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Short Communication

Intraspeci®c variation in nitrogen source utilisation by isolates of

the ericoid mycorrhizal fungus

Hymenoscyphus Ericae


Korf and Kernan

J.W.G. Cairney


*, N.A. Sawyer


, J.M. Sharples


, A.A. Meharg



Mycorrhiza Research Group, School of Science, University of Western Sydney (Nepean), PO Box 10, Kingswood, NSW 2747, Australia


Department of Plant & Soil Science, Cruickshank Building, University of Aberdeen, Aberdeen, AB24 3UU, UK

Received 17 August 1999; received in revised form 7 December 1999; accepted 12 January 2000


Variation in the abilities of 35 isolates of the ericoid mycorrhizal fungal endophyteHymenoscyphus ericaefrom two ®eld sites to utilise inorganic and organic nitrogen sources in axenic culture has been investigated. While most isolates showed a preference for NH4+ as a sole nitrogen source, considerable variation was observed in the abilities of isolates to utilise amino acids and

protein (BSA). In particular, large intraspeci®c variation was observed for glutamine and BSA utilisation, with some isolates thriving on these substrates while others produced little growth. The data suggest that individual isolates ofH. ericaemay vary considerably in their abilities to supply their host plants with nitrogen from di€erent substrates in soil.72000 Elsevier Science Ltd. All rights reserved.

Keywords:Ericoid mycorrhiza; Nitrogen utilisation;Hymenoscyphus ericae

Plants in the family Ericaceae are an important com-ponent of the northern hemisphere heathland ¯ora. These habitats are typi®ed by harsh environmental conditions that hinder decomposition and mineralis-ation processes, resulting in soils that are rich in recal-citrant organic matter, but de®cient in available mineral forms of nitrogen and phosphorus (Read, 1991). Formation of ericoid mycorrhizal associations by Ericaceae is seen as critical to their survival in such conditions (Straker, 1996). While several fungal taxa are probably involved in forming ericoid mycorrhizas with Ericaceae hosts (Hambleton et al., 1998), our cur-rent understanding of the functional importance of eri-coid mycorrhizal fungi relies heavily on data derived from the ascomycete Hymenoscyphus ericae. In par-ticular, the ability of H. ericae to enhance host

nitro-gen status via utilisation of amino acids and simple proteins (Bajwa et al., 1985; Bajwa and Read, 1986; Leake and Read, 1989) has received much attention and is widely regarded as the major functional bene®t conferred on the host by this type of mycorrhizal as-sociation (Smith and Read, 1997).

Physiological investigations of H. ericae have, how-ever, been limited to only a few strains, with most stu-dies relying upon a single isolate (see Cairney and Burke, 1998). We thus have a poor understanding of physiological variation in natural populations of the fungus. Studies of several ectomycorrhizal fungal taxa indicate that extensive intraspeci®c variation exists with respect to, for example, mycelial growth form, sensitivity to toxic metals and nitrogen source utilis-ation (see Cairney, 1999). Such variutilis-ation means that studies based on a single or a few isolates tell us little regarding the functional potential of particular mycor-rhizal fungal taxa and that studies of large numbers of isolates are required in order to give a reasonable

esti-Soil Biology & Biochemistry 32 (2000) 1319±1322

0038-0717/00/$ - see front matter72000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 0 2 5 - 0


* Corresponding author. Tel.: 859903; fax: +61-296-859915.


mate of their broader ecological functioning (Cairney, 1999). For this reason, and given the important roles that the fungus undoubtedly plays in the nitrogen nutrition of its Ericaceae hosts (see Smith and Read, 1997), we have investigated intraspeci®c variation in nitrogen source utilisation by isolates of H. ericae

obtained from ®eld sites in south-west England. Our results indicate that, although all isolates were able to utilise all inorganic and organic nitrogen sources, con-siderable intraspeci®c variation exists within the sampled group of isolates as a whole and that mean growth rates on all nitrogen sources varied signi®-cantly between the isolates from the two sites.

Isolates of Hymenoscyphus ericae (Read) Korf and Kernan were obtained from root systems of Calluna

vulgaris L. Hull at the abandoned Devon Great Con-sols arsenic/copper mine site (DGC), Tamar Valley (18 isolates) and a heathland site at Aylesbeare Common (AC) (17 isolates) (both Devon, UK) during January 1998. Single isolates from Crompton Forest, York-shire, UK (Read 101) and two isolates of Hymenoscy-phus sp. (probably H. ericae) from rhizoids of the leafy liverwort Cephaloziella exi¯ora (Taylor) Stephani collected in New South Wales, Australia and the Windmill Islands, Eastern Antarctica (AUST and ANT respectively; see Chambers et al., 1999) were also included for comparison.

Nitrogen utilisation was assessed by growth in axe-nic liquid cultures (25 ml), with the addition of (NH4)2HPO4, Ca(NO3)2, glutamine or bovine serum

albumen (BSA) and glucose to a basal medium to give a starting nitrogen concentration of 106 mg lÿ1and a C:N ratio of 39:1 (see Anderson et al., 1999), with the ®nal pH adjusted to 5.5. Following incubation for 17 days at 208C in the dark, mycelia were harvested, dried overnight at 808C and biomass determined. Data were corrected for growth due to nitrogen contained in the inoculum discs as described by Anderson et al. (1999). Data for each isolate were analysed separately by one way ANOVA and signi®cant di€erences between nitrogen treatments determined by Fisher's PLSD (Statview1

software). Mean data for each treat-ment and isolate were also used to plot frequency dis-tribution histograms for utilisation of each nitrogen source by the two groups of H. ericae isolates and compared using the Mann±Whitney U test (Statview1 software).

All isolates produced detectable biomass on all nitrogen sources (Table 1), con®rming the ®ndings of Bajwa and Read (1986) that H. ericae can utilise a range of inorganic and organic nitrogen substrates as a sole nitrogen source. The abilities of isolates to utilise individual nitrogen sources, however, varied consider-ably. Most isolates (74%), regardless of origin, pro-duced greatest biomass on NH4+, although in some

cases this did not di€er signi®cantly from NO3ÿ,

gluta-mine and/or BSA (Table 1). Five isolates (DGC12, DGC15, DGC18, AC3, AC10) grew equally well on the four nitrogen sources. The remaining isolates per-formed signi®cantly better on either BSA (DGC6, DGC7), glutamine (DGC11, AC1, AC14, ANT) or both substrates (DGC16, AUST), while HE101 showed a clear preference for growth on NO3ÿ

(Table 1). The majority of isolates (68%) produced sig-ni®cantly less biomass on NO3ÿ, glutamine or both

substrates, however, for some isolates growth on BSA and/or NH4+ was equally poor (Table 1). Isolates

DGC5, DGC9, DGC11, AC1 and HE101 were notable in that they grew signi®cantly more poorly on BSA than the other substrates, while DGC8 and DGC17 showed a signi®cant preference for the inorganic over

Table 1

Mean biomass yield (mg hÿ12se,n= 4) of 38 isolates ofH. ericae

following 17 days growth in axenic liquid cultures containing di€er-ent nitrogen sources. Yields followed by di€erdi€er-ent letters within rows are signi®cantly di€erent (P< 0.05) as determined by Fisher PLSD Isolate NH4


NO3ÿ Glutamine BSA

DGC 1 222.9214.0a 175.125.7ab 122.228.0b 193.4250.3a

J.W.G. Cairney et al. / Soil Biology & Biochemistry 32 (2000) 1319±1322


the organic substrates (Table 1). The extent of vari-ation in nitrogen source utilisvari-ation is similar to that observed previously for populations of ectomycorrhizal fungi (Laiho, 1970; Keller, 1996; Anderson et al., 1999), clearly suggesting that generalisations regarding ericoid mycorrhiza functioning cannot necessarily be inferred directly from observations of single H. ericae

isolates. It is particularly noteworthy that, although most isolates utilised BSA readily, several produced lit-tle biomass on this nitrogen source (Table 1, Fig. 1). While the ability to utilise protein±nitrogen is widely regarded as a key facet of ericoid mycorrhiza function-ing (Smith and Read, 1997), these data indicate that some H. ericae isolates are relatively de®cient in this respect. The extent to which this re¯ects di€erences in extracellular protease expression and regulation or an inability to absorb/assimilate the resulting amino acids remains to be investigated. Frequency distribution his-tograms of nitrogen source utilisation emphasise the

variation within the sampledH. ericae isolates (Fig. 1). The ranges of yields of the isolates as a whole were greatest for glutamine and BSA than for the inorganic nitrogen sources, with some isolates producing con-siderable biomass on the organic substrates, while others produced little or no growth. Isolates of H. eri-cae are thus likely to vary considerably in their ability to supply host plants with nitrogen from di€erent inor-ganic and orinor-ganic substrates in soil and such diversity may be important in maximising host nitrogen acqui-sition. Comparison of the groups of isolates from the two sites revealed that, although there was extensive inter-site overlap, mean biomass yields for the DGC mine site isolates were signi®cantly (P< 0.05) higher than for the AC isolates on all nitrogen substrates, suggesting that selection for isolates that are more e-cient in nitrogen assimilation may be occurring at the mine site. Further investigations of larger groups of isolates will be required to test this hypothesis.

Fig. 1. Frequency distribution (expressed as a percent of the population) of biomass increase on the four nitrogen substrates by isolates ofH. eri-caefrom roots ofC. vulgarisat the Devon Great Consols mine (Q) and Aylesbeare Common (q) sites. Arrows denote mean biomass increase on each nitrogen source for the groups of isolates from the two sites.



We thank Prof. D.J. Read for supplying us with iso-late HE101.


Anderson, I.C., Chambers, S.M., Cairney, J.W.G., 1999. Intra- and interspeci®c variation in patterns of organic and inorganic nitro-gen utilisation by three AustralianPisolithusspecies. Mycological Research 103, 1579±1587.

Bajwa, R, Abuarghub, S., Read, D.J., 1985. The biology of mycor-rhiza in the Ericaceae X. The utilization of proteins, and the pro-duction of proteolytic enzymes by the mycorrhizal endophyte and by mycorrhizal plants. New Phytologist 101, 469±486.

Bajwa, R., Read, D.J., 1986. Utilization of mineral and amino N sources by the ericoid mycorrhizal endophyteHymenoscyphus eri-cae and by mycorrhizal and non-mycorrhizal seedlings of

Vaccinium. Transactions of the British Mycological Society 87, 269±277.

Cairney, J.W.G., 1999. Intraspeci®c physiological variation: impli-cations for understanding functional diversity in ectomycorrhizal fungi. Mycorrhiza 9, 125±135.

Cairney, J.W.G., Burke, R.M., 1998. Extracellular enzyme activities of the ericoid mycorrhizal endophyte Hymenoscyphus ericae

(Read) Korf and Kernan: their likely roles in decomposition of dead plant material in soil. Plant and Soil 205, 181±192.

Chambers, S.M., Williams, P.G., Seppelt, R.D., Cairney, J.W.G., 1999. Molecular identi®cation of Hymenoscyphus sp. from rhi-zoids of the leafy liverwortCephaloziella exi¯orain Australia and Antarctica. Mycological Research 103, 286±288.

Hambleton, S., Currah, R.S., Egger, K.N., 1998. Phylogenetic re-lationships of ascomycetous root endophytes of the Ericaceae inferred from 18S rDNA sequence analysis. In: Ahonen-Jonnarth, U., Danell, E., Fransson, P., KaÊreÂn, O., Lindahl, B., Rangel, I., Finlay, R. (Eds.), Abstracts of the Second International Conference on Mycorrhiza. Swedish University of Agricultural Sciences, Uppsala, p. 78.

Keller, G., 1996. Utilization of inorganic and organic nitrogen sources by high-alpine ectomycorrhizal fungi of Pinus cembrain pure culture. Mycological Research 100, 989±998.

Laiho, O., 1970.Paxillus involutusas a mycorrhizal symbiont of for-est trees. Acta Forfor-estalia Fennica 106, 1±57.

Leake, J.R., Read, D.J., 1989. The biology of mycorrhiza in the Ericaceae XIII. Some characteristics of the extracellular protein-ase activity of the ericoid endophyteHymenoscyphus ericae. New Phytologist 112, 69±76.

Read, D.J., 1991. Mycorrhizas in ecosystems. Experientia 47, 376± 390.

Smith, S.E., Read, D.J., 1997. Mycorrhizal Symbiosis. Academic Press, London, p. 605.

Straker, C.J., 1996. Ericoid mycorrhiza: ecological and host speci-®city. Mycorrhiza 6, 215±225.

J.W.G. Cairney et al. / Soil Biology & Biochemistry 32 (2000) 1319±1322


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