizal and non-mycorrhizal fungi, NeK Phy-totogist 112, (J1-68,
ABi-ztNvmAH, R, A,, FiNL.'VY, R, D. & RE.^D, D , J , ( 1 % 6 ) , The roleof proteins in the nitrogen nutrition of ectomycorrhizal plants,II, Utilization of protein by mycorrhizal plants of Pinuscontorta, Neto Phytohgisi \ti^, 49.S-506.
AHMAD, 1., C.-VRLETON, T . J. ,MAI,I.OCH, D . W , & HEI.LEBUST, J, A,(1990), -Nitrogen metabolism m the ectomycorrbizal fungusLaccaria bicolor (R, Mre,) Orton. New Phytologist 116, 431-441.
BOWEN, G . D . (1973). Mineral nutrition in ectomycorrbizae. In:Ectomycnrrhizae (Ed. by G. C. Marks & T. T. Koslowski) pp.151-205, Academic PresK, New V'ork.
CHLT-CHOL-, M . & GKACE, L . J . (1990). Mycorrhiza] fungi ofradiata pine seedlings in nurseries and trees m forests. SoilBiology and Biochemistry 22, 959-966.
CLARKE. A. L, & BARLEI*, K . P. (1968), The uptake of nitrogenfrom soils in relation to solute diffusion, Australian Journat ofSoil Research 6, 75-88.
DE.'VCON. J , W , , DONALDSON. S, J, & LAST, F , T . (1983),Sequences and interactions of mycorrhizal fungi on birch. Plantand Soil 71, 257-262.
DIGHTON. J, & BODDY, L . (1989). Role of fungi in nitrogen,phosphorus and sulphur cycling in tt-mperate forest ecosystems.In ; Nitrogen, Phosphorus and Sulphur Utilization by Eungi (Ed.by L, Boddy, R. Marchant & D. J. Read), Symposium of theBritish Mycological Society, Cambridge University Press, pp.269-298.
DIGHTON, J., POSKITT, ] . M. & HOWARD, D . (1986). Changes inoccurrence of hasidiomycete fruit bodies during forest standdevelopment: with special reference to mycorrhizal species.Transactions of the British Mycological Society 87, 163-171.
DIGHTON, J., THOMAS, E . D . & LATTER, P. M (1987). Interactions
Utilization of organic and inorganic N by ectomycorrhizal fungi 115
betwet'n tree roots, mycorrhizas, a saprotrophic fungus and thedecomposition of organic substrates in a microcosm. Biologyand Fertility of Soils 4, 145-150.
DlGHTOt<. J. & MASON, P. A. (1985), Mycorrhiza] dynamicsduring forest tree development. In: Development Biology ofHigher Fungi (Ed. by D. Moore, L. A. Casselton, D. A. Wood& J. C. Frankland, Symposium No. 10 of The British Myco-lcgica! Society, Cambridge University Press, Cambridge.
FINLAY, R. D , , EK, H . , OOHAM, G . & S6DERSTB6M, B, (1988).Myeelial uptake, translocation and assimilation of nitrogenfrom '^N-labelled ammonium by Pinus syivestris plantsinfected with four difTerent ectomycorrhizal fungi. New Phy-tologist 110, 59-66.
FiNi-AY, R. D., EK, H , , ODHAM, G . & SODERSTROM, B . (1989).Uptake, translocation and assimilation of nitrogen from '' 'N-labelled ammonium and nitrate sources by intact ectomycor-rbizal systems of Fagus sylvatica infected with Paxillusinvolutus. Nezv Phytologist 113, 47-55.
FiNi-.'VY, R. D. & READ, D . J. (1986). The structure and functionof the vegetative mycelium of ectomycorrhizal plants. I.Translocation of ^^C-labelled carbon between plants inter-connected by a common mycelium. Netv Phvtologist 103,143-15b.
FLEMING, L . V. (1983). Succession of mycorrhizal fungi on birch;infection of seedlings planted around mature trees. Plant andSoil 71. 263-267.
FLEMING, L . V. (1984). Effects of soil trenching and coring on theformation of ectomycorrhizas on birch seedlings grown aroundmature trees. .Vcw Phytologist 98, 143-153.
FRINGE, R. C. & REID, C. P. P. (1983). Interactions in nitrogenand carbon in the physiology of ectomycorrhizae. CanadianJournal of Botany 61, 964-984.
GIBSON, F . & DEACON, J. W. (1990). Establishment of ectomycor-rhizas in aseptic culture: effects of nitrogen and phosphorus inrelation to successions. Mycological Research 94, lAfi-172.
GRIER, C . C , VQGT, K . A . , KEYES, M . R. & EDMONDS, R. L .(1981). Biomass distribution of above- and below-groundproduction in young and mature Abies antabilis zone ecosystemsof the Washington Cascades. Canadian Journal of ForestResearch 11, 155 169.
HARPER, J. E. & WEBSTER, J. (1964). An experimental analysis ofthe coprophiious fungal succession. Transactions of the BritishMycological Society 47, 511-530,
LAIHO, O . (1970). Paxillus involutus as a mycorrhizal symbiont offorest trees. Acta Forestalia Fennica 106, 1-65.
, F. T., MASON, P. A., W'ILSON, J, & DEACON, J. W. (1983).
Fine roots and sheathing mycorrhizas: their formation, functionand dynamics. Plant and Soil 71, 9-21.
LAST, F . T . , M.'LSON, P , A., INGLEDY, K . & FLEMING, L . V.{] 984). Succession of fruitbodies of sheathing mycorrhizalfungi associated with Betula pendula. Forest Ecology andManagement 9, 229-234.
LAST, F . T , , MASON, P A., WILSON, J., INGLEBY, K . , MLNRO,R. C , FLEMING, L . V. & DEAGON, ] . W". (1985). Epidemiologyof sheathing (ecto-)mycorrhizas in unsterile soils: a case studyof Betula pendula. Proceedings of the Royal Society of Edinburgh85B, 299-315.
LEAKE, J, R. & READ, D . J. (1990). Chitin as a nitrogen source fornnycorrhizal fungi. Mycological Research 94, 993-994.
Li.'NDEBERG, J ,R. & READ, D . J . (1990). Utilization of variousnitrogen sources, in particular bound soil nitrogen, by mycor-rhizal fungi. Studia Forestalia Suecica 79, 1-95.
MARTIN, F , , RAMSTEDT, M . & S5DEBHALL, K . (1987). Carbon andnitrogen metabolism in ectomycorrhizal fungi and ecto-mycorrhizas. Biochemie 69, 569-581.
MASON, P. A., LAST, F . T . , PEI.HAM, J. & INGLEBY, K . (J982).Ecology of some fungi associated with an ageing stand ofbirches (Betula pendula and B. pubescens). Forest Ecology andManagement 4, 19-39.
MASON, P. A., WILSON, J., LAST, F . T . & W'ALKER, C . (1983). Theconcept of succession in relation ro the spread of sheathingmycorrhizal fungi on inoculated tree seedlings growing inunsterile soil. Plant and Soil 71, 247-256.
MASON, P. A., LAST, F . T . , WILSON, J,, DEACON, J. W,, FLEMING,L. V. & Fox, F. M, (1987). Fruiting and successions ofeftornycorrbizal fungi. ]n; Fungal fnfection of Plants (Ed. byG. F. Pegg & P. G. Ayers), Cambridge University Press, Cam-bridge.
READ, D . J., FRANCIS, R. & FINLAY, R . D . (1985). Mycorrhiza!mycelia and nutrient cycling in plant communities. In:Ecological Interactions in Soil: Plants, Microbes t=f Animals.British Ecological Society Special Publication No. 4, pp.193 217.
READ, D.J, , LEAKE, J. R . & LANGDALE, A. R. (1989). Nitrogennutrition of mycorrhizas and their hosts. In: Nitrogen, Phos-phorus and Sulphur Utilization by Fungi (Ed. by L. Boddy, R.Marchant & D. J. Read), Symposium of the British Mycologica!Society, Cambridge University Press, pp. 181-204.
UHL, M . (1988). Identifizierung und Characterisierung rowEktomycorrhisen an Pinus sylvestris und von Ektomycorrhizenaud der Gattung Tricholoma. Doctoral Thesis, Ludwig-Maximilians Universitat, Munchen.
